For three men of my life who made it all possible: my Husband, my Father and my Grandfather, although his journey finished right when my PhD journey had started. Also, for my little Sebastian, whose journey has started when

This work would not have been possible without an enormous help of many people.

First and foremost, I would like to thank my supervisor Gwénaёl Rabut for his help, support and encouragement. He was always extremely generous with his time not only devoted to teaching me, both theory and practical work at the bench, but also to discussing and providing useful comments and feedback. I was extremely lucky to have an outstanding mentor like him, who apart from supervising was also advising, and more importantly listening and understanding.

I also thank Claude Prigent for his support, including a strong support in the organization of the Young Life Scientists' Symposium. My special thanks are extended to all the members of Cell Cycle team. It was indeed a privilege to work with all of you.

In particular, I owe a special debt to Gaёlle Le Dez, who was involved in various steps of my PhD project and whose work greatly contributed to the results obtained. I thank Jacek Kubiak and Erwan Watrin for valuable discussions.

There are also two people for who may not be enough words to express my gratitude.

First, I thank my best friend Blanca Gomez-Escoda, who was always there with me during the happiest moments and who was also helping me in the most difficult periods. This is when you know who your true friends are. Second, I am particularly grateful to Stéphane Dréano for doing lots of sequencing for this project but what is more helping me with everyday life situations and teaching me French. Merci car c'est grâce à toi que je peux parler français aujourd'hui.

Szczególne podziękowania składam Rodzicom oraz Mężowi za wsparcie, jakie od nich otrzymałam. Tomkowi również za troskę, opiekę, cierpliwość i wyrozumiałość.

Dziękuję za to, że jesteś. Sebastiankowi, iż pomimo dręczących go kolek pozwolił mi na dokończenie tego manuskryptu.

Last but not least, I thank Ministère de l'Enseignement supérieur et de la Recherche for funding of the three years of my PhD and giving me an opportunity to work on this interesting and perspective project, and La Ligue Contre Le Cancer for additional six months prolongation. I also thank all our international collaborators for their great contribution to the work on the functional part of this project.

ABSTRACT

ABSTRACT

Protein ubiquitylation is a post-translational modification that plays a crucial role in regulating many cellular functions, including cell growth and proliferation. Defects in this control mechanism cause cancer and other diseases. The ubiquitylation process involves a cascade of enzymatic reactions catalyzed by a family of structurally-related enzymes, namely ubiquitin activating enzymes (E1s), ubiquitin conjugating enzymes (E2s) and ubiquitin ligases (E3s). Interactions between E2s and E3s are in the centre of ubiquitylation cascade and it is a combination of particular E2/E3 pairs that determine what types of ubiquitin chains are made, thus determining the regulatory functions of the ubiquitin pathway. To date, only a small fraction of all possible E2/E3 pairs have been investigated, mainly using biochemical and in vitro approaches that may not accurately reflect the conditions that occur in living cells. We aimed to develop a method capable of detecting specific E2/E3 interactions under physiological conditions. Using budding yeast as a model organism, we found that the Bimolecular Fluorescence Complementation (BiFC) enables sensitive detection of the well described Ubc4/Ufd4 pair under endogenous conditions. The assay is specific since the interaction signal is lost in yeasts expressing Ubc4 mutants truncated in its E3 interaction domain.

We then used this system to further analyze the physiological network of E2 and E3 enzymes in living yeast. We performed a microscopy screen to assay all interactions between eleven E2s and 57 E3s/putative E3s. Our results show that approximately 20% of all E2/E3 combinations give a detectable BiFC signal. Few E3s interacted only with a single E2, whereas most E3s produced a BiFC signal with multiple E2s. Ubc13, Ubc1 and Ubc4 were found to be the most frequently interacting E2s. Our results match many examples from current literature but we also detected 95 new E2/E3 interactions. In particular we identified an interaction between the proteins Asi1 and Asi3 and E2s Ubc6 and Ubc7. Asi1 and Asi3 are known to form a complex (the Asi1/3 complex) at the inner nuclear membrane and are involved in the regulation of the response to extracellular amino acids. The Asi1/3 complex was suspected to function as a ubiquitin ligase, since the Asi1 and Asi3 proteins contain a RING domain, but its function as an E3 has previously not been demonstrated. We therefore further characterized it functionally. Prior to the discovery of ubiquitin, Hershko and Tomkins observed that the degradation of the enzyme tyrosine aminotransferase (TAT) in hepatoma culture cells was blocked by potassium fluoride, an inhibitor of cellular ATP production (Hershko and Tomkins, 1971). This had already given the first indication that ATP may be required for the ubiquitin proteolytic system. Ubiquitin itself was discovered by Gideon Goldstein in 1974 and was put forward as a thymopoietic hormone [START_REF] Goldstein | Isolation of bovine thymin: a polypeptide hormone of the thymus[END_REF]. In the following year a complete amino acid sequence of ubiquitin was published and it was suggested that this protein is universal in living cells [START_REF] Goldstein | Isolation of a polypeptide that has lymphocyte-differentiating properties and is probably represented universally in living cells[END_REF][START_REF] Schlesinger | The complete amino acid sequence of ubiquitin, an adenylate cyclase stimulating polypeptide probably universal in living cells[END_REF]. Still at that time, lysosome was assumed to be the organelle that degrades intracellular proteins. However, Etlinger and Goldberg further confirmed in 1977 the existence of a non-lysosomal and ATP-dependent proteolytic system responsible for the degradation of misfolded proteins in reticulocytes. All these laid the basis for seminal work on the ubiquitin proteasome system, dating back to the years 1978-1983 when the components of the ubiquitin system, including ubiquitin activating (E1), ubiquitin conjugating (E2) and ligating (E3) enzymes, were identified in the laboratory of Avram Hershko. Using biochemical fractionation and enzymology, he, together with his student Aaron Ciechanover and collaborator Irwin Rose, discovered that certain proteins are covalently conjugated to ubiquitin (initially named by them as ATP-dependent proteolysis factor 1, APF-1 -subsequently shown by [START_REF] Wilkinson | Ubiquitin is the ATP-dependent proteolysis factor I of rabbit reticulocytes[END_REF] to be indeed ubiquitin) -a process now termed 'ubiquitylation' and that ubiquitylated proteins are destroyed by an ATP-dependent protease in the extract of rabbit reticulocytes (Ciechanover et al., 1978;[START_REF] Hershko | Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATPdependent proteolysis[END_REF]. They went on to examine the characterization of those enzymes and demonstrated ubiquitin involvement in the regulation of protein degradation. For this discovery they were awarded the Nobel Prize in Chemistry in 2004. Later on, the laboratory of Martin Rechsteiner purified and characterized the ATP-dependent protease, now known as the 26S proteasome complex, which mediates destruction of ubiquitin conjugates [START_REF] Hough | Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates[END_REF]. This group of researchers also proposed for the first time that the smaller 20S protease is part of the 26S proteasome [START_REF] Hough | Purification of two high molecular weight proteases from rabbit reticulocyte lysate[END_REF]. In the 1980s, another set of discoveries was made in the laboratory of Alexander Varshavsky concerning the biological functions of the ubiquitin-proteasome system, i.e. its role in the cell cycle, DNA repair pathway, transcriptional regulation and protein synthesis as well as stress responses [START_REF] Finley | Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85[END_REF][START_REF] Finley | Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85[END_REF][START_REF] Varshavsky | The early history of the ubiquitin field[END_REF]. In the 1990s various ubiquitin conjugating (E2s) and ligating enzymes (E3s) were identified and non-proteolytic functions for ubiquitin were discovered, opening up a new era in the ubiquitin field. The aforementioned pioneering discoveries are presented in the timeline below (Figure 1).

RÉSUMÉ

INTRODUCTION 13

eukaryotes. ERAD consists of four steps, namely substrate selection, dislocation across the endoplasmic reticulum membrane, addition and subsequent removal of ubiquitin conjugates, and degradation by the 26S proteasome. Ubiquitin-dependent degradation at the ER in yeast has recently been reviewed by Zattas and Hochstrasser (2014), and in mammals by [START_REF] Olzmann | The mammalian endoplasmic reticulumassociated degradation system[END_REF]. The link between ERAD machinery and human diseases is clearly established and has been reviewed by [START_REF] Guerriero | The delicate balance between secreted protein folding and endoplasmic reticulum-associated degradation in human physiology[END_REF].

In addition to ERAD, several ubiquitin ligases have been implicated in the quality control of cytoplasmic proteins, for example Ubr1 [START_REF] Heck | Cytoplasmic protein quality control degradation mediated by parallel actions of the E3 ubiquitin ligases Ubr1 and San1[END_REF] or Hul5 [START_REF] Fang | Hul5 HECT ubiquitin ligase plays a major role in the ubiquitylation and turnover of cytosolic misfolded proteins[END_REF]. Gardner and colleagues also reported the existence of a protein quality control mechanism in the nucleus of Saccharomyces cerevisiae (henceforth S. cerevisiae). In this compartment, misfolded proteins are recognized and targeted to the proteasome by the San1 ubiquitin ligase, which functions together with the ubiquitin conjugating enzymes Cdc34 and Ubc1 [START_REF] Gardner | Degradation-mediated protein quality control in the nucleus[END_REF].

In addition to its role in quality control, proteasome-mediated degradation of ubiquitylated proteins acts as a regulatory mechanism in a myriad of cellular processes.

The best example is probably the progression through cell cycle, where several Cullin-RING ubiquitin ligases (CRLs) play a dominant role in controlling the degradation of cell cycle proteins. The archetypes here are the anaphase promoting complex/cyclosome (APC/C) and the Skp1/Cullin/F-box protein (SCF complex) ubiquitin ligase. The APC/C is activated during late mitosis and the G1 phase of the cell cycle, eliminating proteins that block mitotic progression and that would have negative consequences for the cell upon accumulation in the G1 phase. The SCF complex ubiquitylates proteins marked by phosphorylation and drives cell cycle progression during G1 and S phases.

Other important classes that have also been shown to play a prominent role in cell cycle regulation are CRL3 and CRL4 complexes. CRL3 complexes, with Aurora B as their best characterized substrate, have been linked to the control of mitosis via proteolytic and non-proteolytic mechanisms. CRL4 complexes, in addition to cell cycle regulation, are implicated in the DNA damage response and DNA replication (recently reviewed by [START_REF] Bassermann | The ubiquitin proteasome system -implications for cell cycle control and the targeted treatment of cancer[END_REF].

It is now known that ubiquitylation also acts as a signal targeting plasma membrane proteins for destruction in lysosomes or vacuoles (reviewed by [START_REF] Hicke | Protein regulation by monoubiquitin[END_REF].

Moreover, ubiquitylation is involved in cellular processes such as cell differentiation, cell signaling, transcription [START_REF] Zhou | Histone H2A monoubiquitination represses transcription by inhibiting RNA polymerase II transcriptional elongation[END_REF], DNA replication, DNA repair [START_REF] Jentsch | The yeast DNA repair gene RAD6 encodes a ubiquitin-conjugating enzyme[END_REF] and DNA damage response [START_REF] Hochstrasser | Protein degradation or regulation: Ub the judge[END_REF].

Examples of diseases associated with defective protein ubiquitylation

Aberrations in protein ubiquitylation have been associated with various diseases such as cancer, neurodegenerative or metabolic disorders, muscular atrophies and certain viral infections (reviewed by [START_REF] Petroski | The ubiquitin system, disease, and drug discovery[END_REF]. For instance, mutations of RINGtype ubiquitin ligases (E3s) or other alterations of their activity have been correlated

with human cancers or inherited genomic instability disorders. Classic examples are the ubiquitin ligase BRCA1 and the FA complex. The former is mutated in familial breast and ovarian cancers [START_REF] Welcsh | BRCA1 and BRCA2 and the genetics of breast and ovarian cancer[END_REF], whereas mutations of the latter lead to defective monoubiquitylation of the substrate FANCD2, which results in Fanconi anaemia [START_REF] Moldovan | How the fanconi anemia pathway guards the genome[END_REF]. Mutations in the gene encoding the Parkin protein, a component of a multiprotein ubiquitin ligase complex, cause an autosomal recessive juvenile Parkinsonism (AR-JP) [START_REF] Kitada | Mutations in the parkin gene cause autosomal recessive juvenile parkinsonism[END_REF]. More examples on how the ubiquitin system is implicated in various diseases are presented in Table 1.

Table 1. Examples of an implication of ubiquitin system in disease pathways INTRODUCTION 15 1.1.4 Ubiquitin Ubiquitin (Ub) is a small 76-amino acids and 8.5 kDa globular, highly stable protein with an overall structure being 'extremely compact and tightly hydrogenbonded'. Thanks to this compact globular structure, with an exposed C-terminal tail (COOH-terminus), Ub can form a covalent linkage with other proteins [START_REF] Vijay-Kumar | Structure of ubiquitin refined at 1.8 A resolution[END_REF]. Its surface is mainly polar, with a large hydrophobic area centered on the leucine 8 (Leu8), isoleucine 44 (Ile44), and valine 70 (Val70) residues [START_REF] Beal | Surface hydrophobic residues of multiubiquitin chains essential for proteolytic targeting[END_REF] (Figure 2). Ubiquitin is present in all types of cells and tissues, with up to 10 8 copies per cell [START_REF] Yewdell | Not such a dismal science: the economics of protein synthesis, folding, degradation and antigen processing[END_REF] and its cellular concentrations as high as ~85 µM [START_REF] Kaiser | Protein standard absolute quantification (PSAQ) method for the measurement of cellular ubiquitin pools[END_REF]. It is both a cytoplasmic and a nuclear protein [START_REF] Lund | Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor[END_REF]. Figure 3. A protein sequence alignment of human and yeast ubiquitin produced by CLUSTALW2. Small, hydrophobic and aromatic amino acids are indicated in red, acidic amino acids in blue and basic amino acids in magenta. Green represents hydroxyl, amine, amide and basic residues. All identical amino acids in human and yeast are marked by asterisk. The differences in the sequence are marked with period (semi-conserved substitution) or colon (conserved substitutions). The sequences for the alignment were retrieved from PDB, accession number: AAA36789.1 and NP_013061.1 for human and yeast, respectively.

Nucleotide sequence analysis has shown that Ub is synthesized in the form of different precursors, which must subsequently be cleaved in order to release functional Ub units [START_REF] Lund | Nucleotide sequence analysis of a cDNA encoding human ubiquitin reveals that ubiquitin is synthesized as a precursor[END_REF] (Figure 4). These precursors are a linear fusion protein consisting of four or more Ub copies (tandem Ub) and fusion proteins between Ub and ribosomal proteins (40S ribosomal protein L40 and 60S ribosomal protein S27) [START_REF] Finley | The tails of ubiquitin precursors are ribosomal proteins whose fusion to ubiquitin facilitates ribosome biogenesis[END_REF][START_REF] Redman | Identification of the long ubiquitin extension as ribosomal protein S27a[END_REF]. The protein is encoded by four genes in mammals: UBC, UBB, UBA52 and UBA80. The first two encode a tandem Ub (9 Ub units and 4 Ub units, respectively) and the last two encode fusions with ribosomal proteins (UBA52 with L40 protein, whereas UBA80 with S27) [START_REF] Redman | Identification of the long ubiquitin extension as ribosomal protein S27a[END_REF]. In yeast there are also four Ub genes: UBI1, UBI2, UBI3 and UBI4 [START_REF] Ozkaynak | The yeast ubiquitin genes: a family of natural gene fusions[END_REF]. UBI1 and UBI2 have non-homologous introns at the same positions and encode identical 52-residue tails. UBI3 encodes a different 76-residue tail, whereas UBI4 encodes a polyubiquitin precursor protein containing five Ub repeats [START_REF] Ozkaynak | The yeast ubiquitin genes: a family of natural gene fusions[END_REF]. Interestingly, in yeast a single UBI4 gene is not required under vegetative conditions. However, cells lacking UBI4 are sensitive to high temperatures, starvation and other amino acid analogue-induced stresses (e.g. canavanine), and they are defective in sporulation [START_REF] Finley | The yeast polyubiquitin gene is essential for resistance to high temperatures, starvation, and other stresses[END_REF]. Ubiquitin is synthesized as a precursor. It is transcribed and translated as a linear fusion consisting of multiple copies of ubiquitin (tandem ubiquitin) or ubiquitin fused with other proteins (L40 or S27 ribosomal proteins). Generation of free ubiquitin is possible due to the action of deubiquitylating enzymes and more precisely ubiquitin C-terminal hydrolases (UCH).

Types of ubiquitylation and ubiquitin chain diversity

Ubiquitin, owing to its highly accessible C-terminal tail (COOH-terminus), is covalently attached via its glycine residue (Gly76) to the amino group (-NH 2 ) of lysine residues (Lys) in substrate proteins by the formation of an isopeptide bond. Types of ubiquitin modifications are immensely diverse (reviewed by [START_REF] Ikeda | Atypical ubiquitin chains: new molecular signals. 'Protein modifications: beyond the usual suspects' review series[END_REF][START_REF] Kulathu | Atypical ubiquitylation -the unexplored world of polyubiquitin beyond Lys48 and Lys63 linkages[END_REF] and form the so-called 'ubiquitin code' [START_REF] Komander | The ubiquitin code[END_REF]. The attachment to a substrate protein can be in either a monomeric or a polymeric form, monoubiquitylation or polyubiquitylation, respectively (Figure 5).

Some substrates are modified with a single ubiquitin on several Lys residues. This is referred to as multiple monoubiquitylation, although the term multi-monoubiquitylation is also used [START_REF] Hicke | Ubiquitination of a yeast plasma membrane receptor signals its ligandstimulated endocytosis[END_REF][START_REF] Haglund | Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation[END_REF] (Figure 5).

Monoubiquitylation

The attachment of a single ubiquitin (monoubiquitin) to the substrate proteins is an important regulatory post-translational modification. It influences the activity of the substrates or their localization, and is therefore called a 'regulator of substrate-protein location and activity' [START_REF] Hicke | Ubiquitination of a yeast plasma membrane receptor signals its ligandstimulated endocytosis[END_REF]. Monoubiquitylation is involved in various cellular functions, including endocytosis, histone regulation, and the budding of retroviruses from the plasma membrane (reviewed by [START_REF] Hicke | Protein regulation by monoubiquitin[END_REF]. For example, an important role played by monoubiquitylation in histone function was first reported by [START_REF] Robzyk | Rad6-dependent ubiquitination of histone H2B in yeast[END_REF]. Using budding yeast S. cerevisiae, they showed that yeast cells carrying the mutated histone H2B (predominantly ubiquitylated histone in yeast) lacking its ubiquitylation site, grow more slowly than wild-type cells and do not sporulate. They therefore concluded that monoubiquitylation of H2B is necessary for normal growth and meiosis [START_REF] Robzyk | Rad6-dependent ubiquitination of histone H2B in yeast[END_REF]. In addition, monoubiquitylation regulates the activity of the plasma membrane proteins by the endocytic pathway. Most of these proteins are then targeted for degradation in the lysosome (reviewed by [START_REF] Hicke | Protein regulation by monoubiquitin[END_REF]. [START_REF] Haglund | Multiple monoubiquitination of RTKs is sufficient for their endocytosis and degradation[END_REF] noted that the epidermal growth factor (EGF) and plateletderived growth factor receptors (PDGFRs) are monoubiquitylated at multiple sites following their ligand-induced activation, and that multi-monoubiquitylation is a main signal driving those receptor tyrosine kinases (RTKs) from the plasma membrane to the lysosome.

A principal model in the ubiquitin field is that contrary to polyubiquitylation, monoubiquitylation and multi-monoubiquitylation do not induce proteasomal degradation. This is due to the fact that an efficient proteasome binding and degradation requires at least four subunit-long ubiquitin chains (tetraubiquitin) [START_REF] Thrower | Recognition of the polyubiquitin proteolytic signal[END_REF]. However, several studies have shown that multi-monoubiquitylation and even monoubiquitylation can be sufficient to target some substrates for proteasomal degradation. For example, it has been demonstrated that it is multiple monoubiquitylation, rather than polyubiquitylation, which is responsible for processing the NF-κB transcription factor precursor p105 to its p50 active subunit by the proteasome. The proteasome recognizes the multiple monoubiquitylated p105 precursor.

Interestingly and exceptionally in this case, the Ub system does not completely destroy its substrate. Nevertheless, this study suggests that not only a poly-Ub chain, but also a cluster of single ubiquitin, can control proteasomal processing [START_REF] Kravtsova-Ivantsiv | Modification by Single Ubiquitin Moieties Rather Than Polyubiquitination Is Sufficient for Proteasomal Processing of the p105 NF-κB Precursor[END_REF]. Other studies of the Pax3 protein, a key regulator of muscle differentiation, have also shown that it is monoubiquitylation which induces the proteasomal degradation [START_REF] Boutet | Taf1 regulates Pax3 protein by monoubiquitination in skeletal muscle progenitors[END_REF]. [START_REF] Carvallo | Noncanonical Wnt signaling induces ubiquitination and degradation of Syndecan4[END_REF] reported that Syndecan4 (SDC4), a cell adhesion receptor required for cell migration, is monoubiquitylated in its cytoplasmic domain in the Wnt signaling pathway receptor Dishevelled (Dsh)dependent manner and it is then degraded by the proteasome. Here however, the induction of the proteasomal degradation by monoubiquitylation has not been fully demonstrated.

Polyubiquitylation

All seven lysine residues (Lys6,Lys11,Lys27,Lys29,Lys33,Lys48 and Lys63) in Ub as well as N-terminal methionine (Met1) (Figure 6) are involved in the assembly of polyubiquitin chains, yielding a wide variety of structures [START_REF] Peng | A proteomics approach to understanding protein ubiquitination[END_REF][START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF]. Current data demonstrate that Lys63, Lys48 and Lys11-linked polyubiquitin chains appear most frequent in budding yeast as well as in mammals. The level of

INTRODUCTION 20

Depending on the linkage, polymeric chains adapt alternative topologies. Chains linked via N-terminus or via one of the seven lysine residues of ubiquitin are homogenous, for example Met1-, Lys11-, Lys48-, or Lys63-linked chains. If different lysine residues are linked at subsequent positions in the chain, then it has a mixed topology (mixed-linkage chains). Moreover, one ubiquitin moiety linked with two or more ubiquitin molecules generates branched chains, whose function is still unknown [START_REF] Komander | The ubiquitin code[END_REF]) (Figure 7). showed that all seven Lys residues can be used to assemble poly-Ub chains in vivo in S. cerevisiae [START_REF] Peng | A proteomics approach to understanding protein ubiquitination[END_REF]. The physiological relevance of all poly-Ub chains combinations remains to be revealed. Another issue that remains to be resolved is the following: where and how are the ubiquitin chains assembled? The majority of experimental evidence suggests that chains are assembled on the substrate sequentially (i.e. initial substrate-attached ubiquitin is added what is followed by a stepwise addition of other single Ub moieties). Nonetheless, there is also evidence for chains being preassembled on E2s or E3s and then transferred as a whole to the substrates [START_REF] Wang | Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis[END_REF][START_REF] Li | A ubiquitin ligase transfers preformed polyubiquitin chains from a conjugating enzyme to a substrate[END_REF][START_REF] Ravid | Autoregulation of an E2 enzyme by ubiquitin-chain assembly on its catalytic residue[END_REF].

Conjugation of polyubiquitin chains to proteins is crucial in regulating functions of many of these proteins (Figure 7). The formation of poly-Ub chains has mainly been implicated in targeting to 26S proteasome and giving a so-called, 'kiss of death' to proteins. A classic example are Lys48-linked chains, which are associated with proteasomal degradation (reviewed by [START_REF] Finley | Recognition and processing of ubiquitin-protein conjugates by the proteasome[END_REF]. On the contrary, Lys63-linked chains have been implicated in a variety of non-proteolytic functions (reviewed by [START_REF] Haglund | Ubiquitylation and cell signaling[END_REF]. [START_REF] Matsumoto | K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody[END_REF] demonstrated that Lys11-chains are involved in the cell cycle and are abundant in mitotic cells during the degradation of the substrates of E3 ligase APC complex. This suggests that the chain assembly can provide cell-specific ubiquitome pattern [START_REF] Matsumoto | K11-linked polyubiquitination in cell cycle control revealed by a K11 linkage-specific antibody[END_REF]. Lys11-linked chains were also shown to play a role in the endoplasmic reticulum-associated degradation (ERAD) pathway [START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF]. In addition, polyubiquitylation has been involved in inflammatory, anti-apoptotic and immune signaling processes [START_REF] Tokunaga | Involvement of linear polyubiquitylation of NEMO in NF-kappaB activation[END_REF].

Mechanism of ubiquitylation

The attachment of ubiquitin to a substrate is catalyzed via a cascade of enzymatic reactions involving E1, E2 and E3s. The first step, the so-called initial activation, consists in the activation of the C-terminal glycine (Gly76) of ubiquitin by a ubiquitin activating enzyme (E1) in an ATP-dependent manner. In the intermediate step the activated ubiquitin is transferred from an E1 to the cysteine (Cys) residue within an active site of a ubiquitin conjugating enzyme (E2). Next, the E2 interacts with a ubiquitin ligase (E3), which recognizes both the E2 and a substrate, subsequently enabling the ubiquitin to become covalently attached to a substrate protein (Figure 8).

During these final step reactions, the carboxyl group (-COOH) of the C-terminus of Ub and the amino group (-NH 2 ) of a lysine (Lys) residue within the substrate are linked by an isopeptide bond. The particular class of E3s, involved in ubiquitylation and more precisely in the elongation of Ub chains via the ligation of Ub to pre-existing poly-Ub chains, was also described and is referred to very often as E4 enzymes [START_REF] Koegl | A novel ubiquitination factor, E4, is involved in multiubiquitin chain assembly[END_REF][START_REF] Pickart | Ubiquitin: structures, functions, mechanisms[END_REF]. The mechanism of ubiquitylation is reversible through the action of so-called deubiquitylating enzymes (DUBs). Apart from a typical attachment to Lys, it has been shown that Ub can also be attached to the substrates through serine (Ser), threonine (Thr) and cysteine (Cys) residues via the formation of oxyester or thioester bonds, respectively. For instance, in yeast a conserved Cys residue is important for Pex4 (Ubc10) protein-dependent ubiquitylation of the peroxisomal import receptor Pex5 [START_REF] Williams | A conserved cysteine is essential for Pex4p-dependent ubiquitination of the peroxisomal import receptor Pex5p[END_REF]. Viral E3 ubiquitin ligases MIR1 and MIR2, which mediate ubiquitylation of major histocompatibility complex class I (MHC I), also target a Cys residue in the MHC I molecules [START_REF] Cadwell | Ubiquitination on nonlysine residues by a viral E3 ubiquitin ligase[END_REF][START_REF] Cadwell | The specificities of Kaposi's sarcoma-associated herpesvirus-encoded E3 ubiquitin ligases are determined by the positions of lysine or cysteine residues within the intracytoplasmic domains of their targets[END_REF]. Another viral E3 ligase mK3 was shown to mediate Ub by its attachment to the Ser and Thr residues, except in the case of the conventional Lys linkage [START_REF] Wang | Ubiquitination of serine, threonine, or lysine residues on the cytoplasmic tail can induce ERAD of MHC-I by viral E3 ligase mK3[END_REF][START_REF] Herr | Role of the RING-CH domain of viral ligase mK3 in ubiquitination of non-lysine and lysine MHC I residues[END_REF].

In addition to conjugation to the amino acid side chains, Ub can also be attached to the amino group (-NH 2 ) at the N-terminal of certain substrate proteins [START_REF] Breitschopf | A novel site for ubiquitination: the N-terminal residue, and not internal lysines of MyoD, is essential for conjugation and degradation of the protein[END_REF][START_REF] Ciechanover | N-terminal ubiquitination: More protein substrates join in[END_REF]. Examples here are the latent membrane proteins LMP1 and LMP2A of the Epstein-Barr virus (EBV) that are ubiquitylated on their N-terminus rather than on the canonical Lys residues [START_REF] Aviel | Degradation of the epstein-barr virus latent membrane protein 1 (LMP1) by the ubiquitin-proteasome pathway. Targeting via ubiquitination of the N-terminal residue[END_REF][START_REF] Ikeda | Lysine-Independent Ubiquitination of Epstein-Barr Virus LMP2A[END_REF]. Interestingly, [START_REF] Scaglione | The ubiquitin-conjugating enzyme (E2) Ube2w ubiquitinates the N terminus of substrates[END_REF] recently reported Ube2w as the first identified E2 that transfers ubiquitin to the N-terminal of substrates.

1.1.7 Ubiquitin activating enzymes (E1s)

The ubiquitin activating enzyme (E1) catalyzes the first step in the ubiquitylation cascade. Two members of the E1 enzymes exist in human and only one in yeast. In human this is Ube1, called Uba1 in yeast, and Uba6 (also called UBE1L2 in human) [START_REF] Pelzer | UBE1L2, a novel E1 enzyme specific for ubiquitin[END_REF]. Both enzymes, Uba1 and Uba6, share only 40% sequence identity [START_REF] Jin | Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging[END_REF]. Uba1 is highly conserved in human, plants and yeast, whereas Uba6 is present only in vertebrates. Uba1 activates Ub and other ubiquitin-like proteins (Ulbs), whereas Uba6 can only activate Ub and the ubiquitin-like protein called FAT10 [START_REF] Pelzer | FAT10 : Activated by UBA6 and functioning in protein degradation[END_REF]). Ub activation rates in vitro between these two enzymes are comparable. In the cell however, Uba6 is around 10-fold less abundant than Uba1 [START_REF] Jin | Dual E1 activation systems for ubiquitin differentially regulate E2 enzyme charging[END_REF]. Uba6 enzyme has been found to function in conjugation with an E2 enzyme Use1, while Uba1 functions with the other E2s [START_REF] Aichem | USE1 is a bispecific conjugating enzyme for ubiquitin and FAT10, which FAT10ylates itself in cis[END_REF][START_REF] Lee | Alternative ubiquitin activation/conjugation cascades interact with N-end rule ubiquitin ligases to control degradation of RGS proteins[END_REF].

To date, the ubiquitin activating enzyme Uba1 (Ube1) has the best characterized mechanism of action (Figure 9). where a fully loaded E1 carries two Ub moieties, one as adenylate and the other as thioester. The double-Ub-bond E1 associates with an E2, and upon transthiolation reaction the Ub is transferred from the catalytic cysteine of E1 onto the catalytic cysteine of E2. Next, the Ub noncovalently attached as adenylate can be transferred to the downstream enzymes [START_REF] Haas | The mechanism of ubiquitin activating enzyme. A kinetic and equilibrium analysis[END_REF][START_REF] Dye | Structural mechanisms underlying posttranslational modification by ubiquitin-like proteins[END_REF]. [START_REF] Vandemark | Structural basis of ubiquitylation[END_REF][START_REF] Burroughs | Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation[END_REF]. Structurally, the UBC domain consists of N-terminal α-helix followed by a four stranded anti-parallel β-sheet.

Some E2s have N-terminal or C-terminal extensions to their catalytic core and/or insertions into it. The E2 enzyme family can therefore be divided up according to the existence of these additional extensions (Table 2). The E2s that consist only of the catalytic UBC domain are in class I. Those in classes II and III have N-or C-terminal extensions, respectively, while class IV contains E2s with extensions on both terminals [START_REF] Van Wijk | The family of ubiquitin-conjugating enzymes (E2s): deciding between life and death of proteins[END_REF]. These sequence extensions result in functional differences between E2s that mainly involve their subcellular localization, stabilization of the E1/E2 interaction and E2/E3 interaction activity modulation. For example, the Cterminal extension of yeast Cdc34 E2 (residues 171-295) is a binding domain for Cdc4 and Cdc53 proteinscomponents of the SCF (Cdc4) ubiquitin ligase complex [START_REF] Mathias | An essential domain within Cdc34p is required for binding to a complex containing Cdc4p and Cdc53p in Saccharomyces cerevisiae[END_REF]. The yeast Ubc6 protein involved in the endoplasmic reticulum-associated degradation (ERAD), in addition to the catalytic core, has its transmembrane domain located at the C-terminus that contains hydrophobic amino acids. This 95 amino acid residues extension determines Ubc6 subcellular location at the cytosolic side of the endoplasmic reticulum membrane [START_REF] Yang | The Transmembrane Domain of a Carboxyl-terminal Anchored Protein Determines Localization to the Endoplasmic Reticulum[END_REF][START_REF] Lenk | A role for mammalian Ubc6 homologues in ER-associated protein degradation[END_REF]. Lenk and colleagues (2002) identified and functionally characterized two distinct families of Ubc6 orthologues in mammals, Ubc6 and Ubc6e (Figure 10). Both families share significant sequence similarities with yeast Ubc6 enzyme and have analogous structural organization with their UBC domains (55.5% sequence identity between yeast and human) and tail-anchored protein motifs, consisting of hydrophobic amino acids [START_REF] Lenk | A role for mammalian Ubc6 homologues in ER-associated protein degradation[END_REF]. All eukaryotes possess several E2s ranging from 8 in organisms such as Giardia lamblia to over 50 in multicellular plants and animals [START_REF] Burroughs | Anatomy of the E2 ligase fold: implications for enzymology and evolution of ubiquitin/Ub-like protein conjugation[END_REF]. There are eleven E2s that form thiolesters with Ub identified in S. cerevisiae [START_REF] Pickart | Ubiquitin: structures, functions, mechanisms[END_REF]) (Table 2). Sequence alignments and phylogenetic analysis of eleven yeast E2s are presented in Figure 11. No enzymes that possess the UBC-folds are present in bacteria [START_REF] Iyer | Unraveling the biochemistry and provenance of pupylation: a prokaryotic analog of ubiquitination[END_REF]. The number variability amongst E2s within different organisms is mainly the result of gene duplication. For instance, the highly similar enzymes Ubc4 and Ubc5 in yeast S. cerevisiae that are involved in the degradation of abnormal or excess proteins are found to be UbcH5A-D (UBE2B1-4), UbcH6, UbcH8

and UbcH9 (UBE2E1-3) in higher eukaryotes [START_REF] Seufert | Ubiquitin conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins[END_REF]. Some E2s seem to be specific to higher eukaryotes. For example, UBE2U has only been identified in mammals, and the enormous 528 kDa E2 BIRC6 (BRUCE) has been found in Drosophila, mice and humans, whereas it is absent in C. elegans and yeast [START_REF] Bartke | Dual role of BRUCE as an antiapoptotic IAP and a chimeric E2/E3 ubiquitin ligase[END_REF][START_REF] Lotz | BRUCE, a giant E2/E3 ubiquitin ligase and inhibitor of apoptosis protein of the trans-Golgi network, is required for normal placenta development and mouse survival[END_REF]. Several of yeast E2s have a high expression level, however, the expression vary amongst different family members (personal observation). Polyubiquitin chain synthesis is associated with the function of specific E2s, and it is the combination of E2/E3 pairs that determines the Ub chains' linkages [START_REF] Kirkpatrick | Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology[END_REF][START_REF] Kim | Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages[END_REF][START_REF] Jin | Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex[END_REF][START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF]. A wellknown example of a single E2 that synthesizes primarily Lys48-linked poly-Ub chains is yeast Cdc34 (Ubc3) [START_REF] Petroski | Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34[END_REF]. [START_REF] Petroski | Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34[END_REF] demonstrated that mutations of an acidic loop region of Cdc34 affect the linkage specificity and the processivity of Lys48 ubiquitin chain synthesis. Yeast Ubc6 mainly synthesizes Lys11-linked chains, and these linkages function in the endoplasmic reticulum-associated degradation (ERAD) pathway [START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF]. Similarly to its yeast homolog, human Ubc6 (called also UBE2J2) was shown to functionally interact with a number of E3, and to be invlved in the regularion of its own proteosomal degradation (Lam et al., 2014). Interestingly, two E2 enzymes can act on a single target protein to synthesize the chain, with one enzyme as an initiator of chain formation and the other extending it. Examples to illustrate this mechanism are provided by yeast Ubc4 and Ubc1, which drive polyubiquitin chain assembly on the anaphase promoting complex/cyclosome (APC/C) targets [START_REF] Rodrigo-Brenni | Sequential E2s drive polyubiquitin chain assembly on APC targets[END_REF]. Similarly, several E2s working with a heterodimeric human E3 ligase BRCA1-BARD1, including UBE2W and UBE2E2, are responsible for Ub chain initiation, while the heterodimer UBE2N-UBE2V1 and UBE2K function specifically in chain elongation [START_REF] Christensen | E2-BRCA1 RING interactions dictate synthesis of mono-or specific polyubiquitin chain linkages[END_REF]. In human, BRCA1, which in complex with BARD1 possesses ubiquitinligase activity, has the ability to catalyze Lys6-linked Ub chains [START_REF] Wu-Baer | The BRCA1/BARD1 heterodimer assembles polyubiquitin chains through an unconventional linkage involving lysine residue K6 of ubiquitin[END_REF].

1.1.9 Ubiquitin ligases (E3s)

The final step of ubiquitylation cascade is the transfer of Ub onto the substrate proteins mediated by ubiquitin ligases (E3s). To mediate this transfer, E3s interact with ubiquitin-loaded E2 enzymes and are able to recognize and bind particular substrates.

Two major mechanistically distinct families of E3s have been identified, RING finger E3s that transfer ubiquitin directly from E2 to the substrate protein, and HECT domain E3s that facilitate ubiquitin transfer to the substrate via the formation of a covalent ubiquitin-E3 thioester intermediate [START_REF] Scheffner | Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade[END_REF]. Moreover, another important family of mechanistically distinct Ub ligases is known as the RBR (RING-Between-RING-RING) or TRIAD (two RING fingers and a DRIL, i.e. double RING finger-linked). This family uses a combination of the RING and HECT ligases mechanisms to mediate the Ub transfer onto the substrate (Wenzel et al., 2011). The mechanism of Ub transfer by different E3 families is presented later in Figure 12.

The human genome encodes more than 600 E3s and substrate recognition subunits of E3 complexes, making the number of putative E3 genes even greater than the number of genes encoded for protein kinases. Approximately 95% of all putative

E3s were found to be RING finger-dependent [START_REF] Li | Genome-wide and functional annotation of human E3 ubiquitin ligases identifies MULAN, a mitochondrial E3 that regulates the organelle's dynamics and signaling[END_REF]. The numbers of putative ubiquitin ligases in both human and yeast are presented in Table 3, and the putative ubiquitin ligases in yeast along with their main functions are presented in Table 4. [START_REF] Deshaies | RING domain E3 ubiquitin ligases[END_REF]. They can form homodimers [START_REF] Zheng | Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases[END_REF], for example, cIAP, BIRC7 and Prp19, or heterodimers, including BRCA1-BARD1 [START_REF] Brzovic | Structure of a BRCA1-BARD1 heterodimeric RING-RING complex[END_REF], and Mdm2-MdmX. Moreover, some exist as multi-subunit assemblies, as in the Cullin RING ligases subfamily. These have recently been reviewed by Metzger et al. (2013).

With respect to the mechanism of action (Figure 12 A), RING-type or U-box ligases do not form the covalent intermediate with ubiquitin, but rather simultaneously bind both substrate and E2~Ub conjugate. They can discharge thioesterfied ubiquitin to its active-site cysteine onto the lysine residue of the substrate protein by acting as molecular scaffolds and positioning both an E2 and a substrate in a 'catalytic favorable orientation' on their domain [START_REF] Petroski | Mechanism of lysine 48-linked ubiquitin-chain synthesis by the cullin-RING ubiquitin-ligase complex SCF-Cdc34[END_REF]. Catalytic active site residues for RING E3s have not been identified [START_REF] Deshaies | RING domain E3 ubiquitin ligases[END_REF]. The RING or RING-like domain of these E3s is responsible for binding to the E2 and stimulates Ub transfer [START_REF] Lorick | RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination[END_REF]. acids in length and is composed of two lobes, an E2-binding N-terminal lobe and a Cterminal lobe that contains the active site cysteine [START_REF] Huibregtse | A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase[END_REF][START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF]. Subsequently, the structures of the seven HECT domains, E6-AP, WWP1, Smurf2, NEDD4L, HUWE1, yeast Rsp5 and NEDD4, were solved by [START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF], [START_REF] Verdecia | Conformational flexibility underlies ubiquitin ligation mediated by the WWP1 HECT domain E3 ligase[END_REF], [START_REF] Ogunjimi | Regulation of Smurf2 ubiquitin ligase activity by anchoring the E2 to the HECT domain[END_REF], [START_REF] Kamadurai | Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex[END_REF][START_REF] Pandya | A structural element within the HUWE1 HECT domain modulates self-ubiquitination and substrate ubiquitination activities[END_REF], [START_REF] Kim | Structure and function of a HECT domain ubiquitin-binding site[END_REF] and [START_REF] Maspero | Structure of the HECT:ubiquitin complex and its role in ubiquitin chain elongation[END_REF], respectively. Furthermore, the C-terminal part of the HECT domain of UBR5 ubiquitin ligase has been published by [START_REF] Matta-Camacho | Structure of the HECT C-lobe of the UBR5 E3 ubiquitin ligase[END_REF]. The crystal structure of the first identified member of the HECT family, E6-associated protein (E6-AP, also known as UBE3A) bound to the UbcH7 ubiquitin-conjugating enzyme revealed that the C-terminal lobe is a loosely packed architecture with its catalytic cysteine residues being 41Å apart [START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF]. Another structural study of the NEDD4L HECT E3 in complex with the ubiquitin-conjugated E2 (UBCH5B~Ub, known as UBE2D2) showed that the Ub Cterminal tail is located between the catalytic centers of the E2 and the HECT domain Cterminal lobe, and that the distance between the catalytic cysteines of E2 and E3 is ~8Å [START_REF] Kamadurai | Insights into ubiquitin transfer cascades from a structure of a UbcH5B approximately ubiquitin-HECT(NEDD4L) complex[END_REF].

Contrary to RING E3s, HECT domain E3s directly catalyze ubiquitylation of proteins due to their defined enzymatic activity in two-step mechanism (Figure 12 B).

The ubiquitin-charged E2 enzyme first transfers ubiquitin to the active site cysteine within the HECT domain in a transthiolation reaction, while preserving the high-energy ubiquitin thioester bond. Substrate ubiquitination then occurs through a subsequent nucleophilic attack of the HECT~Ub thioester bond by a lysine side chain of the target protein [START_REF] Huibregtse | A family of proteins structurally and functionally related to the E6-AP ubiquitin-protein ligase[END_REF][START_REF] Scheffner | Protein ubiquitination involving an E1-E2-E3 enzyme ubiquitin thioester cascade[END_REF].

The Ub-chain linkage specification mainly depends on the catalytic HECT domain. For example, yeast Rsp5 is involved in ubiquitylation of its substrates via Lys48-or Lys63-linked chains [START_REF] Kee | The Rsp5 ubiquitin ligase is coupled to and antagonized by the Ubp2 deubiquitinating enzyme[END_REF]. Members of the Nedd4 family form predominantly Lys63-linked ubiquitin chains but can also assemble Lys48-linked chains, whereas E6-AP E3 has been shown to form Lys48-linked chains [START_REF] Wang | Different HECT domain ubiquitin ligases employ distinct mechanisms of polyubiquitin chain synthesis[END_REF][START_REF] Kim | Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages[END_REF]. The substrate specificity is mainly determined by the N-terminal extensions. On this basis, HECT E3s were divided into the following three subfamilies: NEDD4/NEDD4-like E3s with double tryptophan residue (WW) domains, HERC (HECT and RCC1-like domains) with RLDs domains (RCC1-like domains), and others that contain neither WW nor RLDs domains (recently reviewed by Martin [START_REF] Scheffner | Mammalian HECT ubiquitin-protein ligases: biological and pathophysiological aspects[END_REF]. WW domains, shared by many HECT E3s, are involved in protein-protein interactions, and play a role in targeting certain substrates for degradation [START_REF] Hesselberth | Comparative analysis of Saccharomyces cerevisiae WW domains and their interacting proteins[END_REF]. Additionally, HECT E3s have a N-terminal C2 domain, responsible for plasma membrane translocation in response to increasing levels of intracellular Ca 2+ . In yeast, Rsp5 is the only HECT E3 to have a C2-WW domain, and is capable of ubiquitylating different proteins in various cellular compartments [START_REF] Rotin | Ubiquitination and endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of ubiquitin-protein ligases[END_REF].

1.1.9.3 RBR E3s

The RBR family includes the parkin protein, whose dysfunction is associated with early-onset Parkinson's disease [START_REF] Dawson | The role of parkin in familial and sporadic Parkinson's disease[END_REF], HOIP (HOIL-1interacting protein) and HOIL-1 (haem-oxidized IRP2 ubiquitin ligase 1) [START_REF] Kirisako | A ubiquitin ligase complex assembles linear polyubiquitin chains[END_REF], which both belong to the multiprotein LUBAC (linear ubiquitin chain assembly complex), as well as other complex multidomain enzymes. This family is characterized by the presence of the RING-Between-RING domain, which was first identified in a Drosophila gene, ariadne-1 (ari-1), which is required for a proper differentiation of all cell types in an adult fly [START_REF] Aguilera | Ariadne-1: a vital Drosophila gene is required in development and defines a new conserved family of ring-finger proteins[END_REF]. This E3 family was named 'the RBR ubiquitin ligases' after the RBR domains that were also identified in several other proteins using sequence alignment methods. It was shown that the RING domains in the RBR region contain cysteine and histidine residues with the ability to bind metal ions. It was suggested that the N-terminal RING domain (RING 1) binds two zinc ions and folds into a classical ring finger, whereas the second C-terminal RING domain (RING 2) binds only one ion and forms a hydrophobic core different from that of the classical ring fingers [START_REF] Capili | Structure of the C-terminal RING finger from a RING-IBR-RING/TRIAD motif reveals a novel zinc-binding domain distinct from a RING[END_REF]. Additionally, the third domain with a central cysteine/histidine cluster is also likely to form a ring-finger type structure. This domain was named IBR (in between ring) [START_REF] Morett | A novel transactivation domain in parkin[END_REF] or DRIL (double ring finger linked) (van der [START_REF] Van Der Reijden | TRIADs: a new class of proteins with a novel cysteine-rich signature[END_REF]. The RBR family was therefore given, a second name as TRIAD (two ring fingers and DRIL) (van der [START_REF] Van Der Reijden | TRIADs: a new class of proteins with a novel cysteine-rich signature[END_REF].

Recently, however, [START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights, new questions[END_REF] [START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights, new questions[END_REF].

RBR ligases use the mechanism of both RING and HECT ligases to catalyze the ubiquitylation, a process termed as 'RING-HECT hybrid mechanism' [START_REF] Wenzel | UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids[END_REF] (Figure 12 C). It was demonstrated that these ligases bind E2s through a RING domain but then transfer Ub via a thioester linked Ub in a transthiolation reaction, requiring a catalytic cysteine in the RING 2 domain [START_REF] Wenzel | UBCH7 reactivity profile reveals parkin and HHARI to be RING/HECT hybrids[END_REF], thereafter renamed the Rcat domain [START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights, new questions[END_REF]. It was, however suggested that the recruitment of the E2 is not as straight-forward mechanism as in the case of the canonical RING E3s [START_REF] Budhidarmo | RINGs hold the key to ubiquitin transfer[END_REF]. The RBR ligases can recruit mono-Ub or be involved in the poly-Ub chain formation thanks to the presence of regions with different domains, such as UBA, NZF and ZnF (described in more detail in the chapter on Ubiquitin binding proteins). Interestingly, a multisubunit ubiquitin ligase complex, LUBAC, was shown to efficiently assemble linear (Met1) ubiquitin chains [START_REF] Kirisako | A ubiquitin ligase complex assembles linear polyubiquitin chains[END_REF]. [START_REF] Atanassov | The role of deubiquitinating enzymes in chromatin regulation[END_REF].

With regard to the catalytic mechanism, cysteine proteases 'perform a nucleophilic attack' on the carbonyl of a peptide bond via their cysteine (Figure 13). [START_REF] Nijman | A genomic and functional inventory of deubiquitinating enzymes[END_REF]. The main function of DUBs is the generation of free ubiquitin from ubiquitin precursors. The is performed by UCHs, which cleave Ub preferentially from peptides and small adducts, unlike USPs which cleave Ub from protein substrates [START_REF] Kinner | The yeast deubiquitinating enzyme Ubp16 is anchored to the outer mitochondrial membrane[END_REF]. Moreover, by cleaving Ub-protein bonds, DUBs regulate ubiquitin pathways and are thereby involved in a broad range of cellular functions. They have been shown to play a role in membrane trafficking, protein quality control, and cell signaling by the establishment of protein-protein interactions. Moreover, they are implicated in the regulation of nuclear events such as DNA damage repair and transcription (reviewed by [START_REF] Clague | Cellular functions of the DUBs[END_REF]. An example of a well characterized yeast DUB is Doa4 (Ubp4), significant for both proteasome-dependent degradation as well as Ub homeostasis [START_REF] Amerik | Analysis of the deubiquitinating enzymes of the yeast Saccharomyces cerevisiae[END_REF].

In order to control ubiquitin-dependent signaling, DUBs have to deal with Ub chains that have distinct linkages, show different topology, and vary in length [START_REF] Komander | The ubiquitin code[END_REF]. Therefore, they display specificity on several levels, which enables to distinguish isopeptides or linear peptides and different types of Ub linkage or chain structures, as well as to discriminate particular chain linkages. Due to the difficulties in Ub chain preparation (other than linear), we still lack in-depth knowledge on DUBs specificity. However, within the USP and OUT DUBs families, Lys48-and Lys63-linkage specific members are well described [START_REF] Komander | Breaking the chains: structure and function of the deubiquitinases[END_REF].

For example, USP14 -the 26S proteasome associated DUB (the yeast homologue of Ubp6) -shows specificity for Lys48-linked Ub chains [START_REF] Hu | Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14[END_REF], as does otubain 1 (OTUB1) [START_REF] Wang | Evidence for bidentate substrate binding as the basis for the K48 linkage specificity of otubain 1[END_REF]. Interestingly, USP14 has so-called exo-activity and cleaves the chains from the distal end only, thus generating monoubiquitin [START_REF] Hu | Structure and mechanisms of the proteasome-associated deubiquitinating enzyme USP14[END_REF][START_REF] Komander | Breaking the chains: structure and function of the deubiquitinases[END_REF]. DUBs positioning on the ubiquitin chain can also be internal, which consequently releases longer chains from the substrates. Examples of DUBs that show endo-activity are those that regulate ubiquitin-mediated signaling, for instance CYDL [START_REF] Komander | The structure of the CYLD USP domain explains its specificity for Lys63-linked polyubiquitin and reveals a B box module[END_REF] and A20 [START_REF] Lin | Molecular basis for the unique deubiquitinating activity of the NF-kappaB inhibitor A20[END_REF]. Among the members of the OTU family, DUBA (also known as OTUD5) and Trabid are Lys63linkage specific DUBs [START_REF] Kayagaki | DUBA: a deubiquitinase that regulates type I interferon production[END_REF][START_REF] Tran | Trabid, a new positive regulator of Wnt-induced transcription with preference for binding and cleaving K63-linked ubiquitin chains[END_REF]. Lys11-linked ubiquitin chains are preferentially hydrolyzed by the deubiquitylating enzyme Cezanne, which also belongs to the OTU family [START_REF] Bremm | Lys11-linked ubiquitin chains adopt compact conformations and are preferentially hydrolyzed by the deubiquitinase Cezanne[END_REF]. The Josephin family belonging DUB, Ataxin3, edits Lys63 linkages in mixed-linkage chains [START_REF] Winborn | The deubiquitinating enzyme ataxin-3, a polyglutamine disease protein, edits Lys63 linkages in mixed linkage ubiquitin chains[END_REF]. [START_REF] Schaefer | Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes[END_REF] reported that long Lys48-linked chains are resistant to many DUBs. They showed that the yeast Ubp15 deubiquitylating enzyme (and its human orthologue USP7) rapidly cleaves mono-and di-ubiquitin from the substrates, but that the removal of longer Lys48-linked chains is slow. On the contrary, the yeast Ubp12 easily cleaves the ends of long Lys48 chains on substrates, and like Ubp15 can also efficiently hydrolyze the short chains [START_REF] Schaefer | Protein-linked ubiquitin chain structure restricts activity of deubiquitinating enzymes[END_REF].

Ubiquitin binding proteins

As described in previous chapters, ubiquitin not only has a proteolytic function but also acts as a cellular signal, controlling a myriad of biological processes. The specificity of ubiquitin signaling is determined by its interactions with ubiquitin binding proteins, so-called ubiquitin receptors that recognize and decode the ubiquitylated signals into different biochemical cascades in the cell [START_REF] Ikeda | What determines the specificity and outcomes of ubiquitin signaling?[END_REF][START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF]. In other words, ubiquitin binding proteins are the readers that interpret 'the ubiquitin code' and translate it in biological outcomes. Importantly, they have the abilty to read either Lys63-or Lys48-linked chains. This is thanks to various ubiquitin binding domains (UBDs). They noncovalently and weakly bind monoubiquitin and/or polyubiquitin chains (the most common binding affinities of monoubiquitin are with K d > 100 µM) [START_REF] Hurley | Ubiquitin-binding domains[END_REF]. UBDs are usually small (20-150 amino acids) but structurally diverse, and can have various biological functions [START_REF] Hicke | Ubiquitin-binding domains[END_REF].

Ubiquitin binding domains have been extensively reviewed by Dikic andcolleagues (2009), and[START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF][START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF]. The main classes: UBA (ubiquitin associated), CUE (coupling of ubiquitin conjugation to endoplasmic reticulum degradation), UIM (ubiquitin interacting motif), GAT (GGA and TOM1), NZF (Np14 Zn-finger)belonging to the largest class of ZnF (Zinc finger) domains, A20 -also belonging to the ZnF class, and UEV (Ubc E2 variant), which are briefly presented below.

UBA domain

The Ub-associated domain was described by Hofmann and Bucher in 1996, and it was the first UBD described. It is present among members of the E2 and E3 families and ubiquitin binding protein superfamily, or proteins that contain domains similar to Ub itself. The size of the UBA domain is ~55 residues, with about 45 residues (generally nonpolar) as the conserved core region [START_REF] Hofmann | The UBA domain: a sequence motif present in multiple enzyme classes of the ubiquitination pathway[END_REF]. The UBA domain has a preference for polyubiquitin chain binding, but also binds monoubiquitin.

Pioneering work on the chain recognition of a number of UBA domains in the laboratory of Cecile Pickard showed that UBA domains recognize ubiquitin chains that are formed via different linkage types. Budding yeast Rad23 (human orthologue hHR23A) that contains an internal UBA1 domain and a C-terminal UBA2 domain preferentially binds Lys48 tetraubiquitin. The human ubiquitin-conjugating enzyme (E2-25K) is namely an example of Lys63 binding [START_REF] Raasi | Diverse polyubiquitin interaction properties of ubiquitin-associated domains[END_REF]. Binding to the Lys29-linked chains was also demonstrated [START_REF] Rao | Recognition of specific ubiquitin conjugates is important for the proteolytic functions of the ubiquitin-associated domain proteins Dsk2 and Rad23[END_REF]. Recently, [START_REF] Walinda | Solution Structure of the Ubiquitin-associated (UBA) Domain of Human Autophagy Receptor NBR1 and Its Interaction with Ubiquitin and Polyubiquitin[END_REF] have shown that the UBA domain of human autophagy receptor NBR1 interacts with both monoubiquitin and polyubiquitin.

CUE domain

CUE domains are ~50 amino acids binding motifs, structurally related to UBA domains (both being three-helix bundles) despite their negligible sequence identity. They are found in proteins involved in the ubiquitylation pathways and trafficking. The CUE domain was first detected in the bioinformatics analysis of proteins in the endoplasmic reticulum-associated degradation (ERAD) pathway [START_REF] Ponting | Proteins of the endoplasmic-reticulum-associated degradation pathway: domain detection and function prediction[END_REF]. Subsequently, yeast studies have revealed the function of the CUE domain in the monoubiquitin binding [START_REF] Davies | Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic[END_REF][START_REF] Donaldson | Ubiquitin Signals Protein Trafficking via Interaction with a Novel Ubiquitin Binding Domain in the Membrane Fusion Regulator, Vps9p[END_REF][START_REF] Shih | A ubiquitinbinding motif required for intramolecular monoubiquitylation, the CUE domain[END_REF]. The CUE domain of yeast Vsp9 protein (the orthologue of mammalian Rabex-5), involved in the yeast endocytic pathway, is required for monoubiquitin binding. The CUE domain of Vsp9 promotes monoubiquitylation by the HECT domain of Rsp5 ubiquitin ligase [START_REF] Davies | Vps9p CUE domain ubiquitin binding is required for efficient endocytic protein traffic[END_REF][START_REF] Shih | A ubiquitinbinding motif required for intramolecular monoubiquitylation, the CUE domain[END_REF]. CUE domains were named after the yeast Cue1

protein that plays a role in the ERAD pathway and recruits to the ER the ubiquitin conjugating enzyme Ubc7, which is necessary for the degradation of misfolded proteins (Biederer et al., 1997). [START_REF] Bagola | Ubiquitin binding by a CUE domain regulates ubiquitin chain formation by ERAD E3 ligases[END_REF] have shown that the CUE domain of Cue1 binds ubiquitin chains. It is important for an efficient formation of Lys48 polyubiquitin chains in vitro by the ERAD E3s and promotes the ubiquitylation of substrates [START_REF] Bagola | Ubiquitin binding by a CUE domain regulates ubiquitin chain formation by ERAD E3 ligases[END_REF].

UIM domain

The UIM is a conserved motif, present mainly in proteins involved in ligand-activated receptor endocytosis and degradation that recognizes the S5a proteasome subunit (known as Rpn10 in yeast) and variety of other proteins involved in ubiquitylation. It is a short motif of around 20 amino acid residues, and can recognize both mono-and polyubiquitylated proteins [START_REF] Hofmann | A ubiquitin-interacting motif conserved in components of the proteasomal and lysosomal protein degradation systems[END_REF]. It can recognize both Lys48and Lys63-linked chains [START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF].

GAT domain

GAT domains occur in GGA proteins (Golgi-localized, gamma-ear-containing, ADPribosylation-factor-binding proteins) as well as TOM1 and TOM1-like proteins [START_REF] Suer | Structure of the GAT domain of human GGA1: a syntaxin amino-terminal domain fold in an endosomal trafficking adaptor[END_REF]. [START_REF] Puertollano | Interactions of GGA3 with the ubiquitin sorting machinery[END_REF] first discovered the GAT domain involvement in ubiquitin binding and suggested that GGA proteins may play further roles in sorting of ubiquitylated cargos. Structural studies using X-ray crystallography have shown that the GAT domain has a three-helix bundle structure, and that within its C-terminal there are several conserved hydrophobic residues [START_REF] Collins | The Structure of the GGA1-GAT Domain Reveals the Molecular Basis for ARF Binding and Membrane Association of GGAs[END_REF]. [START_REF] Bilodeau | The GAT domains of clathrin-associated GGA proteins have two ubiquitin binding motifs[END_REF] have shown that the GAT domain can have two distinct Ub binding sites, suggesting that it could not only bind monoubiquitin but also promote the binding multi-monoubiquitylated and/or polyubiquitylated proteins [START_REF] Hurley | Ubiquitin-binding domains[END_REF].

NZF domain

The NZF domains are compact zinc-binding modules of approximately 30 amino acid residues that have been found in many proteins involved in Ub-dependent processes.

Ubiquitin binding by the NZF domain was discovered in studies of mammalian Npl4 protein (nuclear protein localization 4) [START_REF] Meyer | Direct binding of ubiquitin conjugates by the mammalian p97 adaptor complexes, p47 and Ufd1-Npl4[END_REF]. Several NZF domains bind to both mono-and poly-Ub in vitro. For instance, the NZF domain of the HOIL-1L subunit of LUBAC (linear ubiquitin chain assembly complex) has been shown to bind linear polyubiquitin chains [START_REF] Sato | Specific recognition of linear ubiquitin chains by the Npl4 zinc finger (NZF) domain of the HOIL-1L subunit of the linear ubiquitin chain assembly complex[END_REF]. However, certain NZF domains, for example Ran-binding protein 2, do not bind to ubiquitin. This is plausibly due to a lack of the hydrophobic residue responsible for ubiquitin recognition [START_REF] Alam | Ubiquitin interactions of NZF zinc fingers[END_REF].

A20 ZnF domain

The A20 domain belongs to a class of zinc finger (ZnF) domains. It was first found to function as a ubiquitin ligase domain in the NF-κB (nuclear factor κB) signaling pathway. It is the C-terminal domain of the A20 protein, composed of seven zinc fingers, that catalyzes the formation of the Lys48 chain, thereby triggering the substrates for proteasomal degradation [START_REF] Wertz | De-ubiquitination and ubiquitin ligase domains of A20 downregulate NF-kappaB signalling[END_REF]. Interestingly, the A20 ZnF domain of the A20 protein can contact three ubiquitins through Ile-44 and Asp-58 residues as well as via another surface of Ub, the so-called TEK-box [START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF]. The A20 domain can also directly bind mono-Ub, as shown in the Rabex-5 protein (the mammalian orthologue of yeast Vps9), which has its A20 domain on the N-terminus (residues 1-76) [START_REF] Mattera | The Rab5 guanine nucleotide exchange factor Rabex-5 binds ubiquitin (Ub) and functions as a Ub ligase through an atypical Ubinteracting motif and a zinc finger domain[END_REF]. The A20 ZnF domain of Rabex-5 binds ubiquitin with ~22 μM affinity within a polar region centered on the Asp-58 residue [START_REF] Lee | Structural basis for ubiquitin recognition and autoubiquitination by Rabex-5[END_REF].

UEV domain

UEV domains are structurally similar to E2 enzymes, but lack the active site cysteine.

This prevents them from catalyzing the Ub transfer as they cannot form a thioester bond with the C-terminus of Ub [START_REF] Ponting | The breast cancer gene product TSG101: a regulator of ubiquitination[END_REF][START_REF] Pornillos | Structure and functional interactions of the Tsg101 UEV domain[END_REF]. A well-known example of a UEV domain protein is the budding yeast Mms2 protein, which forms a complex with the E2 enzyme Ubc13 and facilitates the assembly of poly-Ub chains.

In vitro Mms2 forms 1/1 complexes with a Ub dissociation constant of 98 +/-15 µM, whereas the Ubc13-Mms2 heterodimer shows significantly stronger Ub-binding than Mms2 alone, with a dissociation constant of 28 +/-6 µM [START_REF] Mckenna | Energetics and specificity of interactions within Ub.Uev.Ubc13 human ubiquitin conjugation complexes[END_REF]. The UEV domain binds monoubiquitin via the Ile-44 patch [START_REF] Husnjak | Ubiquitin-Binding Proteins: decoders of ubiquitin-mediated cellular functions[END_REF].

The network of E2/E3 interactions

Different combinations of E2/E3 pairs catalyze either monoubiquitylation or the polyubiquitin chain formation [START_REF] Kirkpatrick | Quantitative analysis of in vitro ubiquitinated cyclin B1 reveals complex chain topology[END_REF][START_REF] Kim | Certain pairs of ubiquitin-conjugating enzymes (E2s) and ubiquitin-protein ligases (E3s) synthesize nondegradable forked ubiquitin chains containing all possible isopeptide linkages[END_REF][START_REF] Jin | Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex[END_REF][START_REF] Xu | Quantitative proteomics reveals the function of unconventional ubiquitin chains in proteasomal degradation[END_REF][START_REF] Wickliffe | The mechanism of linkagespecific ubiquitin chain elongation by a single-subunit E2[END_REF]. This 'combinatorial nature' of protein ubiquitylation networks appears in all eukaryotic systems [START_REF] Markson | Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network[END_REF].

Several physiological E2/E3 pairs have been revealed to date and it is known that a given E2 may interact with several E3s and vice versa. However, the exact mechanism of E2/E3 pairs function as well as their selectivity and specificity are still not fully understood [START_REF] Weissman | Themes and variations on ubiquitylation[END_REF]. Major steps towards revealing the complex E2/E3 network have been made by two large-scale studies, both published in 2009, and based on the global screening of interactions between human E2s and RING domains of E3s [START_REF] Markson | Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network[END_REF][START_REF] Van Wijk | A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system[END_REF].

Hence, van Wijk and colleagues ( 2009) performed a global yeast two-hybrid screen to investigate the specificity of the interactions between the catalytic domains (UBC-folds) of the 35 human E2s with the 250 RING-type E3s. The researchers uncovered 346 high-confidence E2/E3 interactions and found that multiple E2s can interact with multiple E3s, and conversely. The human UBE2U conjugating enzyme, which belongs to the UbcH5 family (members of this family are highly conserved and homologous to the yeast Ubc4 and Ubc5 enzymes), showed the highest number of interactions (52 interactions), followed by UBE2D2 ( 35), UBE2D3 and UBE2D4 [START_REF] Le ´on | Ear1p and Ssh4p are new adaptors of the ubiquitin ligase Rsp5p for cargo ubiquitylation and sorting at multivesicular bodies[END_REF], UBE2N [START_REF] Nakamura | The Role of the transmembrane RING finger proteins in cellular and organelle function[END_REF] and UBE2D1 [START_REF] Beck | Snapshots of nuclear pore complexes in action captured by cryo-electron tomography[END_REF]. Some of these interactions, including UBE2U with the ligase MDM2, were verified by independent GST-pull-down analysis [START_REF] Van Wijk | A comprehensive framework of E2-RING E3 interactions of the human ubiquitin-proteasome system[END_REF]. The second study by [START_REF] Markson | Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network[END_REF] combined yeast two-hybrid screens, using full-length 39 human E2s, with homology modeling methods to show a map of E2/E3-RING interactions. They generated a set of 568 reproducible positive human E2/E3-RING interactions. 80 different E2/E3 pairs were tested to eliminate the falsepositive interactions. To do so, they used either single-or double-point mutants with the mutated conserved amino acids involved in E2 binding. Approximately 92% of those interactions were abolished, indicating the specificity of the interactions. A large number of RING E3s were observed to interact with members of the UBE2D and UBE2E families of E2s [START_REF] Markson | Analysis of the human E2 ubiquitin conjugating enzyme protein interaction network[END_REF].

The aforementioned studies have provided a genome-wide view of the E2/E3 interactions network in the human ubiquitin-proteasome system. Other studies by [START_REF] Christensen | E2-BRCA1 RING interactions dictate synthesis of mono-or specific polyubiquitin chain linkages[END_REF] looked at possible E2 interactors of the human RING ligase BRCA1/BARD1 and showed that it can interact with ten different E2s, including UbcH5a, UbcH5b, UbcH5c, UbcH6, UbcH7, Ube2e2, UbcM2, Ubc13, Ube2k, andUbe2w. Moreover, numbers of both large and small scale studies in human as well as yeast have expanded the E2/E3 network (data concerning interactions in yeast are presented in the chapter Physiological E2/E3 pairs, Table 5). So far, however, many

E3s remain lone with no known interacting E2s and it is not possible to easily predict their functional E2 partners.

Canonical interaction between the E2 UBC domain and the E3 RING, HECT domains

Experimental structures have shown that interactions with E3s occur in the UBC domain of E2s [START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF][START_REF] Zheng | Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases[END_REF]. Although the E2 core domain shows high sequence conservation, E3 enzymes interact physically and functionally only with particular E2 enzymes. Again, the key questions are how this selectivity is obtained and how it then determines the substrates specificity [START_REF] Winkler | An altered-specificity ubiquitin-conjugating enzyme/ubiquitin-protein ligase pair[END_REF].

The first structural information on E2/E3 interaction specificity came from the crystal structures of the human HECT ligase E6-AP bound to the ubiquitin conjugating enzyme UbcH7 [START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF] and from the c-Cbl proto-oncogene, which belongs to a RING family E3s, also bound to UbcH7 [START_REF] Zheng | Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases[END_REF] (Figure 14). Both complexes had similar structures concerning hydrophobic residues located in the loop regions of UbcH7 and hydrophobic regions in the HECT or RING domains of E3s. The structural studies by comparison of E2/E3 interfaces showed that these E3s interacted with almost identical regions of the UbcH7 E2 (loops L1 and L2), despite the E3s belonging to two functionally and structurally distinct E3 families. The residues in the UbcH7 responsible for the most extensive contact are Phe-63, Pro-97, and Ala-98 [START_REF] Huang | Structure of an E6AP-UbcH7 complex: insights into ubiquitination by the E2-E3 enzyme cascade[END_REF][START_REF] Zheng | Structure of a c-Cbl-UbcH7 complex: RING domain function in ubiquitin-protein ligases[END_REF]. In the majority of E2s, the residues involved in the specificity of the interactions are not located in a single point. They are, however, dispersed over the N-terminal helix flexible and divergent loop regions (L1 and L2) [START_REF] Van Wijk | Dynamic control of selectivity in the ubiquitination pathway revealed by an ASP to GLU substitution in an intra-molecular salt-bridge network[END_REF].

'Backside' interactions

Although structural studies using recombinant proteins have demonstrated that all E2s interact with E3s by using a conserved interaction surface close to their active site, some E2s have in fact been shown to also contact proteins with the opposite surface (Figure 16). This so-called 'backside' was first identified by [START_REF] Brzovic | A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination[END_REF] as a site for noncovalent ubiquitin binding of the human UbcH5c conjugating enzyme (UbcH5s are yeast homologues of Ubc4). [START_REF] Brzovic | A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination[END_REF] found that Ub can recognize a surface on UbcH5c comprised of the β sheet, which is separate from the catalytic center. Upon this interaction, UbcH5c~Ub self-assemble into high molecular weight complexes, which is required for the BRCA1-directed polyubiquitin chain formation [START_REF] Brzovic | A UbcH5/ubiquitin noncovalent complex is required for processive BRCA1-directed ubiquitination[END_REF].

INTRODUCTION 49

More recently, Metzger et al. (2013) reported the crystal structure of the yeast Ubc7 conjugating enzyme, a homologue of Ube2g2, with a C-terminal Ubc7-binding region (U7BR) of the Cue1 protein (Cue1 recruits Ubc7 to the ER and is required for Ubc7 stability) (Figure 17). This structural data revealed that the U7BR has a domain that includes three α-helices that interact extensively with the 'backside' of Ubc7. Furthermore, the study of yeast ubiquitylation enzymes revealed that the E2 Rad6 uses its 'backside' to interact with RING E3s Rad18, Ubr1 and Bre1. Rad18 monoubiquitylates the target proliferating cell nuclear antigen (PCNA), and binds the E2 not only through its RING domain but also via a distinct α-helix which interacts with the 'backside' [START_REF] Bailly | Domains required for dimerization of yeast Rad6 ubiquitinconjugating enzyme and Rad18 DNA binding protein[END_REF][START_REF] Notenboom | Functional characterization of Rad18 domains for Rad6, ubiquitin, DNA binding and PCNA modification[END_REF]. Worthy of note, the Rad6/Rad18 'backside' interaction has a low affinity and competes with a noncovalent Ub binding on Rad6, giving the Rad6 the ability to form free Ub chains in the absence of Rad18, and directing monoubiquitylation [START_REF] Hibbert | E3 ligase Rad18 promotes monoubiquitination rather than ubiquitin chain formation by E2 enzyme Rad6[END_REF]. Recently, Turco et al.

(2014) demonstrated that the Bre1 RING ligase interacts with Rad6, both canonically via the RING domain as well as through its Rad6 binding domain (RBD) using a 'backside' of Rad6. This E2/E3 pair plays a role in the monoubiquitylation of the histone H2B during transcription. Although the 'backside' interaction is nonessential for histone monoubiquitylation, it has been shown that this binding strongly enhances Rad6 activation, thereby accelerating the overall reaction [START_REF] Turco | Monoubiquitination of histone H2B is intrinsic to the Bre1 RING -Rad6 interaction and augmented by a second Rad6 binding site on Bre1[END_REF]. The anaphase promoting complex (APC), was similarly found to recruit its E2 UBCH10 using both canonical interactions with the RING domain of APC11 and backside interactions with APC2 [START_REF] Brown | RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex[END_REF]. Also, Ubr1 E3 was shown to interact with the Rad6 INTRODUCTION 51

Identification of E2/E3 interactions

The identification of functional E2/E3 pairs is hindered by two main factors. First, it is clear that the E2/E3 complexes have a modest affinity and are transient in nature (Wenzel et al., 2011). This makes the interactions difficult to study using conventional biochemical approaches, as the weak complexes very often do not survive, for example in case of pull down assays or co-immunoprecipitation [START_REF] Christensen | Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination[END_REF].

Although the binding affinities between E2/E3 enzymes can vary substantially, they are relatively low for most active E2/E3 complexes (often in the µM range). Recently, a difference in affinity of nearly 50-fold was reported in the binding of the E3 Rbx1 to Cdc34∼Ub conjugate over free Cdc34 [START_REF] Spratt | RBR E3 ubiquitin ligases: new structures, new insights, new questions[END_REF]. The second main factor that hinders the identification of E2/E3 pairs is that a significant number of E3s function as dimers (homo-or heterodimeric E3s are known) or as multicomponent protein complexes. In many cases, these E3s can only function within those contexts [START_REF] Christensen | Dynamic interactions of proteins in complex networks: identifying the complete set of interacting E2s for functional investigation of E3-dependent protein ubiquitination[END_REF] and it is therefore not always easy to reconstitute these complexes in vitro.

Physiological network of E2/E3 interactions

David Komander and Michael Rape (2012) concluded in their excellent review on the ubiquitin code: 'Given the importance of E2s in determining specificity, it is surprising how little we know about physiological E2-E3 pairs'. Indeed, our current knowledge on E2/E3 pairs is mainly based on individual studies and E2/E3 interactions have rarely been investigated systematically (see Table 5 for a list of E2/E3 interactions reported in budding yeast). Hence, our knowledge on how E2s and E3s operate at a network level is still preliminary. How do E3s collaborate with multiple E2s to assemble specific ubiquitin signals on their substrate? Do E2s function redundantly or do they have distinct activities (e.g. to prime or elongate specific chains)? Do they serve to ubiquitylate identical or different substrates? Can they assemble heterologous ubiquitin chains with particular functions? These questions have been addressed only for few E3s (e.g. [START_REF] Christensen | E2-BRCA1 RING interactions dictate synthesis of mono-or specific polyubiquitin chain linkages[END_REF][START_REF] Petroski | Substrate modification with lysine 63-linked ubiquitin chains through the UBC13-UEV1A ubiquitinconjugating enzyme[END_REF][START_REF] Rodrigo-Brenni | Sequential E2s drive polyubiquitin chain assembly on APC targets[END_REF][START_REF] Wu | UBE2S drives elongation of K11-linked ubiquitin chains by the anaphase-promoting complex[END_REF][START_REF] Meyer | Enhanced protein degradation by branched ubiquitin chains[END_REF] but remain completely unanswered for the vast majority of E3s. [START_REF] Howell | An evaluation of in vitro proteinprotein interaction techniques: assessing contaminating background proteins[END_REF][START_REF] Berggård | Methods for the detection and analysis of protein-protein interactions[END_REF].

However, all these techniques remove proteins from their native physiological environment. The monitoring and visualization of PPIs in living cells and in organisms, as well as the characterization of the entire interaction networks, are necessary to understand precisely the molecular mechanisms that underlay biological processes.

A large set of data on PPIs in S. cerevisiae comes from the yeast two-hybrid analysis (Y2H) [START_REF] Uetz | A comprehensive analysis of protein-protein interactions in Saccharomyces cerevisiae[END_REF][START_REF] Ito | A comprehensive twohybrid analysis to explore the yeast protein interactome[END_REF], a very widely used approach in screening for PPIs in living yeast cells, designed by [START_REF] Fields | A novel genetic system to detect protein-protein interactions[END_REF]. Y2H relies on monitoring the complex formation via transcriptional activation of reporter genes.

Upon the interaction of two proteins, the DNA binding and activation domains of the transcription factor ('bait' and 'prey', respectively) are brought together, thereby producing a functional transcription activator. The detection is made possible by the expression of reporter genes that results in yeast viability or growth on selective media (Chien et al., 1991;[START_REF] Parrish | Yeast two-hybrid contributions to interactome mapping[END_REF]. However, in the Y2H system proteins are usually overexpressed and thus present at concentrations far higher than under physiological conditions. This in turn often results in false positives [START_REF] Bartel | Elimination of false positives that arise in using the two-hybrid system[END_REF][START_REF] Serebriiskii | Approaches to detecting false positives in yeast two-hybrid systems[END_REF]. It was also noted that the Y2H method is inappropriate for studying the interactions between proteins that cannot be transported to the nucleus, since for the reporter gene to be activated the interaction must occur in the nucleus [START_REF] Sung | Genomewide bimolecular fluorescence complementation analysis of SUMO interactome in yeast[END_REF]. Another limitation of Y2H is that proteins overexpressed out of their physiological environment are prone to inappropriate folding. Advantages and As stated in the preceding chapter, several methods exist for studying PPIs in living cells. One of the most popular and frequently applied methods is Förster resonance energy transfer, often referred to as fluorescence resonance energy transfer (FRET) analysis [START_REF] Sorkin | Interaction of EGF receptor and Grb2 in living cells visualized by fluorescence resonance energy transfer (FRET) microscopy[END_REF][START_REF] Sekar | Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations[END_REF]. This method relies on the use of two fluorophores that are either genetically fused or chemically linked to two proteins of interest, and is based on the transfer of excitation energy between them [START_REF] Förster | Transfer mechanisms of electronic excitation[END_REF]. The energy is transferred from an excited state of one fluorophore, which is a donor, to the other fluorophore which is an acceptor. FRET allows for realtime detection of a complex formation and dissociation, and can detect PPIs occuring in different cellular compartments. However, it often requires the expression of proteins at high levels for energy transfer to be detectable. Moreover, it relies on the proper orientation and distance of the donor and the acceptor fluorophores. In practice, 10 nm (100 Å) is the maximum distance over which significant energy transfer can be detected between donor and acceptor fluorophores. Therefore, the detection of PPIs by FRET often requires extensive optimization and is restricted to relatively small proteins and complexes [START_REF] Piehler | New methodologies for measuring protein interactions in vivo and in vitro[END_REF][START_REF] Piehler | Spectroscopic techniques for monitoring protein interactions in living cells[END_REF].

Bioluminescence resonance energy transfer (BRET)

Another method for studying protein-protein interactions in real-time in vivo is bioluminescence resonance energy transfer (BRET) (Pfleger et al., 2006b), first used to study the bacterial KaiB circadian clock protein dimerization (Xu et al., 1999b). BRET relies on the same principle as FRET, with the exception that the donor here is not a fluorophore but a bioluminescent molecule (typically Renilla luciferase). The transfer of energy from the donor enzyme to the acceptor fluorophore, such as GFP or YFP, occurs upon substrate oxidation. The main limitation of BRET is that Renilla luciferase generates a broad emission peak that considerably overlaps with the emission of the yellow fluorescent protein (YFP), contributing to the low signal-to-noise ratio when

INTRODUCTION 57
YFP or its variants are used as an acceptor molecule (Xu et al., 1999b). BRET might be applicable to studying dynamic PPIs in a cell population, however the visualization of PPIs in the different subcellular compartments of a single living cell is more challenging due to the low intensity of luciferase light emission [START_REF] Boute | The use of resonance energy transfer in high-throughput screening: BRET versus FRET[END_REF]. 1.3.1.3 Fluorescence cross-correlation spectroscopy (FCCS)

In recent years, cross-correlation techniques that monitor correlated movement of interacting partners prior to protein complex formation have been widely developed,

with fluorescence cross-correlation spectroscopy (FCCS) applied to the quantitative and selective monitoring of interactions in living cells (reviewed by [START_REF] Bacia | Fluorescence cross-correlation spectroscopy in living cells[END_REF] recently by [START_REF] Ma | Fluorescence cross-correlation spectroscopy (FCCS) in living cells[END_REF]. This technique is an extension of fluorescence correlation spectroscopy (FCS) that measures the fluctuations in the number of fluorescent molecules in a confocal volume. FCCS detects the fluctuations of two spectroscopically separated fluorescent labels (two dyes with well-separated emission wavelengths, for example green and red) [START_REF] Schwille | Dual-color fluorescence cross-correlation spectroscopy for multicomponent diffusional analysis in solution[END_REF], and enables to quantify the formation of potential protein complexes. However, the absolute quantification is technically challenging, as it requires very precisely overlapping confocal volumes [START_REF] Foo | Factors affecting the quantification of biomolecular interactions by fluorescence cross-correlation spectroscopy[END_REF]. A limiting factor for FCCS, as well as fluorescence correlation spectroscopy in general, is that the particles under study have to be sufficiently mobile in the detection volume. Moreover, FCCS is only applicable at relatively low fluorophores concentration (otherwise the fluctuations in fluorescence intensity are difficult to detect), and therefore cannot detect weak interactions that occur at higher concentrations [START_REF] Bacia | Practical guidelines for dual-color fluorescence cross-correlation spectroscopy[END_REF]. Some FCCS variants and improvement have been reported

recently for studying PPIs in living cells [START_REF] Padilla-Parra | Dual-color fluorescence lifetime correlation spectroscopy to quantify protein-protein interactions in live cell[END_REF][START_REF] Singh | SPIM-FCCS: A novel technique to quantitate protein-protein interaction in live cells[END_REF][START_REF] Krieger | Dual-color fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS)[END_REF][START_REF] Sadaie | Quantitative in vivo fluorescence crosscorrelation analyses highlight the importance of competitive effects in the regulation of proteinprotein interactions[END_REF].

Protein-fragment complementation assays (PCAs)

The first protein-fragment complementation assay (PCA) for in vivo protein interactions was demonstrated by [START_REF] Johnsson | Split ubiquitin as a sensor of protein interactions in vivo[END_REF] using split-ubiquitin as a sensor. Here, the inactive N-and C-terminal fragments of ubiquitin with an attached reporter protein (C-terminal attachment) were fused to proteins of interest and upon their interaction the Ub function was restored. Ub was then recognized by ubiquitin specific proteases (USPs) and the C-terminal attached reporter protein was cleaved, leading to the activation of gene expression [START_REF] Johnsson | Split ubiquitin as a sensor of protein interactions in vivo[END_REF].

The general principle was further applied in the laboratory of Stephen Michnick, starting with the enzyme murine dihydrofolate reductase (mDHFR) as a reporter. The group showed that the reporter enzyme folds into its native structure from its two inactive fragments, and its activity is reconstituted upon the interaction of two proteins of interest. The restored function was then detected by reconstituted enzyme activity.

They demonstrated the principle on two PPIs in vivo, namely those of p21 ras GTPase with its target the ras-binding domain (RBD) of the Ser/Thr kinase raf, and the rapamycin-mediated interaction of the immunophillin FKBP with the S. cerevisiae target of rapamycin (TOR2) [START_REF] Pelletier | Oligomerization domain-directed reassembly of active dihydrofolate reductase from rationally designed fragments[END_REF]. Since then a number of enzymes, including β-lactamase [START_REF] Galarneau | Beta-lactamase protein fragment complementation assays as in vivo and in vitro sensors of protein protein interactions[END_REF][START_REF] Wehrman | Protein-protein interactions monitored in mammalian cells via complementation of beta -lactamase enzyme fragments[END_REF], firefly (Luker et al., 2004), Renilla [START_REF] Kim | High-throughput sensing and noninvasive imaging of protein nuclear transport by using reconstitution of split Renilla luciferase[END_REF][START_REF] Stefan | Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo[END_REF] and Gaussia luciferases [START_REF] Remy | A highly sensitive protein-protein interaction assay based on Gaussia luciferase[END_REF], as well as fluorescent proteins such as green fluorescent protein (GFP) [START_REF] Magliery | Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism[END_REF], yellow fluorescent protein (YFP) [START_REF] Nyfeler | Capturing protein interactions in the secretory pathway of living cells[END_REF], Venus [START_REF] Nakagawa | Improvement of a Venus-based bimolecular fluorescence complementation assay to visualize bFos-bJun interaction in living cells[END_REF][START_REF] Ohashi | Visualization of cofilin-actin and Ras-Raf interactions by bimolecular fluorescence complementation assays using a new pair of split Venus fragments[END_REF] or mCherry [START_REF] Fan | Split mCherry as a new red bimolecular fluorescence complementation system for visualizing proteinprotein interactions in living cells[END_REF] BiLC relies on the ability of split nonluminescent protein fragments to reconstitute functional luminescent proteins when these are brought into close contact by fused interacting partners (Figure 18). Among the BiLC are luciferase-based systems with various luciferases as aforementioned, and Renilla luciferase as the major reporter protein used to date in living cells and rodents. Luciferase complementation involves fusing the inactive N-and C-terminal luciferase fragments to two proteins of interests, which bring the fragments together upon their interaction and reconstitute an active enzyme. Luciferase then catalyses the oxidation of their cell membrane-permeable substrate coelenterazine, and the activity is detected by emitted bioluminescence in the form of blue light (400-500 nm). The bioluminescent signal is emitted only at the sites and times of interaction occurrence in cells [START_REF] Kaihara | Locating a Protein-Protein Interaction in Living Cells via Split Renilla Luciferase Complementation[END_REF]. Luciferase-based assays can be applied to quantify regulation of protein-protein complexes in response to various signaling events, chemical probes or drugs [START_REF] Luker | Split Gaussia luciferase for imaging ligand-receptor binding[END_REF].

Fluorescent protein fragments used in BiFC analysis

A considerable number of different fluorescent protein fragments have been used

for BiFC analysis. The green fluorescent protein (GFP) from the jelly fish Aequorea victoria has emerged as a versatile tool in biology since its cloning and sequencing in 1992 [START_REF] Prasher | Primary structure of the Aequorea victoria green-fluorescent protein[END_REF], and has been extensively used in various complementation assays. The first split GFP assay to detect PPIs was described by [START_REF] Ghosh | Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein[END_REF].

In their study they used antiparallel leucine zippers, which were attached via a linker sequence to two split (between residues 157 and 158) GFP N-and C-terminal fragments called NGFP and CGFP, containing 157 and 81 residues respectively. Leucine zippers were required to produce fluorescence, suggesting that the split fragments could not reassemble GFP on their own [START_REF] Ghosh | Antiparallel leucine zipper-directed protein reassembly: application to the green fluorescent protein[END_REF]. A number of GFP variants have similarly been used in BiFC assays, including EGFP, EBFP, ECFP, yellow fluorescent protein (YFP) or its enhanced version (EYFP), Venus and Cerulean (Shyu et al., 2006; reviewed by [START_REF] Kodama | Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives[END_REF]. In addition, other proteins that show different spectral properties and stabilities have also been used, including mCherry [START_REF] Fan | Split mCherry as a new red bimolecular fluorescence complementation system for visualizing proteinprotein interactions in living cells[END_REF], mRFP1 [START_REF] Jach | An improved mRFP1 adds red to bimolecular fluorescence complementation[END_REF] or photoswitchable Dronpa [START_REF] Lee | Development of bimolecular fluorescence complementation using Dronpa for visualization of protein-protein interactions in cells[END_REF].

Currently the most widely used fluorescent protein fragments are derived from EYFP and Venus. EYFP-based BiFC was first reported to visualize PPIs in living mammalian cells (Hu et al., 2002). However, these assays were sensitive to high temperatures. Venus (Figure 20), a YFP variant with an extra point mutation F46L, was

shown to be less sensitive to the environment [START_REF] Nagai | A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications[END_REF]. Moreover, Venus was identified by [START_REF] Nagai | A variant of yellow fluorescent protein with fast and efficient maturation for cell-biological applications[END_REF]

Use of BiFC to investigate protein-protein interactions in living cells

The BiFC approach has been extensively used to study PPIs in various cell types and organisms. PPIs have been visualized in bacteria, for example E. coli [START_REF] Magliery | Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism[END_REF][START_REF] Pazos | Interactions among the early Escherichia coli divisome proteins revealed by bimolecular fluorescence complementation[END_REF] or Agrobacterium tumefaciens [START_REF] Cascales | Agrobacterium tumefaciens oncogenic suppressors inhibit T-DNA and VirE2 protein substrate binding to the VirD4 coupling protein[END_REF], in fungilargely in S. cerevisiae [START_REF] Blondel | Degradation of Hof1 by SCF(Grr1) is important for actomyosin contraction during cytokinesis in yeast[END_REF][START_REF] Cole | Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae[END_REF][START_REF] Sung | Bimolecular fluorescence complementation analysis system for in vivo detection of protein-protein interaction in Saccharomyces cerevisiae[END_REF], nematode C. elegans [START_REF] Hiatt | Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in Caenorhabditis elegans[END_REF], plants [START_REF] Bracha-Drori | Detection of protein-protein interactions in plants using bimolecular fluorescence complementation[END_REF][START_REF] Walter | Visualization of protein interactions in living plant cells using bimolecular fluorescence complementation[END_REF]) and mammalian cells (Hu et al., 2002;[START_REF] Hynes | Visualization of G protein betagamma dimers using bimolecular fluorescence complementation demonstrates roles for both beta and gamma in subcellular targeting[END_REF].

However, it is interesting to note that molecular oxygen is required in the chemistry of fluorophore formation, rendering the approach ineligible for use in anaerobic organisms [START_REF] Kerppola | Design and implementation of bimolecular fluorescence complementation (BiFC) assays for the visualization of protein interactions in living cells[END_REF]. Several studies have revealed that BiFC enables simple and direct visualization of PPIs (Hu et al., 2002;[START_REF] Hu | Simultaneous visualization of multiple protein interactions in living cells using multicolor fluorescence complementation analysis[END_REF][START_REF] Hudry | Visualization of protein interactions in living Drosophila embryos by the bimolecular fluorescence complementation assay[END_REF]. This can be performed directly in living cells, eliminating possible artifacts inherent in cell lysis or fixation. PPIs using BiFC can be detected without the addition of fluorogenic or chromogenic agents, thereby avoiding cells perturbation by those agents. Aside from determining whether two proteins can interact, BiFC provides information on the localization of protein complexes. Previous studies have reported visualization of BiFC complexes in various subnuclear structures (Hu et al., 2002), lysosomes [START_REF] Fang | Ubiquitin-mediated fluorescence complementation reveals that Jun ubiquitinated by Itch/AIP4 is localized to lysosomes[END_REF], plasma membrane [START_REF] Remy | Regulation of apoptosis by the Ft1 protein, a new modulator of protein kinase B/Akt[END_REF], endoplasmic reticulum (ER) [START_REF] Anderie | Direct interaction between ER membrane-bound PTP1B and its plasma membrane-anchored targets[END_REF] and lipid droplets [START_REF] Granneman | Analysis of lipolytic protein trafficking and interactions in adipocytes[END_REF]. BiFC has been also applied to examine cell cycle regulated PPIs. Examining the individual stages of the cell cycle remains however still a challenging subject for BiFC imaging [START_REF] Blondel | Degradation of Hof1 by SCF(Grr1) is important for actomyosin contraction during cytokinesis in yeast[END_REF][START_REF] Cole | Use of bimolecular fluorescence complementation to study in vivo interactions between Cdc42p and Rdi1p of Saccharomyces cerevisiae[END_REF].

Advantages and limitations of BiFC

BiFC is an attractive method for investigating PPIs. Its main advantage is the capacity to detect transient and weak interactions. Indeed, the association of two fluorescent protein fragments is irreversible and stabilizes interactions between the binding partners (transient interactions can be trapped in a complex). Therefore not only weakly associating proteins but also interactions with short half-lives can be detected [START_REF] Magliery | Detecting protein-protein interactions with a green fluorescent protein fragment reassembly trap: scope and mechanism[END_REF]. This irreversible complex formation is however a drawback as it might potentially influence the function or activity of the complex. It is now known that BiFC assay can be used to detect interactions within minutes after the complexes being formed, but the limitation here is that it does not provide real-time measurement of the amount of formed complexes and does not provide information on complex dissociation [START_REF] Kerppola | Design of Fusion Proteins for Bimolecular Fluorescence Complementation (BiFC)[END_REF]. Another advantage however is that proteins those interactions are to be examined are not overexpressed but rather are expressed in their cellular context, ideally at similar levels as the endogenous proteins. This helps to avoid the falsepositive results derived from a self-association of two fluorescent protein fragments, when expressed at sufficiently high concentrations [START_REF] Cabantous | Protein tagging and detection with engineered self-assembling fragments of green fluorescent protein[END_REF] in the absence of the interaction. It is important therefore to test the fluorescence complementation using fusion proteins with the mutated interaction interface. The mutant proteins should be fused in the same manner to the wild-type proteins (Hu et al., 2002). Another benefit of this method is that the fluorescence of BiFC complex can be measured using a widely available fluorescence microscope or a flow cytometer without the need for any specialist equipment.

AIM OF THIS STUDY

Interactions between ubiquitin conjugating enzymes and ubiquitin ligases are in the center of ubiquitylation cascade. E2 enzymes constitute a passage between the first step of activation and the final covalent conjugation, and the combination of particular E2/E3 pairs determine what types of Ub chains are made, thus determining the regulatory functions of the Ub pathway. To date, only a small fraction of all possible E2/E3 pairs have been investigated, mainly using biochemical and in vitro approaches that often may not accurately reflect the conditions that occur in living cells.

The primary goal of my PhD project was therefore to establish a method suitable to systematically assay E2/E3 interactions under near physiological conditions and then screen for new E2/E3 pairs in living cells, using yeast Saccharomyces cerevisiae as a model organism. Looking at the E2/E3 interactions under near physiological conditions is of great importance to preserve features that influence biologically relevant interactions such as protein localization, concentration, posttranslational modifications and incorporation into interacting partners as well as E2~Ub conjugates, and other regulatory mechanisms.

This project was set with the following objectives:

1. Identification and optimization of a method to detect and quantify E2/E3 interactions in living yeast cells.

2. Construction of an array of yeast strains to systematically assay E2/E3 interactions.

3. Unbiased screening for new putative E2/E3 pairs in budding yeast.

4. Functional characterization of one candidate E2/E3 pair.

MATERIALS AND METHODS

Materials

All Protect from light as it can affect the breakdown rate of this antibiotic;

 Coelenterazine and coelenterazine analogs solutions -dissolved in ethanol and prepared under indirect (dimmed) light. Aliquots should be prepared fresh, optimally one day prior use or on the day of use. For longer storage aliquots can be frozen at -20°C and need to be adjusted to RT prior using for at least 1h.

CRITICAL: exposure of coelenterazine or its analogs to light and ambient oxygen must be avoided;

 Amino acid stock solution -0.2 g L -1 uracil, 0.2 g L -1 L-histidine, 0.3 g L -1 Llysine HCl, 0.6 g L -1 L-leucine, 0. CRITICAL: Autoclaved and cooled to 50°C before adding antibiotics, the desired amino acids as well as canavanine and thialysine. Note: For any synthetic media containing G418 or hygromycin monosodium glutamic acid is added as a nitrogen source instead of ammonium sulfate [START_REF] Cheng | Controlling gene expression in yeast by inducible site-specific recombination[END_REF]; AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR AmpR



Antibodies

S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C S288C
3.2 Methods

Molecular biology

Plasmid construction

Plasmid constructions were produced using standard molecular biology techniques as described by [START_REF] Sambrook | Molecular Cloning: A Laboratory Manual[END_REF]. Plasmid DNA was amplified in E. coli and isolated using Macherey-Nagel kits according to the manufacturer's instructions. Plasmid digestions were performed using restriction enzymes from New

England Biolabs (NEB), according to the instruction. DNA fragments were assembled 7. The amplification of DNA templates was performed using a T100™ Thermal Cycler from Bio-Rad.

Agarose gel electrophoresis

Agarose gel electrophoresis was performed in order to separate the DNA fragments and determine their length (bp). 5 µL of each PCR product was run on 0.8% (for DNA fragments expected >700 bp) or 2% (for DNA fragments expected 50-2,000 bp) agarose gel, with TAE (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH~8.3) as a running buffer. 5 µL of GeneRuler DNA ladder mix (Thermo Scientific) was used as a standard. The gel was stained with GelRed (Biotium) and visualized using a UV transilluminator imaging system.

Transformation of heat-competent E. coli cells 100 μL of DH5α chemically competent E. coli cells was thawed on ice for each transformation. 1-2.5 μL of DNA was added and incubated on ice for 10 min. The mixture was plated on pre-warmed LB-Amp plates and incubated O/N at 37°C.

Yeast genetics

Isolation of yeast genomic DNA A 10 ml yeast culture grown overnight in YPD was harvested by centrifugation (3000 rpm for 5 min). The cell pellet was washed with ddH2O and transferred to DNA pellet without supernatant was then dried for 5 min at 50°C and the DNA was resuspended in 100 μL of TE buffer (pH 8.0).

Endogenous tagging

Many of the yeast strains used in this study were genetically modified to express that harbor E2 interacting domains at their N-terminal were generated using N-terminal tagging. Among these are E3s such as Tfb3, Prp19, Mot2, Psh1, Nam7 and Ste5.

Yeast transformation

Transformation of yeast cells using plasmids and PCR products was achieved using a slightly modified method of [START_REF] Gietz | Yeast transformation by the LiAc/SS Carrier DNA/PEG method[END_REF]. In brief, yeast cells were cultured overnight in 10 mL of YPD at 30°C. The next day, cells were diluted 1:20 into fresh YPD and grown for an additional 2-3 h until the culture reached an OD 600 of 0.4-0.8. Cells were harvested by centrifugation (3200 rpm for 5 min at RT). The cell pellet was resuspended in 5 mL of LiAc mix and again harvested by centrifugation (3200 rpm for 5 min at RT). Cells were resuspended in 100 μL of 1M LiAc mix (one transformation) and transferred to a microcentrifuge tube. For each transformation reaction, the following reagents were added to the cell pellet:

-700 μL of PEG mix (autoclaved) -30 μL of salmon sperm DNA (10 mg/mL), incubated at 95°C for 5 min, placed on ice prior to being added to the mixture. Salmon sperm DNA is used as a carrier in yeast transformations.

-2 μL of a plasmid DNA preparation (for replicative plasmids), 25 μL of previously digested plasmid in case of integrative plasmids, or 20 μL of PCR product.

The components were mixed gently by inverting the tube several times. The mix was heat shocked in a thermomixer at 42°C for 40 min under agitation. After a centrifugation step (3200 rpm, 3 min), the pellet was resuspended in 100 μL of sterile ddH 2 O and the cell suspension was plated with sterile glass beads (∅ 3 mm) on selective media. For drug selection on the plates containing antibiotics, cells were allowed to recover prior plating and were therefore incubated in the thermomixer, in liquid YPD at 30°C for 2-3 h. Transformants were restreaked on the new plates and screened for the proper integration by PCR. PCR was performed using a Taq polymerase and 20 base pairs primers. The forward primer binds to the region 300-400 bp from the stop codon of a targeted gene, whereas the reverse primer binds to the resistance cassette.

Generation of yeast strains

The deletion and tagging of particular genes of interest were performed according to the methods described by [START_REF] Güldener | A new efficient gene disruption cassette for repeated use in budding yeast[END_REF] and [START_REF] Knop | Epitope tagging of yeast genes using a PCR-based strategy: more tags and improved practical routines[END_REF], respectively.

For the initial tests of BiFC optimization, the strain BY4741 (MATa ura3Δ0 leu2Δ0 his3Δ1 met15Δ0) was transformed using PCR products generated after the amplification of pFA6a_VC155-kanMX6, carrying Escherichia coli kanamycin-resistance gene [START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF] plasmid DNA. The primers designed for this PCR were to amplify the genes of interest Ubr1 and Ufd4, coding for the ubiquitin ligases. The strains obtained had the following genotypes BY4741 ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 ubr1::UBR1-VenusF[C]-KAN and BY4741 ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 ufd4::UFD4-VenusF[C]-KAN, respectively. Since strains derived from BY4741 and Y7092 carry the kanamycin-resistance (kanMX6), the resistance of strains derived from BY4741 was switched to nourseothricin resistance (natMX4). The strain Y7092 (MATα ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 can1::STE2pr-Sphis5 lyp1Δ) was transformed using PCR products generated after amplification of pFA6a_VN173-kanMX6 plasmid DNA.

The primers designed for this PCR were to amplify the genes of interest Rad6 and Ubc4, coding for ubiquitin conjugating enzymes. The strains obtained had the genotype BY4745 Y7092 ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 can1::STE2pr-Sphis5 lyp1Δ rad6::RAD6-VenusF[C]-KAN and BY4745 Y7092 ura3Δ0 leu2Δ0 his3Δ met15Δ0 can1::STE2pr-Sphis5 lyp1Δ ubc4::UBC4-VenusF[C]-KAN, respectively. The strains obtained carry a query mutation linked to a dominant selectable marker, such as the kanamycin-resistance (kanMX6) module, and can1::STE2pr-Sphis5 lyp1Δ of the CAN1 gene that normally confers sensitivity to canavanine. The strain also has lyp1 gene deletion that confers resistance to thialysine. These genetic elements are necessary for the selection of haploid double mutant progeny. For BiFC optimization we generated four endogenously tagged haploid yeast strains, which were then manually mated to one another by mixing them together on YPD media and leaving overnight at 30°C, to produce diploid yeast that expressed the desired C-terminally tagged proteins of interest.

We confirmed the successful tagging by PCR and sequencing. Upon mating, the yeast were restricted on double selection plates with kanamycine (G418) and nourseothricin (clonNAT), grown for 2 days at 30°C and then sporulated in ZnSPO media for 3-4 days.

Double mutant MATa was selected directly after sporulation on SED medium lacking histidine, arginine and lysine (-His/Agr/Lys), with addition of G418 (350 µg/ml), clonNAT (100 µg/ml), canavanine (50 mg/ml) and thialysine (50 mg/ml). The strains for luciferase experiments were generated in a similar manner. ORFs of interest, Rad6, Ubc4 (E2s) and Ubr1, Ufd4 (E3s), were tagged with Renilla luciferase fragments, RlucF [START_REF] Mekhail | The nuclear envelope in genome organization, expression and stability[END_REF] and RlucF [START_REF] Zattas | Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope[END_REF] respectively.

The construction of an array of yeast strains, in which each endogenous E2 gene was fused to the C-terminal fragment of Venus and the library construction of haploid yeast cells with E2/E3 pair combinations and C-and N-terminal fragments of Venus, was carried out as follows. The respective yeast strains, where the ORF of each E2 had been replaced with a kanamycin resistance module, were transformed using plasmids containing E2s that were tagged with a C-terminal fragment of Venus and a nourseothricin resistance cassette. The correct integration of these plasmids into the yeast was verified by PCR. All the tagged E2 strains were then crossed with a modified scEB0028 strain (Y7092 rpn7::RPN7-tdimer2( 12)-LEU). This strain expresses Rpn7 proteasome subunit fused to the red fluorescent protein tdimer2, which enables to quantify the fluorescent signal not only in the cytoplasm but also in the nucleus. The genetic elements of the background strain Y7092 (MATα ura3Δ0 leu2Δ0 his3Δ1 met15Δ0 can1::STE2pr-Sphis5 lyp1Δ) enable the selection of haploid double mutant progeny. The strain scEB0028 was additionally modified to enable selection of MATα progeny using a plasmid pEB0053 (pUC57, ProteoGenix). This is the plasmid used for the integration of the yeast a-factor receptor (Ste3p), which is expressed only in MATα cells and is resistant to hygromycin (HPH), into the lyp1 locus. All the E2 strains obtained were then ready for random spore selection after crossing with E3 strains. The strain crossing procedure, based on synthetic genetic array (SGA) technology [START_REF] Tong | Synthetic genetic array analysis in Saccharomyces cerevisiae[END_REF], was performed in 96-well plates containing liquid media. Unique well coordinates were assigned to each tagged E2 and E3. Strains were mated in YPD with the addition of tetracycline (10 mg/mL) to avoid possible contamination. After diploid selection, followed by sporulation, the haploid double mutant MATα was directly selected. All crossing and selection steps were carried out in a horizontal shaker at 20°C, as during our initial tests we observed stronger fluorescence signal than at 30°C.

Immunoblot

To determine the expression levels of the proteins of interest and to determine whether the fusion of PCA fragments to the respective N-or C-terminal of the proteins of interest do interfere with their normal expression levels, strains expressing fusion proteins were analyzed by immunoblot using specific antibodies (see Table 6, Materials). Total yeast protein extracts were prepared from the whole cells according to standard protocols. In brief, cells were grown overnight in 10 mL of YPD medium at 30°C in a horizontal shaker. The following day, the cultures were used to inoculate fresh 10 mL cultures at cell density of 0.5 OD 600 and grown for additional 3 h. OD at 600 nm was measured again for each sample and was adjusted if necessary prior to the extraction procedure. The cells were pelleted for 3 min at 4000 rpm and resuspended in 300 µL of 20% trichloroacetic acid (TCA). Samples were then transferred to 1.5 mL Eppendorf tubes containing approximately 1 ml of glass beads (1.25 mm diameter).

Cells were homogenized for 2 min in a cell disruptor (Disruptor Genie). The tubes were then pierced with an 18 G needle and the broken cells collected in fresh tubes by centrifugation (5 s at 5000 rpm in a tabletop centrifuge). The cells were then spun at maximum speed to precipitate proteins, TCA was removed and each pellet was resuspended in 90 µL of TCA-sample buffer. 10 µL of dithiotretiol (DTT) was added to each sample as a reducing agent, and samples were denatured for 5 min at 95°C. The samples were pelleted to remove debris for 5 min at maximum speed in a tabletop centrifuge. Supernatant was collected in fresh tubes and 5 µL of each sample was loaded on sodium dodecyl sulphate (SDS)-PAGE mini gels. The samples were run on 4-15% polyacrylamide gels (Mini-PROTEAN® TGX™ Precast Gels, BioRad) in a Tris-Glycine-SDS 1x buffer at 200 V for approximately 30 min. The proteins were then transferred from the gel to the PVDF membranes using a semi-dry transfer method.

After the transfer, the membrane was blocked with 5% milk solution in PBS-T (PBS with 0.1% Tween 20) for 1 h at RT. The blots were probed with respective primary antibodies in 5% milk solution in PBS-T at 4°C O/N with gentle shaking. The blots were then washed five times with PBS-T, each wash lasting 10 min at RT on a rocker.

After the washes they were incubated with respective secondary antibody in 5% milk solution in PBS-T for 1 h at RT. They were washed again five times prior to the distribution of chemiluminescent reagent (non-commercial ECL solution or Super Signal West Femto from Thermo Scientific), and then exposed to the radiography films.

Luciferase assay

Yeast transformed using the plasmids p41HPHtef_nNOS-L-RlucF (neuronal nitric oxide synthase domain, nNOS) and p41NATtef_aSYN-L-RlucF (aSyntrophin domain, aSYN) as well as untransformed yeast (as a control to determine the background ratio)

were picked from the selection plates and grown O/N at 30°C in 10 ml of YPD medium in a horizontal shaker. The cultures of transformed strains were supplemented with both nourseothricin (100 µg/L) and hygromycin b (50 mg/mL). The following morning, the cultures were diluted in a fresh YPD medium. The above-mentioned antibiotics were again added to the transformed strains. Yeast was grown at 30°C in a horizontal shaker until they reached an OD 600 of 1 (exponential phase of growth). 100 µl of each culture was then distributed to white 96-well plates. Luciferase activity was measured using a Centro LB 960 Microplate Luminometer (Berthold Technologies). The luciferase substrate used for this study was the coelenterazine analogcoelenterazine h (CTZ h),

as it has previously been shown by [START_REF] Zhao | Characterization of coelenterazine analogs for measurements of Renilla luciferase activity in live cells and living animals[END_REF] to increase the luminescence intensity more than the native coelenterazine. CTZ h aliquots were freshly prepared at the desired concentration prior to each experiment. The plate was briefly shaken and the measurements were performed immediately after the addition of CTZ h. Rluc PCA signal was integrated for 15 s. In a single experiment, the signal was measured in triplicates for each sample and experiments were repeated independently three times. In order to determine Rluc PCA signal from the background autoluminescence of CTZ h, the luminescence signal of medium and substrate alone was subtracted from all measured signals combined to obtain the net luminescence. Opaque white plates were used for measurements since they gave the best performance; they maximized the signal in luciferase assays, whereas black plates reduced the signal (personal observation, data not shown). However, white plates absorb energy from the ambient light in the room and emit the energy as light during luciferase measurement. Therefore, they should preferably be stored in a dark and also exposed to reduced light while pipetting the samples. Alternatively, they can be left in the dark for approximately 10 min prior to measurements. All BiLC tests were performed at least in two independent experiments in duplicates.

Confocal microscopy

For microscopic analysis of BiFC yeast cells were grown to mid-logarithmic phase in a YPD medium. The cells were transferred to 96-well optical quality clear bottom plates containing 100 µL of visualization medium in each well. The cells were left to sit at the bottom of the wells for 30 min. Images were acquired on the Leica TCS SP8 MP confocal microscope using water-immersed objective at x 63 magnification and an argon laser. To image and process the 96-well plates the matrix screener mode was applied. Excitation and emission spectra for Venus fluorescent protein were 515 nm and 528 nm, respectively. Image acquisition was carried out using the Leica Application Suite X (LAS X) software. Data management and analysis was performed using ImagJ software.

Quantification of the efficiency of BiFC complex formation and statistical analysis

BiFC screen measurements were analyzed statistically using the GraphPad software with standard statistical test available. BiFC signals in cells co-expressing VCtagged E2s and VN-tagged E3s were quantified in both the cytoplasm and the nucleus of individual cells. Kurtosis measurements were incorporated as a measure of the distribution of the fluorescent pixels that enables to detect localized BiFC signals.

RESULTS

RESULTS

Bioluminescent versus fluorescent protein complementation assays to study E2/E3 interactions in living cells

As outlined in the introduction, our initial objective was to establish a suitable method to systematically assay interactions between ubiquitin conjugating (E2) and ligating (E3) enzymes in living cells. This was carried out in budding yeast, a model organism, in which it is easy to express tagged proteins from their endogenous chromosomal locus. It was challenging however, since E2/E3 interactions are weak and transient (typical Kds of E2/E3 interactions are in the low µM range) [START_REF] Deshaies | RING domain E3 ubiquitin ligases[END_REF][START_REF] Ye | Building ubiquitin chains: E2 enzymes at work[END_REF] and therefore require very sensitive detection methods. Protein-fragment complementation assays (PCAs) have been proven suitable to study weak and transient protein-protein interactions without the need to disrupt cell integrity [START_REF] Remy | A highly sensitive protein-protein interaction assay based on Gaussia luciferase[END_REF][START_REF] Ozbabacan | Transient protein-protein interactions[END_REF]. Here, we aimed to test, optimize and compare bioluminescent versus fluorescent PCAs in order to study E2/E3

interactions. These PCAs are known as Bimolecular Luminescence Complementation (BiLC) and Bimolecular Fluorescence Complementation (BiFC), respectively. We evaluated the ability to detect E2/E3 interactions using these methods, exemplified by the interaction of well described E2/E3 pairs in yeast, namely Ubc4/Ufd4 and Rad6/Ubr1.

Bimolecular Luminescence Complementation (BiLC)

One of the method belonging to protein fragment complementation assays (PCAs) that we decided to test was Bimolecular Luminescence Complementation (BiLC) (Luker et al., 2004). We chose to test BiLC, which is based on the Renilla luciferase (Rluc) due to its reported simplicity and sensitivity. Rluc PCA relies on the reconstitution of the enzymatic activity of luciferase from two split fragments upon the interaction of two proteins to which they are genetically fused [START_REF] Stefan | Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo[END_REF].

Luciferase utilizes molecular oxygen and catalyzes the oxidation of its cell membranepermeable substrate coelenterazine, emitting photon of light [START_REF] Sherf | Dual-Luciferase® reporter assay: An advanced co-reporter technology integrating firefly and Renilla luciferase assays[END_REF]. The

RESULTS

permeability of coelenterazine differs among the cells, which might impact the results.

Therefore, we started by testing different yeast cells for their substrate permeability (Figure 21 A). Among these were pdr1, pdr5 and erg6 mutants. These were previously shown to be more permeable for different chemicals [START_REF] Jensen-Pergakes | Sequencing, disruption, and characterization of the Candida albicans sterol methyltransferase (ERG6) gene: drug susceptibility studies in erg6 mutants[END_REF][START_REF] Emter | ERG6 and PDR5 regulate small lipophilic drug accumulation in yeast cells via distinct mechanisms[END_REF]. Wild type cells (BY4741) served as a control. The strains used here were transformed using control plasmids containing two luciferase fragments fused to the neuronal nitric oxide synthase (nNOS) and aSyntrophin (aSYN) PDZ domains that are known to form a heterodimer, p41HPHtef_nNOS-L-RlucF [START_REF] Mekhail | The nuclear envelope in genome organization, expression and stability[END_REF] and p41NATtef_aSYN-L-RlucF [START_REF] Zattas | Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope[END_REF], respectively. Erg6Δ cells generated a luminescence signal ~2.5 times higher than that of the wild type cells, whereas the signal of pdr5Δ cells was reduced by half (Figure 21 A). No difference in signal was observed for the pdr1 mutant in comparison with the wild type cells. Since erg6Δ cells had the highest luminescence signal-to-background ratio, we used these cells for BiLC assay optimization. We also compared different luminometers (data not shown), the FLUOstar Omega (BMG Labtech) and Centro LB 960 Microplate Luminometer (Berthold Technologies), with the latter performing best and therefore used in further analysis.

In our first tests, using erg6Δ cells, we compared different concentrations (10 µM and 50 µM) of the Renilla luciferase substrate, coelenterazine h (CTZ h) (Figure 21 B).

Untransformed erg6Δ cells served as a negative control. The bioluminescence signal was integrated every 15 s for 150 s after the addition of CTZ h. The highest signal was observed 45 s after substrate addition. As expected, an addition of 50 µM of CTZ h increased the luciferase activity ~2.2 times more than an addition of 10 µM (45 s after the addition of CTZ h). Nonetheless, the concentration of 10 µM was sufficient to observe ~10 times more signal than the negative control (45 s after the addition of CTZ h). For assay calibration we used a zero control, which was YPD media alone with no biological samples. Coelenterazine is a substrate known to be oxidized and produce light even when luciferase is absent. This is known as autoluminescence and creates a background signal which in turn reduces the assay sensitivity. We, therefore, additionally tested various autoluminescence reducing agents such as KI, Na 2 S 2 O 3 and vitamin C [START_REF] Harry | Methods, reagents and kits for luciferase assay[END_REF]. We noticed that the addition of these compounds results in a slight increase in substrate stability and therefore an increase in luminescent signal (Figure 21 C). This can be especially helpful for weak interactions. However, as there was not a significant reduction in the CTZ h autoluminescence, we decided to perform further analysis in the most natural cellular context. Omitting any exogenous reagents would definitely provide more accurate information on the E2/E3 interaction network in the cells.

RESULTS

Finally, we studied the detection of E2/E3 interactions using BiLC with two well described E2/E3 pairs in yeast, Ubc4/Ufd4 and Rad6/Ubr1, which were previously shown to interact and form a complex. Ubc4/Ufd4 are involved in the degradation of abnormal or excess Ub-fusion proteins by the so-called UFD pathway, whereas Rad6/Ubr1 in the polyubiquitylation of proteins containing non-acetylated N-terminal residues as part of the Ac/N-end rule pathway (Varshavsky, 2011). The erg6Δ strain with plasmids containing two luciferase fragments, p41HPHtef_nNOS-L-RlucF [START_REF] Mekhail | The nuclear envelope in genome organization, expression and stability[END_REF] and p41NATtef_aSYN-L-RlucF [START_REF] Zattas | Ubiquitin-dependent protein degradation at the yeast endoplasmic reticulum and nuclear envelope[END_REF], served as a positive control whilst wild type strain BY4741 served as a negative control (Figure 22). The experiment was performed in triplicates using 10 µM of CTZ h. We assessed the interactions between endogenously tagged Ubc4 or Rad6 and Ufd4 or Ubr1 in a erg6Δ strain background. No signal was detected in the four pairs studied in comparison with a positive control. Although Ubc4/Ufd4 and Rad6/Ubr1 were expected to interact, the absence of luminescent signal may also be due to the fact that the BiLC is reversible assay and may not capture these transient interactions. Additionally, the geometry of the fragments may prevent them from coming into close proximity with one another. Low BiLC signal could also be masked by the background autoluminescence. [START_REF] Kodama | Bimolecular fluorescence complementation (BiFC): a 5-year update and future perspectives[END_REF]. We noticed that expressing, for instance, the VC-tag alone (not fused to Ufd4) from the Ufd4 promoter was not a proper strategy, because its level of expression was much weaker than that of the Ufd4-VC. This could probably be due to the fact that VC-tag expressed on its own does not fold properly in the cell (data not shown). To test the specificity of the BiFC signal observed between Ubc4 and Ufd4 proteins, we therefore decided to generate Ubc4 mutants that could not interact with Ufd4. We first generated point Ubc4 point mutants in its canonical E3 interaction surface (ubc4(F63A) and ubc4(A97D)), but those were still able to produce a BiFC signal. We therefore decided to more dramatically mutate Ubc4 and truncate its N-terminal helix (which is involved in the canonical E2/E3 interface) and few amino acids in its C-terminal helix (which could be involved in 'backside' interactions (Figure 23 B). We checked by Western blot that those mutants are properly expressed and folded, since they can be charged with ubiquitin (Figure 23 C). Interestingly, the deletion mutants reduced the BiFC signal (Figure 23 D-E). The expression levels of the deletion mutants were higher than those of the wild type (not shown). Furthermore, the BiFC signal was almost completely abolished when Ubc4 was truncated in both its Nand C-terminus (Figure 23 D). Although this form of Ubc4 could not be loaded with

RESULTS

ubiquitin, it was still expressed to a level comparable to the other truncated mutants Ubc4 (Figure 23 C) and was therefore probably not completely unfolded.

While we were optimizing our imaging conditions to monitor the BiFC signal, we noticed that Ubc4/Ufd4 test pair generated a higher BiFC signal when cells were grown at lower temperatures (20°C) in comparison with standard growth conditions at 30°C

(data not shown). Therefore, we used 20°C as standard growth conditions for all experiments performed. Furthermore, to minimize the background fluorescence of yeast cells and therefore increase the assay sensitivity, we visualized the cells in a home-made visualization media prepared as described by Sheff and Thorn, (2004).

The results obtained with the truncation mutants of Ubc4 suggested that the BiFC signal produced between Ubc4 and Ufd4 is at least in part due to a bona fide E2/E3 interaction, and therefore convinced us to use this method to more systematically assay E2/E3 interactions in yeast. Since we detected signal by BiFC and not BiLC with the same E2/E3 pair (Ubc4/Ud4), we decided not to do further experiments using BiLC.

However, it is still possible that BiLC could be suitable to detect certain E2/E3 interactions, depending on the geometry and the level of expression of the proteins.

RESULTS

Construction and use of an array of yeast strains to assess E2/E3 interactions

To enable the application of the BiFC assay in screening for E2/E3 interactions in yeast under physiological conditions we constructed an array of eleven strains in which each endogenous E2 gene was C-terminally tagged with a C-terminal fragment of Venus (VC), with the exception of the Ubc1 and Ubc6 genes, which were tagged Nterminally. This was because the strain with C-terminally tagged Ubc1 was not viable (personal observation), whereas Ubc6 is an E2 that possesses a transmembrane domain and is a C-terminal membrane-anchored protein [START_REF] Yang | The Transmembrane Domain of a Carboxyl-terminal Anchored Protein Determines Localization to the Endoplasmic Reticulum[END_REF]. Simultaneously, we acquired 55 strains with endogenously tagged genes that encode E3s/putative E3s from a genome-wide library of yeast strains, where most yeast ORFs have been C-terminally fused to VN (S. cerevisiae VN-Fusion Library, Bioneer Corporation). Additionally, we generated E3 strains of interest that were missing in the library (such as Far1 and Rsp5)

or that were fused N-terminally to VN. These E3 genes include Tfb3, Prp19, Mot2, Psh1, Nam7 and Ste5 and they encode E3s that harbor their E2 interacting domain in their N-terminus. Indeed, since it is estimated that BiFC can occur when two Venus fragments are fused with a distance no greater than ~10 nm (Hu et al., 2002), it is possible that the position of the VN-tag greatly influence the possibility to reform a properly folded Venus protein. We also used as a control, the E1 enzyme Uba1, which is expected to interact with all eleven ubiquitin E2s. In total we therefore had 63 VNtagged strains representing 57 different E3s (six E3s were tagged both N-and Cterminally). The E2 strains were then crossed systematically with the E3 strains and the Uba1 strains to produce an array of 704 haploid yeast strains, with 627 strains expressing a unique combination of tagged E2 and E3 ORFs. The strain crossing procedure was performed in 96-well plates and the strains were then subjected to live cell imaging and analyzed for fluorescence upon BiFC complex formation in two independent experiments (Figure 24). We generated all these strains with an Rpn7 proteasome subunit fused to the red fluorescent protein tdimer2 to visualize the signal in the nucleus. This allowed not only to detect the particular E2/E3 interactions but also provided information on the subcellular localization of E2/E3 pairs. When measuring the BiFC signal, we also measured the Kurtosis of the fluorescent signal, which give information on the distribution of the fluorescent pixels that enables to more easily detect localized BiFC signals.

RESULTS

Figure 24. BiFC array construction to assay E2/E3 interactions. Yeast strains with the E2s tagged with VC and the E3s tagged with VN were incubated in liquid cultures. The strains were then mated in rich medium (YPD). Resulting haploid and diploid mixture strains were transferred onto the plates with diploid selective medium. Following diploid selection, strains were sporulated and haploid cells were selected in medium containing desired antibiotics, auxotrophic markers, canavanine and thialysine. E2/E3 interactions were assayed using quantitative microscopy. Fluorescence intensity was visualized both in the cytoplasm and in the nucleus. Rpn7 proteasome subunit, fused to the red fluorescent protein tdimer2, served as a nuclear marker.

Since yeast can easily be manipulated with plasmids that integrate into the genome by homologous recombination, their genes can be tagged endogenously. The endogenous tagging enables each fusion protein to be expressed from its own native promoter [START_REF] Janke | A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes[END_REF]. To assay the expression level of the tagged E2s and verify that they are not overexpressed, we compared the expression level of a few of them (tagged with VC) with the endogenous E2s across the generated strains (Figure 25).

RESULTS

Detection and localization of E2/E3 pairs with BiFC

Overall, our results enabled us to identify 128 putative E2/E3 interactions, 33 of which were previously reported in the literature (approximately 57% of already known interactions), and 95 constitute new putative E2/E3 pairs. Few E3s interacted only with a single E2, whereas others produced a BiFC signal with multiple E2s. Ubc13, Ubc1 and Ubc4 were found to be the most frequently interacting E2s. The ubiquitin activating enzyme Uba1 served as a positive control in BiFC assay, and there was a BiFC signal detected across all E2s crossed with the strain with VN tagged Uba1. The summary of the screen result are presented in Figure 26, which is then summarized by Table 10.

RESULTS

E3s

Since the reconstruction of a functional Venus fluorescent protein is constrained by the topology of the interacting proteins, it is expected that BiFC can give different results when using N-and C-terminally tagged proteins. In our screen we used six E3s

that were tagged at their N-terminal, in addition to the C-terminally tagged strains that were available from the commercial collection (Bioneer). These E3s, namely Tfb3, Prp19, Mot2, Psh1, Nam7 and Ste5, have their E2 interaction domain in their Nterminus. Indeed, we did observe different BiFC signals for some of these E3s, suggesting that the reconstitution of Venus is subject to topological constrains. In the figures below those results are illustrated with the three E3s, namely Tfb3, Prp19 and Psh1. In the case of Psh1 and Tfb3, we observed that the N-terminally tagged E3s gave much stronger signal that the C-terminally tagged E3s, suggesting that the interaction signal is specific and strongly dependent on the close proximity of the VN-tag to the E2 interaction domain of the E3. In contrast, in the case of Prp19, the difference in signal produced by N-and C-terminally tagged Prp19 suggests that in this case the signal could be non-specific or that this E3 is subject to different topological constraints. Indeed, yeast Prp19 is known to form head-to-tail tetramers (Ohi et al., 2005), which could explain why tagging this E3 at the N-or its C-terminus does not strongly influence the BiFC signal. Note that due to difficulties in detecting the VN-tag of those proteins by Western blot, we were not able to compare the expression level of the N-and C-terminally tagged proteins. Examples of E3s that interact with a single E2 Few E3s showed specificity only for a single E2. This was for instance the case of the E3s Rad5 and Irc20, which produced a clear BiFC signal in the nucleus with Ubc1 and Ubc13, respectively . The fact that no signal was detected with other E2s, such as Cdc34 which is expressed at a similar level as Ubc1 or Ubc13 (Figure 25), further indicate that the BiFC signal produced with these E3s is likely to be specific.

RESULTS

Rad5 is indeed known to function with Ubc13 and to mediate polyubiquitylation of PCNA [START_REF] Parker | Mechanistic analysis of PCNA poly-ubiquitylation by the ubiquitin protein ligases Rad18 and Rad5[END_REF]. In contrast, Irc20 has not yet been described to function as a ubiquitin ligase. It is an adenosine triphosphatase (ATPase) protein with a RING domain and it is involved in DNA repair [START_REF] Richardson | Physical and genetic associations of the Irc20 ubiquitin ligase with Cdc48 and SUMO[END_REF]. Our results thus suggest that it could indeed function as an E3 and it would be really interesting to further investigate its function in response to DNA damage. Note however that at this stage we cannot exclude that its interaction with Ubc1 reflects an E2/substrate interaction rather than a bona fide E2/E3 interaction. Figures 33 and 34 also present a single E3 interacting with a single E2, namely interactions of ubiquitin conjugating enzyme Ubc8, called also Gid3, with ubiquitin ligases Gid9 and Rmd5 (or Gid2). These results are fully consistent with the current literature on these proteins. Indeed, it has previously been shown that Gid9 and Rmd5 form a complex (the so-called 'Gid complex') that functions with Ubc8 to polyubiquitylate gluconegenic proteins, being therefore required for catabolite degradation [START_REF] Braun | Gid9, a second RING finger protein contributes to the ubiquitin ligase activity of the Gid complex required for catabolite degradation[END_REF]. Interestingly, our data show that Ubc8 is rather weakly expressed E2 (Figure 25), suggesting that the BiFC is indeed a sensitive assay to detect E2/E3 interactions. In addition, we note that although Ubc8 was shown to function with the Gid complex by genetic means, it had never been shown to physically interact with this complex. Again, this illustrates the power of BiFC to investigate weak proteinprotein interaction in the context of living cells.

Examples of E3s that interact with several E2s

Below are included some examples of E3s that produced a BiFC signal with several E2s. The most prominent of such E3 is Rsp5, which we found to interact with all E2s, except the weekly expressed Ubc7 and Ubc11 (Figure 35). Despite the fact that this E3 interact with many E2s, the subcellular localization of the BiFC signal suggest that all E2s might be equivalent for this E3. For instance, the BiFC signal produced with Rad6

and Cdc34 was enriched in the nucleus, while the other E2s produced a stronger fluorescence signal in the cytoplasm rather than in the nucleus. This suggests that different E2s could function with Rsp5 in different subcellular compartments.

Other examples of E3s that interacted with several E2s are Ufd2, Ufd4 and the peroxisomal E3s Pex2, Pex10 and Pex12. Both Ufd2 and Ufd4 produced a clear BiFC signal with Ubc1, Ubc4, Ubc5 and Ubc13 (Figure 36). This result is intriguing since Ubc1 and Ubc13 are E2s dedicated to the assembly of Lys48-and Lys63-linked ubiquitin chains, respectively, while Ubc4 and Ubc5 assemble different kinds of ubiquitin chains (Rodrigo-Brenni et al., 2010;Eddins et al., 2006). It would be interesting to determine whether and how those different E2s contribute to the modification of the substrates of Ufd2 and Ufd4. The Pex2, Pex10 and Pex12 proteins are known to form a complex at the peroxisomes where they have been described to function with Ubc10 (also called Pex4) and Ubc4. Surprisingly, Pex2 and Pex10 also produced a clear BiFC signal with Ubc1 and Ubc13 (Figures 37 and38). Moreover, Pex10 BiFC signal produced with Ubc1 and Ubc13 (but not Pex4) was not localized at the peroxisomes (which gives a strong kurtosis measurement in the quantification data) but at the vacuole (Figure 38). This result could indicate that Pex10 has a unique function at the vacuole, independent of Pex2 and Pex12.

RESULTS

Functional characterization of the candidate E2/E3 pair

Interestingly, while performing the BiFC screen, we identified an interaction between the proteins Asi1 and Asi3 and the ubiquitin conjugating enzymes Ubc6 and Ubc7 (Figure 39). The fluorescence signal was localized at the nuclear rim and was specific to these E2s. Together with another protein, Asi2, Asi1 and Asi3 are known to form a complex (the Amino acid Signaling Independent, known as the 'Asi complex') at the inner nuclear membrane (INM) [START_REF] Zargari | Inner nuclear membrane proteins Asi1, Asi2, and Asi3 function in concert to maintain the latent properties of transcription factors Stp1 and Stp2[END_REF]. Both Asi1 and Asi3 contain a RING domain and were therefore postulated to function as E3s (Forsberg et al., 2001) but such an activity had never been demonstrated. Furthermore, the activity of the Asicomplex had been well characterized to function in the Ssy1-Ptr3-Ssy5 (SPS) amino acid-sensing pathway of S. cerevisiae. This is a nutrient induced signal transduction pathway via which yeast responds to the extracellular amino acids (reviewed by [START_REF] Ljungdahl | Amino-acid-induced signalling via the SPS-sensing pathway in yeast[END_REF]. In this pathway, the function of the Asi complex was described to inhibit the activity of Stp1 and Stp2, two transcription factors required for the expression of amino-acid permeases. These transcription factors were therefore candidate to be ubiquitylation substrates of Asi1 and Asi3. Since Asi3 has a RING domain and was considered as a putative ubiquitin ligase, we decided to further test the specificity of the detected interaction with Ubc6 by deleting the RING domain of Asi3. This dramatically decreased the BiFC signal, suggesting that the interaction is indeed specific. However, we could not verify that the two forms of Asi3 have the same expression level (we did not manage to detect endogenously expressed Asi3-VN by Western blot) and this data is therefore not conclusive. We further tested whether the deletion of the Asi complex components in the Asi3-VN/VC-Ubc6 strain would influence the BiFC signal. Our results showed that deletion of Asi1 dramatically reduces BiFC signal, while the deletion of Asi2 causes only a slight decrease in the fluorescence intensity. This result again suggests that the detected Asi3/Ubc6 is specific but the interpretation of this data is difficult because we could not verify that Asi3-VN is properly expressed in asi1 or asi2 cells.

To further analyse the interaction of Asi1 and Asi3 with Ubc6 and Ubc7 we turned to in vitro experiments, which were mainly performed by an engineer, Gaëlle Le Dez. We purified the RING domains of Asi1 and Asi3, as well as those of Ubc6 and Ubc7 (alone or in complex with a fragment of its partner protein Cue1) produced recombinantly. As control, we also used the RING domain of Hrd1 which is known to function with Ubc7 but not Ubc6. The interaction between those proteins was tested using a method called MicroScale Thermophoresis. Our results demonstrated that both Asi1 and Asi3 can interact with Ubc7 and also with Ubc6, but with a weaker Kd. These results thus confirmed the in vivo BiFC data and showed that Ubc6 and Ubc7 can directly interact with the RING domains of Asi1 and Asi3.

To determine whether the interaction between the Asi complex and the E2s Ubc6 Those results strongly suggest that the Asi complex ubiquitylates Stp2 in a Ubc6 and Ubc7 dependent manner, with Ubc7 being the primary E2 for Stp2 ubiquitylation. It is possible that other E2s, such as Ubc4 which we found to weakly interact with Asi3 in the absence of Ubc6, also contributes to Stp2 ubiquitylation in vivo.

RESULTS

While working on this project, we started to collaborate with the laboratories of Per Ljungdahl from Stockholm University and Michael Knop from the University of Heidelberg. Most importantly, the laboratory of Michael Knop was able to demonstrate that in addition to Stp2, the Asi-complex and Ubc7 target for degradation several transmembrane proteins that normally localize to the endomembrane system.

Altogether, our work demonstrates the existence of a novel quality control pathway associated with the inner nuclear membrane (Inner Nuclear Membrane Associated Degradation, INMAD) and suggests that the primary function of the Asi proteins is to act as a shield against proteins that mislocalize to the nucleus. This pathway is distinct from the previously described ER associated degradation pathway and since Asi proteins do not have clear orthologues in higher eukaryotes, it is not yet known whether a similar INMAD pathway exists outside yeasts.

Publication

This part of the results has been included in a publication which is joined at the end of the manuscript. *equal contribution

DISCUSSION

The initial aim of this study was to establish a method to assay E2/E3 interactions in living cells. We have compared two methods based on protein-fragment complementation, as this has been shown to detect weak and transient interactions in vivo (reviewed by [START_REF] Morell | Detection of transient protein-protein interactions by bimolecular fluorescence complementation: the Abl-SH3 case[END_REF]. Among the PCAs that we tested were BiFC assay, based on the fluorescent protein complementation and BiLC assay, based on the luminescent protein complementation. Fluorescent protein fusions have been particularly useful, as they allow fluorescence microscopy of living cells with a minimal perturbations (Sheff and Thorn, 2004). Similarly BiLC assays, with the luciferase assay as an example, also display minimal cell perturbation in the system under investigation [START_REF] Zhang | Effect of perturbation of ATP level on the activity and regulation of nitrogenase in Rhodospirillum rubrum[END_REF].

Here we report that the BiFC assay can detect E2/E3 interactions in living yeast.

First, we have shown that BiFC can detect the interaction between Ubc4 and Ufd4, a well characterized E2/E3 pair in yeast. To evaluate whether this method reliably detects this interaction, a BiFC experiment was performed using deletion mutants. We generated a series of deletion mutants with truncated N-terminal or C-terminal domains.

The various deletion constructs yielded a significant decrease in the BiFC signal, indicating that the detected BiFC signal is likely due to the E2/E3 interaction and not due to unspecific reconstitution of the Venus protein. Interestingly, these results suggest that Ufd4 utilizes not only the canonical E2/E3 interacting surface but also the 'backside' of Ubc4, since it is necessary to truncate not only the N-terminus but also the C-terminus of Ubc4 to abolish the BiFC signal. However, due to the fact that BiFC can lead to false positive results, the putative interactions have to be assayed using other methods before being able to draw definitive conclusions. BiLC, which has to our knowledge never been reported to lead to the false positive signals, does not have this drawback. However, despite several attempts, we did not detect the Ubc4/Ufd4 interaction using BiLC. This could be for various reasons and we could probably optimize this assay even more. However, it seems likely that the difference with BiFC comes mainly from the fact that BiLC is reversible and does not trap transient interactions contrary to irreversible BiFC (once the Venus is reconstituted, it cannot unfold into the VN and VC fragments), which 'locks' the protein-protein interactions (Hu et al., 2002). This irreversibility is a limitation of BiFC when dynamic changes in protein complexes need to be examined [START_REF] Vidi | Fluorescent and bioluminescent protein-fragment complementation assays in the study of G protein-coupled receptor oligomerization and signaling[END_REF]. However, in the case of weak and transient interactions it seems to be advantageous over BiLC. In a way, the sensitivity of BiFC is at the cost of specificity and secondary assays are required to further make sure that the detected interactions are biologically relevant. Finally, it is still possible that BiLC could detect certain E2/E3 interactions, depending on the geometry and the level of expression of the proteins. Since we detected an interaction signal using BiFC with the same E2/E3 pair as for the BiLC experiments, we decided not to perform further tests with the later method but rather to screen for E2/E3 interactions using BiFC.

With this limitation of BiFC in mind, we decided to perform a systematic screen for new E2/E3 interactions. As aptly noted by [START_REF] Kerppola | Bimolecular fluorescence complementation (BiFC) analysis as a probe of protein interactions in living cells[END_REF] 'the challenge for implementing a screen for interaction partners is that the levels of expression of different fusion proteins in a library is likely to vary over the level of expression of the corresponding endogenous proteins. Thus the differences in BiFC signal are likely to be affected by a variety of factors unrelated to the efficiency of the protein interaction'. In our case, all proteins except Rsp5 were expressed from their endogenous chromosomal loci (Rsp5-VN was cloned in a centromeric plasmid containing its endogenous promoter). For some E2s, for which we had suitable antibodies, we compared the level of expression of the VC-tagged and untagged E2s. Surprisingly, we noted a lower expression level of the tagged versus endogenous E2s. This is probably due to the fact that the VC-tag is mainly unfolded in the cells and targets the fusion protein for degradation. In any case, we did not observe any overexpression of the tagged-E2s, which limits the risk that the fusion proteins are mislocalized to cellular compartments, where they do not reside in normal cells. Despite this lower expression level, we noticed that the different E2s were expressed at different levels, which likely reflects differences in the abundance of the endogenous E2s. We noticed that weaker expressed E2s produced less detectable interactions than stronger expressed E2s. This observation is consistent with recent research by [START_REF] Levy | High-resolution mapping of protein concentration reveals principles of proteome architecture and adaptation[END_REF] who reported that PPIs depend greatly on protein abundance. It is also likely that the interactions of proteins with relatively low expression levels are difficult to detect. BiFC can therefore suffer from false negatives, and some interactions can be missed using this method. However, we detected some E2/E3 interactions with the less abundant E2s such as Rad6. The only E2 and more data in the literature indicating that many E3s engage multiple interactions with E2s (see for instance [START_REF] Brown | RING E3 mechanism for ubiquitin ligation to a disordered substrate visualized for human anaphase-promoting complex[END_REF]. Therefore, designing simple mutations in the canonical E3 interaction surface of E2s might not be sufficient to abolish the observed BiFC signal. Conversely, mutating RING or HECT domains of E3s may not abolish E2 interactions in vivo if other domains or partners of the E3s contribute to the interaction in the cell. Therefore, the best way to determine whether an E2/E3 pair is really active in the cell is to assay the ubiquitylation of its substrate(s).

CONCLUSIONS AND PERSPECTIVES

Despite all the limitations of BiFC discussed above, we are convinced that our approach can help to uncover the range of E2s that function in the cell with certain E3s.

This opens a perspective on analyzing how the different E2s, which function with an E3, collaborate to assemble specific ubiquitin signals on their substrates. Performing such an analysis however would require efficient tools to determine the architecture of ubiquitin chains. Such assays are still being developed and one of the most promising way to analyze the type of ubiquitin signals assembled on the ubiquitylation substrates is the UbiCRest method, which makes use of chain specific deubiquitylating enzymes and has been developed in the laboratory of David Komander [START_REF] Hospenthal | Deubiquitinase-based analysis of ubiquitin chain architecture using Ubiquitin Chain Restriction (UbiCRest)[END_REF].

It would be very interesting to use this method to, for instance determine if Ubc13, which is dedicated to the assembly of Lys63-linked ubiquitin and which we found to produce a BiFC signal with numerous E3s, indeed assembles Lys63 ubiquitin chains on the substrates of these E3s.

Another appealing perspective of our work is the possibility to use our collection of strains expressing 627 unique combinations of VC-tagged E2s and VN-tagged E3s/putative E3s to assay how the ubiquitin machinery responds to specific growth or stress conditions. Indeed we have developed the pipeline for high-throughput imaging and image analysis of BiFC and it would be possible to identify new E2/E3 pairs that only interact under specific conditions, for example, upon DNA damage.

LETTER doi:10.1038/nature14096

Protein quality control at the inner nuclear membrane The nuclear envelope is a double membrane that separates the nucleus from the cytoplasm. The inner nuclear membrane (INM) functions in essential nuclear processes including chromatin organization and regulation of gene expression 1 . The outer nuclear membrane is continuous with the endoplasmic reticulum and is the site of membrane protein synthesis. Protein homeostasis in this compartment is ensured by endoplasmic-reticulum-associated protein degradation (ERAD) pathways that in yeast involve the integral membrane E3 ubiquitin ligases Hrd1 and Doa10 operating with the E2 ubiquitinconjugating enzymes Ubc6 and Ubc7 (refs 2, 3). However, little is known about protein quality control at the INM. Here we describe a protein degradation pathway at the INM in yeast (Saccharomyces cerevisiae) mediated by the Asi complex consisting of the RING domain proteins Asi1 and Asi3 (ref. 4). We report that the Asi complex functions together with the ubiquitin-conjugating enzymes Ubc6 and Ubc7 to degrade soluble and integral membrane proteins. Genetic evidence suggests that the Asi ubiquitin ligase defines a pathway distinct from, but complementary to, ERAD. Using unbiased screening with a novel genome-wide yeast library based on a tandem fluorescent protein timer 5 , we identify more than 50 substrates of the Asi, Hrd1 and Doa10 E3 ubiquitin ligases. We show that the Asi ubiquitin ligase is involved in degradation of mislocalized integral membrane proteins, thus acting to maintain and safeguard the identity of the INM.

To identify components of INM quality control, we focused on the ubiquitin-conjugating enzyme Ubc6. Ubc6 is an integral membrane protein that localizes to the endoplasmic reticulum and the INM where it targets for degradation soluble and integral membrane proteins together with Ubc7 and Doa10 (refs 6, 7). We established a microscopy-based bimolecular fluorescence complementation (BiFC) assay 8 to screen for new E3 ubiquitin ligases interacting with Ubc6 (Fig. 1a). In total, 10 out of 54 known or putative E3s, including Doa10, interacted with Ubc6 at distinct subcellular locations (Fig. 1b and Extended Data Fig. 1a). Among these, Asi1 and Asi3 displayed a BiFC signal restricted to the nuclear rim (Fig. 1b). Despite their colocalization at the endoplasmic reticulum, no interaction was detected between Ubc6 and Hrd1 (Extended Data Fig. 1a), suggesting a low rate of false-positive interactions in our BiFC assay.

Asi1 and Asi3 are integral membrane RING domain proteins of the INM and form the Asi complex 4,9,10 . Together with the INM protein Asi2, the Asi complex functions in the Ssy1-Ptr3-Ssy5 (SPS) aminoacid-sensing pathway, where it is involved in the degradation of Stp1 and Stp2 transcription factors 11 . We tested the interactions of Asi1 and Asi3 with all E2 ubiquitin-conjugating enzymes using the BiFC assay. In addition to Ubc6, Asi1 and Asi3 interacted with Ubc7 and weakly with Ubc4 (Extended Data Fig. 1b-d). We validated these interactions in microscale thermophoresis experiments 12 with recombinant proteins (Fig. 1c and Extended Data Fig. 1e). The Ubc7-binding region of Cue1 (Cue1 U7BR ) 13 , a protein that tethers Ubc7 to the endoplasmic reticulum membrane 14 , was included in the assays. A carboxy-terminal fragment of Hrd1 (Hrd1 CT ) expected to interact with Ubc7 but not Ubc6 served as control 2,3 . The RING domains of Asi1 and Asi3 (Asi1 RING and Asi3 RING ) interacted with Ubc7, provided it was bound to Cue1 U7BR , with affinities similar to Hrd1 CT . Asi1 RING and Asi3 RING , but not Hrd1 CT , also interacted weakly with Ubc6 lacking its transmembrane domain (Ubc6 DTM ) (Fig. 1c).

The Asi proteins maintain the SPS pathway in the 'off state' in the absence of inducing amino acids, and do so by targeting for proteasomal degradation the low levels of Stp1 and Stp2 that inadvertently mislocalize into the nucleus 11 . Consequently, asi mutants exhibit aberrant constitutive Stp1/Stp2-dependent transcription 9 . We observed that ubc7D and, to a lesser extent, ubc6D mutants exhibited increased expression of Stp1/Stp2-regulated genes similar to the asi1D and asi3D mutants (Fig. 1d and Extended Data Fig. 1f). These effects were not due to inactivation of Hrd1 or Doa10 ubiquitin ligases (Extended Data Fig. 1f), thus implicating Ubc6 and Ubc7 in the SPS pathway.

Next, we assayed the ubiquitylation of an artificial Asi substrate based on the first 45 amino acids of Stp2 (Stp2 N ). This fragment of Stp2 contains a degron that is recognized by the Asi complex 11 . Ubiquitylation of Stp2 N fused to the tandem affinity purification (TAP) tag was reduced in ubc6D and severely impaired in asi3D and ubc7D mutants (Fig. 1e). In addition, ubiquitylation of Stp1 and Stp2 mutants with constitutive SPSindependent nuclear localization was impaired in asi1D and asi3D strains (Extended Data Fig. 1g). Together, these results establish the Asi complex as an E3 ubiquitin ligase of the INM that functions with Ubc6 and Ubc7.

Functionally related genes can be identified by similarity of genetic interaction profiles 15 . We searched for novel functions of the Asi ubiquitin ligase by mining a genome-scale genetic interaction map 16 . In this data set, the fitness of 5.4 million double-mutant combinations was measured by colony size, generating genetic interaction profiles for ,75% of all S. cerevisiae genes. We calculated correlation coefficients between genetic interaction profiles of ASI genes and the other 4,458 genes in the genetic interaction map. In this analysis, the genetic interaction profiles of ASI genes correlated with each other and, to a similar extent, with HRD1, DOA10, UBC6, UBC7 and CUE1 among others (Fig. 2a and Supplementary Table 1), suggesting that Asi and ERAD E3 ubiquitin ligases are functionally related. We sought to determine whether they work in the same or parallel pathways. Strains lacking HRD1 and the unfolded protein response genes IRE1 or HAC1 show impaired growth at increased temperature 17 . Additional deletion of ASI1 resulted in a synthetic lethal phenotype under these conditions 18 (Fig. 2b and Extended Data Fig. 2), suggesting that Asi1 and Hrd1 function in parallel pathways.

We used a tandem fluorescent protein timer (tFT) approach 5 to perform unbiased proteome-wide screens for substrates of the Asi, Hrd1 and Doa10 ubiquitin ligases. A tFT is a tag composed of two fluorescent proteins (mCherry and superfolder green fluorescent protein (sfGFP)) with distinct fluorophore maturation rates. The mCherry/sfGFP intensity ratio is a measure of protein degradation kinetics in steady state (Fig. 3a), with a dynamic range and sensitivity that exceed conventional cycloheximide chase experiments 5 (Supplementary Note 1). We constructed a genome-wide library of yeast strains each expressing a different tFT-tagged protein (Supplementary Methods). Library construction relied on a seamless tagging strategy that minimizes the influence of the tag on gene expression 19 (Extended Data Fig. 3a). In total, 4,044 proteins were successfully tagged to create a tFT library covering ,73% of verified or uncharacterized open reading frames in the S. cerevisiae genome (Supplementary Table 2). We introduced asi1D, asi3D, hrd1D, doa10D, ubc6D and ubc7D deletion alleles into the tFT library using high-throughput genetic crosses 20 . The effect of each gene deletion on the stability of each protein in the library was examined with high-throughput fluorescence measurements of colonies 5 (Extended Data Fig. 3b) and quantified as a z-score. More proteins were stabilized (positive z-score) than destabilized in the six mutants (Extended Data Fig. 3c and Supplementary Table 3), in agreement with the role of Asi, Hrd1 and Doa10 ubiquitin ligases in protein degradation. Hierarchical clustering of top hits recapitulated known E2-E3 interactions and revealed three clusters of 20, 30 and 9 potential substrates for the Asi, Hrd1 and Doa10 ubiquitin ligases, respectively (Fig. 3b). Hrd1 substrates, including the known substrate Der1 (ref. [START_REF] Zattas | N-terminal acetylation of the yeast Derlin Der1 is essential for Hrd1 ubiquitin-ligase activity toward luminal ER substrates[END_REF], were stabilized only in the ubc7D mutant, whereas Doa10 substrates were stabilized in both ubc6D and ubc7D mutants. Most Asi substrates, including the recently identified Erg11 (ref. [START_REF] Foresti | Quality control of inner nuclear membrane proteins by the Asi complex[END_REF], were stabilized in the ubc7D mutant with only weak effects of the ubc6D mutant (Fig. 3b). Stp1 and Stp2 were not identified as Asi substrates in the screen, probably the consequence of their efficient targeting for degradation by the E3 ubiquitin ligase SCF Grr1 in the cytoplasm 11 . The vast majority of potential substrates in each set were integral membrane or secretory proteins distributed along the endomembrane system and the Hrd1 and Asi substrates were enriched in endoplasmic reticulum and vacuolar proteins (Fig. 3c,d and Extended Data Fig. 3d,e). These findings are consistent with the organization and functions of endoplasmicreticulum-associated ubiquitin ligases, thus establishing the tFT library as a valuable resource for studies of protein degradation (Supplementary Note 2), and indicate that the Asi complex is involved in degradation of a distinct set of integral membrane proteins. a, Histograms of Pearson correlation coefficients calculated between the genetic interaction profiles of each ASI gene and ,75% of all yeast genes, obtained from a previously published genome-scale genetic interaction map 16 . Asterisks mark the dubious open reading frame YMR119W-A, which overlaps with the ASI1 gene. b, Tenfold serial dilutions of strains grown on synthetic complete medium for 2 days at 30 or 37 uC.

proteins mislocalize to the endoplasmic reticulum and nuclear rim only on overexpression or C-terminal tagging (Extended Data Fig. 6).

Whether the Asi ubiquitin ligase recognizes such mislocalized proteins through specific degrons, as is the case with Stp1 and Stp2 transcription factors 11 , or other features such as compartment-specific properties of transmembrane domains 23 is an open question.

The nuclear pore complex establishes a barrier between the cytoplasm and the nucleoplasm. However, increasing evidence suggests that not only small soluble proteins but also integral membrane proteins with cytoplasmic domains of up to 60 kilodaltons (kDa) can passively diffuse past the nuclear pore, the latter through a ,10 nm side channel 6,24-28 . We propose that the Asi ubiquitin ligase targets such mislocalized and potentially harmful proteins for degradation. Although the Asi proteins are not obviously conserved outside of yeast, the general importance of membrane-associated protein degradation mechanisms and the large diversity of integral membrane RING domain proteins in mammalian cells 29 suggest that dedicated E3 ubiquitin ligases functioning in INM-associated protein degradation exist also in metazoans.

Online Content Methods, along with any additional Extended Data display items and Source Data, are available in the online version of the paper; references unique to these sections appear only in the online paper.

Received 21 September; accepted 20 November 2014.

METHODS

Yeast methods and plasmids. Yeast genome manipulations (gene deletions and tagging) were performed using PCR targeting, as described 30 . Yeast strains and plasmids used in this study are listed in Supplementary Tables 4 and5, respectively. b-galactosidase activity assay. Cells were grown in synthetic minimal medium and b-galactosidase activity was measured in N-lauroyl-sarcosine-permeabilized cells as described 31 . RNA isolation and qRT-PCR. Strains with auxotrophies complemented by plasmids pRS316 (URA3), pRS317 (LYS2) and pAB1 (HIS3, MET15 and LEU2) were grown in synthetic minimal medium to 10 7 cells ml 21 and collected by centrifugation. RNA was isolated using the RiboPure Yeast Kit and treated with Turbo-DNase (Ambion). The quality of RNA preparations was assessed by electrophoresis on a 1% agarose gel with 10 mM guanidine thiocyanate, and the lack of DNA contamination was confirmed by PCR. One microgram of RNA was used for complementary DNA synthesis with oligo (dT) [START_REF] Wienken | Protein-binding assays in biological liquids using microscale thermophoresis[END_REF][START_REF] Kostova | Ubc7p-binding domain in Cue1p activates ER-associated protein degradation[END_REF][START_REF] Biederer | Role of Cue1p in ubiquitination and degradation at the ER surface[END_REF][START_REF] Costanzo | Charting the genetic interaction map of a cell[END_REF][START_REF] Costanzo | The genetic landscape of a cell[END_REF][START_REF] Friedlander | A regulatory link between ER-associated protein degradation and the unfolded-protein response[END_REF][START_REF] Foresti | Quality control of inner nuclear membrane proteins by the Asi complex[END_REF][START_REF] Khmelinskii | Seamless gene tagging by endonuclease-driven homologous recombination[END_REF] using SuperScript III Reverse Transcriptase (Life Technologies). Quantitative reverse transcriptase PCR (qRT-PCR) reactions were prepared using Kapa SybrFast qPCR Master Mix (Kapa-Biosystems). cDNA mixtures were diluted 1:40 and 5 ml were used in a reaction volume of 20 ml with the following primer pairs: AGP1fwd 59-CTGCCGTGCG TAGGTTTT-39 and AGP1rev 59-AGAAGAAGGTGAGATAGCCGA-39; GNP1fwd 59-CACCACAAGAACAAGAACAGAAAC-39 and GNP1rev 59-ACCGACCAG CAAACCAGTA-39; TAF10fwd 59-ATATTCCAGGATCAGGTCTTCCGTAGC-39 and TAF10rev 59-CAACAACAACATCAACAGAATGAGAAGACTAC-39.

The levels of gene expression in three biological replicates were determined in two separate amplifications with triplicate technical replicates of each of the three genes analysed using the comparative DC T method (RotorGene 6000, Corbett Life Science). Relative levels of AGP1 and GNP1 messenger RNA were normalized with respect to the levels of the invariant reference gene TAF10; the levels of AGP1 and GNP1 in strains carrying the indicated mutations were subsequently averaged and normalized to the levels of expression in the corresponding isogenic wild-type strains. Purification of decahistidine-ubiquitin protein conjugates. Ubiquitylated proteins were purified from 1 3 10 9 exponentially growing yeast cells expressing 103His-tagged ubiquitin using a protocol adapted from ref. [START_REF] Becuwe | A molecular switch on an arrestin-like protein relays glucose signaling to transporter endocytosis[END_REF]. Cell pellets were resuspended in 2 ml 20% trichloroacetic acid and lysed for 2 min using glass beads in a Disrupter Genie homogenizer (Scientific Industries). After precipitation, proteins were resuspended in 3 ml guanidium buffer (6 M guanidinium chloride, 100 mM Tris-HCl, pH 9, 300 mM NaCl, 10 mM imidazole, 0.2% Triton X-100 and 5 mM chloroacetamide), clarified at 30,000g and incubated for 1.5 h at room temperature with TALON Metal Affinity Resin (Clontech). The beads were then washed with wash buffer (8 M urea, 100 mM sodium phosphate, pH 7.0, 300 mM NaCl, 5 mM imidazole, 0.2% Triton X-100 and 5 mM chloroacetamide) containing 0.2% SDS (twice) and lacking SDS (twice). 103His-ubiquitin conjugates were finally eluted with 200 ml elution buffer (8 M urea, 100 mM sodium phosphate, pH 7.0, 300 mM NaCl, 250 mM imidazole, 0.2% Triton X-100 and 5 mM chloroacetamide). Total extracts (1% of the amount used for purification) and ubiquitin conjugate eluates were analysed by SDS-PAGE and immunoblotting with antibodies against the TAP tag (PAP, 1:1,000, Sigma). As controls, levels of ubiquitin conjugates and Pgk1 were assessed with anti-ubiquitin (P4D1 horseradish peroxidase (HRP) conjugate, 1:1,000, Santa Cruz) and anti-Pgk1 antibodies (clone 22C5D8, 1:10,000, Invitrogen), respectively. Immunogenic proteins were detected by chemiluminescence using SuperSignal West Femto Substrate (Thermo Scientific) and recorded using autoradiographic films (CP-BU, Agfa) processed with a Curix 60 developing machine (Agfa). Purification of hexahistidine-ubiquitin protein conjugates. Ubiquitylated proteins were purified from 5 3 10 8 exponentially growing yeast cells expressing 63His-tagged ubiquitin as previously described 33 . 63His-ubiquitin conjugates were retained on nickel-nitrilotriacetic acid Sepharose beads (Qiagen) and eluted in the presence of 300 mM (Stp1-HA, Stp1-RI 17-33 -HA ) or 500 mM (Stp2-HA, Stp2D 2-13 -HA) imidazole. Total extracts, flow-through and eluate fractions were precipitated with 10% trichloroacetic acid, analysed by SDS-PAGE and immunoblotting with antibodies against the haemagglutinin tag (1:5,000, Roche) and the signals were recorded using autoradiographic film (CL-Xposure, Thermo Scientific). As controls, levels of ubiquitin conjugates and Pgk1 were assessed with anti-His 5 (1:5,000,Qiagen) and anti-Pgk1 antibodies (1:10,000, InVitrogen), respectively, and detected by chemiluminescence using SuperSignal West Dura Extended Duration Substrate (Thermo Scientific) and a Molecular Imager ChemiDoc XRS1 with Image Lab v3 build 11 software (BioRad). Loaded total and flow-through fractions correspond to 2% (Stp1-HA or Stp1-RI 17-33 -HA) and 0.7% (Stp2-HA or Stp2D 2-13 -HA) of the amount used for purification of ubiquitin conjugates. Bimolecular fluorescence complementation. BiFC interaction assays were performed using E2 and E3 proteins tagged with the VC173 and VN155 fragments (VC and VN) of the Venus fluorescent protein, respectively 34 . All E2 and E3 proteins were tagged C-terminally, with the following exceptions that were N-terminally tagged: Ubc6, because the C terminus of Ubc6 faces the endoplasmic reticulum lumen 35 ; Ubc7, to preserve its interaction with Cue1 (ref. 36); Ubc1, because the growth of strains expressing Ubc1 endogenously tagged at the C terminus with VC appears compromised; the E3 proteins Far1, Mot2, Nam7, Prp19, Ste5 and Tfb3, as they all have their E2 binding domain at the N terminus. All fusions were expressed from their endogenous chromosomal loci, with the exception of Rsp5-VN, which was expressed from its endogenous promoter on the centromeric plasmid pGR703 (Supplementary Table 5).

Strains expressing VC-tagged E2 proteins were constructed in the scEB115 background. scEB115 carries markers for selection of haploid progeny in automated crosses (can1::STE2pr-spHIS5 and lyp1::STE3pr-HPH) and expresses the proteasomal subunit Rpn7 fused to the red fluorescent protein tDimer2 as nuclear marker (Supplementary Table 4). Strains expressing VN-tagged E3 proteins were either obtained from a commercially available collection (Bioneer Corporation) or constructed by homologous recombination in the BY4741 background. Expression of VC-and TAP-tagged fusions was validated by immunoblotting with mouse anti-GFP (clones 7.1 and 13.1, Roche) and peroxidase anti-peroxidase (Sigma) antibodies to detect the VC and TAP tag, respectively, and mouse anti-actin (clone c4, Merck Millipore) for loading controls.

Strains expressing individual E2 and E3 protein fusions were crossed to produce an array of yeast strains each expressing Rpn7-tDimer2 and a unique combination of tagged E2 and E3 proteins, as described 20 . The resulting strains were cultivated overnight at 20 uC in YPD medium and diluted in low fluorescence medium 37 3-4 h before imaging. Imaging was performed in 8-well LabTek chambers or 96-well plates (Imaging plates CG, Zell-Kontakt) using an inverted Leica SP8 confocal microscope. Images of the BiFC signal were collected using a 514 nm laser and a narrow band-pass filter (525-538 nm) around the emission peak of the Venus fluorescent protein to reduce the contribution of cellular autofluorescence. Rpn7-tDimer2 was imaged simultaneously using a 580-630 nm filter. Cellular autofluorescence was imaged separately using the same band-pass filter as for BiFC images, but with a 458 nm excitation. Rpn7 localizes to the nucleus throughout the cell cycle in growing cells and relocalizes to cytoplasmic structures when cells enter quiescence 38 . Rpn7-tDimer2 images were visually inspected before image processing to verify that cells are not quiescent. Rpn7-tDimer2 and autofluorescence images were used to segment the BiFC images into nuclear and cytoplasmic (whole cell minus nucleus) regions and to unmix the BiFC signal. Image segmentation and single-cell fluorescence measurements were performed using custom plugins in ImageJ 39 (available on request). To enable comparison of data from different experiments, the quantification results were rescaled so that BiFC signals of control cells had a mean of zero and a standard deviation of one. Statistical analysis and graphical representation were performed with GraphPad Prism software. Statistically significant differences from control cells were identified by one-way ANOVA followed by Bonferroni post-hoc tests to correct for multiple comparisons. No statistical method was used to predetermine sample size. Recombinant protein expression and purification. Escherichia coli BL21(DE3) were transformed with plasmids encoding MBP-Hrd1 CT (Hrd1 residues 321-551), MBP-Asi1 RING (Asi1 residues 559-624), MBP-Asi3 RING (Asi3 residues 613-676), glutathione S-transferase (GST)-Ubc6 DTM (Ubc6 residues 1-230), GST-Ubc7 or Cue1 U7BR (Cue1 residues 151-203) and were cultivated in LB medium. Cue1 U7BR was coexpressed with GST-Ubc7. Protein expression was induced by addition of 1 mM isopropyl-b-D-thiogalactoside (IPTG) during 4 h at 25 uC. Cells were pelleted, resuspended in PBS, and lysed by sonication. Lysates were rotated with glutathione (GE Healthcare) or amylose beads (New England Biolab) for 1 h at 4 uC. Beads were washed with PBS containing 1 mM dithiothreitol (DTT). E2s were cleaved from GST using thrombin (Stago). MBP-E3s were eluted using 10 mM amylose and dialysed against PBS plus 1 mM DTT. All recombinant proteins were concentrated using spin filters (3 kDa,Amicon). Protein purity was tested by Coomassie staining after SDS-PAGE. Protein concentration was estimated by absorbance at 280 nm. Microscale thermophoresis. Microscale thermophoresis analysis was performed essentially as described 12 using MBP-Asi1 RING , MBP-Asi3 RING or MBP-Hrd1 CT fluorescently labelled with the fluorescent dye NT-647 (labelling was performed with the Monolith Protein Labelling Kit RED-NHS according to the instructions of the supplier) and high precision standard treated capillaries. MBP-E3s were diluted to 100 nM in PBS, 5% glycerol, 0.1% Tween 20, 1 mM DTT, 10 mM ZnAc and titrated with varying concentrations of unlabelled E2s before loading into capillaries. The difference of the thermophoretic properties of MBP-E3s were measured using a Monolith NT.115 instrument (NanoTemper Technologies GmbH) and a laser power of 60%. A nonlinear fit with the law of mass action was used to derive the dissociation constant (K d ) of the interaction as well as the theoretical thermophoretic properties of the MBP-E3 in its fully bound and unbound states. Those values were then used to normalize the measurements and calculate the fraction of E3 bound at each E2 concentration. Data were plotted and fitted with the GraphPad Prism software. tFT library construction. A total of 4,081 verified or uncharacterized S. cerevisiae open reading frames were selected for tagging based on structural and functional criteria (detailed in Supplementary Methods) to increase the probability that the C-terminal tFT tag would not affect protein functionality, and to avoid exposing the tag to an environment that could affect folding and maturation of the fluorescent proteins. Protocols for strain construction and validation are described in the Supplementary Methods. In brief, strain manipulations were automated and performed in 96-well format whenever possible. Using PCR targeting 30 and lithium acetate transformation of yeast 40 , the module for seamless protein tagging with the mCherry-sfGFP timer (pMaM168 in Supplementary Table 5) was integrated into each selected genomic locus in the strain yMaM330 (Supplementary Table 4), a strain compatible with automated yeast genetics that carried a construct for conditional expression of the I-SceI meganuclease from the GAL1 promoter integrated into the leu2 locus. Correct integration of the tagging module into each locus and expression of tFT protein fusions was verified by PCR and whole colony fluorescence measurements for 4,044 open reading frames, with two independent clones validated for 3,952 open reading frames (Supplementary Table 2). tFT library screening. Haploid array strains carrying deletions of individual components of the ubiquitin-proteasome system were obtained from the genome-wide heterozygous diploid yeast deletion library 41 by sporulation and tetrad dissection. Screens were conducted in 1536-colony format. Using pinning robots (BioMatrix, S&P Robotics), tFT query strains (before marker excision) were mated with array mutants. Selection of diploids, sporulation and selection of haploids carrying simultaneously a tFT protein fusion and a gene deletion were performed by sequential pinning on appropriate selective media, as described 20 , followed by seamless marker excision 19 . In each screen, a single tFT strain was crossed to a set of mutants in the ubiquitin-proteasome pathway (including the asi1D, asi3D, hrd1D, doa10D, ubc6D and ubc7D mutants) (A.K. et al., manuscript in preparation) with four technical replicates of each cross. Technical replicates were arranged next to each other. Fluorescence intensities of the final colonies were measured after 24 h of growth on synthetic complete medium lacking histidine at 30 uC using Infinite M1000 or Infinite M1000 Pro plate readers equipped with stackers for automated plate loading (Tecan) and custom temperature control chambers. Measurements in mCherry (587/10 nm excitation, 610/10 nm emission, optimal detector gain) and sfGFP (488/10 nm excitation, 510/10 nm emission, optimal detector gain) channels were performed at 400 Hz frequency of the flash lamp, with ten flashes averaged for each measurement.

Measurements were filtered for potentially failed crosses based on colony size after haploid selection. Fluorescence intensity measurements were log-transformed and the data were normalized for spatial effects on plates by local regression. To estimate the changes from normal protein stability, median effects for tFT and deletion strains were subtracted from log-ratios of mCherry and sfGFP intensities. To avoid variance-mean dependences, standard deviations were regressed against the absolute fluorescence intensities. Changes in protein stability were divided by the regressed standard deviations, yielding a measurement comparable to a z-score, and tested against the hypothesis of zero change. A moderated t-test implemented in the R/Bioconductor package limma 42 was used to compute P values. P values were adjusted for multiple testing by controlling the false discovery rate using the method of Benjamini-Hochberg.

Crosses with additional mutants were performed with independently constructed deletion strains using identical procedures on a RoToR pinning robot (Singer). Whole colony fluorescence intensities were corrected for autofluorescence using measurements of corresponding mutant colonies crossed to strain yMaM344-2 (Supplementary Table 4) expressing a truncated non-fluorescent mCherry DN protein. For each tFT fusion, mCherry/sfGFP intensity ratios in each mutant were compared to a control cross with a wild-type strain carrying the kanMX selection marker in the his3D locus. Fluorescence microscopy. Strains were grown at 30 uC in low fluorescence medium (synthetic complete medium prepared with yeast nitrogen base lacking folic acid and riboflavin; CYN6501, ForMedium) to 0.4-1.2 3 10 7 cells ml 21 and attached to glass-bottom 96-well plates (MGB096-1-2-LG-L, Matrical) using Concanavalin A (C7275, Sigma) as described 43 . Single plane images were acquired on a DeltaVision Elite system (Applied Precision) consisting of an inverted epifluorescence microscope (IX71; Olympus) equipped with an LED light engine (SpectraX, Lumencor), 475/28 and 575/25 nm excitation, and 525/50 and 624/40 nm emission filters (Semrock), a dual-band beam splitter 89021 (Chroma Technology), using either a 1003 numerical aperture (NA) 1.4 UPlanSApo or a 603 NA 1.42 PlanApoN oil immersion objective (Olympus), an sCMOS camera (pco.edge 4.2, PCO) and a motorized stage contained in a temperature-controlled chamber. Image correction and quantification were performed in ImageJ 39 . Dark signal and flat field corrections were applied to all images as described 43 . Image deconvolution was performed with Softworx software (Applied Precision) using the conservative ratio algorithm with default parameter settings. Individual cell, perinuclear region and cytoplasm segmentation masks were manually defined in deconvolved images and applied to non-deconvolved images. Mean single-cell fluorescence measurements were corrected for cellular autofluorescence. Mean perinuclear fluorescence measurements were corrected for cytoplasmic fluorescence of each individual cell.

Strains expressing N-and C-terminally tagged Vtc1 and Vtc4 were imaged with exposure setting adjusted to the expression levels: 3.3-fold longer exposure time for C-terminally tagged fusions. Representative deconvolved images were scaled identically. Cycloheximide chases. Strains were grown at 30 uC in synthetic complete medium to ,0.8 3 10 7 cells ml 21 density before addition of cycloheximide to 100 mg ml 21 final concentration. One-millilitre samples taken at each time point were immediately mixed with 150 ml of 1.85 M NaOH and 10 ml b-mercaptoethanol, and flash frozen in liquid nitrogen. Whole-cell extracts were prepared as previously described 40 , separated by SDS-PAGE followed by semi-dry blotting and probed sequentially with mouse anti-HA (12CA5) and mouse anti-Pgk1 (22C5D8, Molecular Probes) antibodies. A secondary goat anti-mouse antibody (IgG (H1L)-HRP, Dianova) was used for detection on a LAS-4000 system (Fuji). ubiquitin ligases. a, Tagging approach used to construct the tFT library in a strain carrying the I-SceI meganuclease under an inducible promoter. First, a module for seamless C-terminal protein tagging with the mCherry-sfGFP timer is integrated into a genomic locus of interest using conventional PCR targeting. Subsequent I-SceI expression leads to excision of the heterologous terminator and the URA3 selection marker, followed by repair of the doublestrand break by homologous recombination between the mCherry and mCherry DN sequences. A tFT fusion protein is expressed under control of endogenous promoter and terminator in the final strain. b, Workflow of screens for substrates of E3 ubiquitin ligases involved in protein degradation. Each tFT query strain is crossed to an array of mutants carrying different gene deletion alleles. The resulting strains are imaged with a fluorescence plate reader to identify proteins with altered stability in each mutant. c, Volcano plots of the screens for proteins with altered stability in the indicated mutants. Plots show z-scores for changes in protein stability on the x axis and the negative logarithm of P values adjusted for multiple testing on the y axis. The number of proteins with increased (red) or decreased (blue) stability at 1% false discovery rate is indicated. d, Fraction of proteins in the tFT library and in the three clusters in Fig. 3b mapped to the full yeast slim set of component GO terms.

Note that the GO term cytoplasm contains all cellular contents except the nucleus and the plasma membrane. e, The three clusters in Fig. 3b are enriched for proteins in the indicated component GO terms. Bar plot shows 2log 10transformed P values of significant enrichments.

RESEARCH LETTER

Macmillan Publishers Limited. All rights reserved ©2014 52 ,57,40 57,53,73 53,56,52 50,50,58 51,52,50 55,58,51 54,55,57 83,85,80 n = 35,46,42 35,44,39 40,40,40 40,40,40 40,38,38 35,41,40 32,38,37 65,60,60 35,35,35 WT asi1∆ hrd1∆

Extended Data Figure 4

2

 2 Overview of the cellular functions of protein ubiquitylation 1.1.3 Examples of diseases associated with defective protein ubiquitylation 1.1.4 Ubiquitin 1.1.5 Types of ubiquitylation and ubiquitin chain diversity 1

RÉSUMÉL

  'ubiquitylation des protéines est une modification post-traductionnelle qui correspond à l'ajout d'une protéine d'ubiquitine sur d'autres protéines de la cellule et joue un rôle capital dans la régulation de nombreuses fonctions cellulaires, en particulier la croissance et la prolifération cellulaire. Des dysfonctionnements de ce mécanisme sont à l'origine de cancers et d'autres maladies. Le processus d'ubiquitylation met en jeu une cascade des réactions enzymatiques catalysées par 3 familles d'enzymes : des enzymes d'activation de l'ubiquitine (E1s), des enzymes de conjugaison de l'ubiquitine (E2s) et des ubiquitine ligases (E3s). L'interaction entre une E2, qui porte l'ubiquitine activée et une E3, qui recrute la protéine à modifier, permet le transfert de l'ubiquitine sur sa cible. Les hétérodimères E2/E3 sont donc au coeur de la réaction d'ubiquitylation et dictent la manière précise dont les substrats sont modifiés. A ce jour, un nombre limité de couples E2/E3 ont été décrits, en particulier grâce à des approches biochimiques. L'objectif de ce doctorat était d'étudier les interactions E2/E3 dans des cellules vivantes afin de conserver les conditions physiologiques du fonctionnement de ces enzymes. Ce travail s'est déroulé en 4 étapes qui ont consisté à : -identifier et optimiser une méthode capable de détecter les interactions E2/E3 dans une cellule vivante en utilisant la levure de boulanger (Saccharomyces cerevisiae) comme organisme modèle -construire une collection de souches de levure permettant de tester les interactions entre E2s et E3s -réaliser un crible systématique afin d'identifier de nouvelles paires E2/E3 -caractériser fonctionnellement une des paires E2/E3 nouvellement identifiées. L'étude des interactions protéine-protéine dans des cellules vivantes est relativement difficile, en particulier lorsqu'il s'agit de détecter des interactions faibles et transitoires comme les interactions entre E2s et E3s. Nous avons testé deux techniques différentes et avons choisi la complémentation bimoléculaire de fluorescence, (BiFC, Bimolecular Fluorescence Complementation). Le principe de cette technique repose sur la fusion des partenaires d'interaction avec deux fragments complémentaires (N-et C-terminaux) d'une protéine fluorescente (ici Venus). L'interaction entre les protéines d'intérêt va positionner les deux fragments de la protéine Venus (VN et VC) à proximité l'un de RÉSUMÉ l'autre, ce qui leur permet de reformer la structure native de Venus qui peut ensuite être détectée par microscopie à fluorescence. Pour tester cette technique, nous avons construit une souche de la levure exprimant les enzymes Ubc4 et Ufd4 fusionnées respectivement avec VN et VC et sous contrôle de leur promoteur endogène. Ubc4 est une E2 et Ufd4 une E3 qui sont relativement abondantes et bien connues pour fonctionner ensemble dans l'ubiquitylation de protéines fusionnées à l'ubiquitine. Nous avons observé que des levures exprimant à la fois Ubc4-VN et Ufd4-VC étaient 10 à 15 fois plus fluorescentes que des levures exprimant Ubc4-VN portant des mutations empêchant son interaction avec Ufd4-VC. Ces résultats démontrent que la technique de BiFC permet de révéler de manière spécifique des interactions entre une E2 et une E3 dans les cellules vivantes. Afin de procéder à une analyse systématique de paires potentielles E2/E3, nous avons construit 11 souches de levures exprimant chacune une E2 fusionnée à VC sous contrôle de son promoteur endogène. En parallèle, nous avons obtenu une collection de souches de levures où la majorité des gènes avaient été fusionnés à VN. A partir de cette collection nous avons pu obtenir 56 souches exprimant des E3s fusionnées à leur extrémité C-terminale avec VN. Cependant certaines E3s ont leur domaine d'interaction avec les E2s dans leur région N-terminale. Pour maximiser nos chances de détecter des interactions avec certaines de ces E3s, nous avons construit de nouvelles souches exprimant des E3s fusionnées avec VC à leur extrémité N-terminale. Nous avons également construit des souches de levures exprimant des E3s qui n'étaient pas présentes dans la collection initiale. Au total nous avons ainsi obtenus 63 souches exprimant des E3s fusionnées avec VC. Ces souches ont été systématiquement croisées avec les souches exprimant des E2s, ce qui nous a permis d'obtenir 704 souches dont 627 expriment une combinaison unique d'E2-VN et E3-VC. Toutes ces souches expriment également la sous-unité Rpn7 du protéasome fusionnée avec une protéine fluorescente tdimer2 qui nous a permis de segmenter les images obtenues par microscopie et de quantifier la fluorescence de Venus dans le noyau et dans le cytoplasme des cellules. Ces souches ont été soumises à l'imagerie BiFC dans deux expériences indépendantes, ce qui nous a permis, après analyse des images, d'identifier 128 interactions E2/E3 dont 33 avaient déjà été décrites dans la littérature. La grande majorité des E3s pour lesquelles nous avons pu détecter des interactions avec des E2s sont visiblement capables d'interagir avec plusieurs E2s et seules quelques E3s RÉSUMÉ semblent interagir avec une seule E2. Parmi les E2s, Ubc13, Ubc1 et Ubc4 sont celles qui interagissent avec le plus d'E3s. Parmi les 95 interactions E2/E3 nouvellement identifiées, nous nous sommes particulièrement intéressés aux interactions entre les E3s Asi1 et Asi3 et les E2s Ubc6 et Ubc7. Asi1 et Asi3 sont deux protéines transmembranaires formant un complexe protéique localisé dans la membrane interne du noyau et qui étaient connues pour inhiber Stp1 et Stp2, deux facteurs de transcriptions régulant l'expression de perméases permettant le transport des acides aminés dans les levures. Asi1 et Asi3 contiennent un domaine RING caractéristique d'ubiquitine ligases, mais il n'avait pas été montré qu'elles fonctionnaient effectivement comme des ubiquitine ligases. En utilisant des expériences in vitro, nous avons pu démontrer que les domaines RING de Asi1 et Asi3 interagissent directement avec Ubc6 et Ubc7 et que le complexe Asi1/Asi3 est nécessaire pour l'ubiquitylation nucléaire de Stp2. En conclusion ce travail a permis d'établir une carte des interactions E2/E3 chez la levure et servira de base pour des études ultérieures qui permettront de mieux comprendre le fonctionnement du réseau E2/E3 dans les cellules.

Figure 1 .

 1 Figure 1. A timeline of some key discoveries in the ubiquitin field (adapted with permission from: Ciechanover, Nature Reviews Molecular Cell Biology, 2005).

Figure 2 .

 2 Figure 2. Ubiquitin structure (PDB ID: 1 UBQ) generated with PyMOL software (http://www.pymol.org/) with its hydrophobic patch (indicated in red) centered on the leucine 8 (Leu8), isoleucine 44 (Ile44), and valine 70 (Val70) and its C-terminal tail (C-term).

Figure 4 .

 4 Figure 4. Ubiquitin is synthesized as a precursor. It is transcribed and translated as a linear fusion consisting of multiple copies of ubiquitin (tandem ubiquitin) or ubiquitin fused with other proteins (L40 or S27 ribosomal proteins). Generation of free ubiquitin is possible due to the action of deubiquitylating enzymes and more precisely ubiquitin C-terminal hydrolases (UCH).

Figure 5 .

 5 Figure 5. The different types of ubiquitylation. The attachment of a single ubiquitin to a substrate is known as monoubiquitylation, whereas multi-monoubiquitylation occurs when several single ubiquitin moieties are attached. The attachment of a series of ubiquitin molecules to a substrate protein known as polyubiquitylation.

Figure 7 .

 7 Figure 7. The different topologies of polyubiquitin chains with their main cellular functions.

Figure 8 .

 8 Figure 8. The schematic representation of the ubiquitylation process. Ubiquitin is activated by a Ub activating enzyme (E1) in an ATP-dependent manner, and then transferred into a ubiquitin conjugating enzyme (E2). E2 interacts with a ubiquitin ligase (E3), which enables Ub to be covalently attached to the substrate protein. The process is reversible through the action of deubiquitylating enzymes (DUB).

Figure 9 .

 9 Figure 9. The mechanism of action of the ubiquitin conjugating enzyme (E1). SH refers to the free form of the catalytic cysteine, S~Ub represents the thioester bond between the catalytic cysteine and the Cterminal of ubiquitin, and PPi refers to inorganic pyrophosphate.

Figure 10 .

 10 Figure 10. Phylogenetic analysis demonstrating two families of Ubc6 enzymes and protein sequence alignment of yeast mouse and human Ubc6. A) Two classes of Ubc6-related proteins, Ubc6 and Ubc6e. Ubc6e is not found in yeast and D. melanogaster. B) Comparison of the amino acid sequences of S. cerevisiae Ubc6, mouse Ubc6 and human Ubc6e. The CLUSTAL alignment shows the well-conserved N-terminal UBC domain (marked by a dotted line) and the less conserved C-terminal tail region containing the transmembrane segment in both Ubc6 families. Asterisk indicates the conserved active site cysteine. The region of the membrane spanning domains is underlined (adapted with permission from: Lenk et al., Journal of Cell Science, 2002).

Figure 12 .

 12 Figure 12. The mechanism of Ub transfer by different E3 families. A) RING E3 binds to the Ub conjugating enzyme (E2), from which Ub is transferred directly onto a substrate protein. B) HECT E3 binds to E2, from which Ub is first transferred to the catalytic cysteine of the ligase and is the transferred onto the substrate protein. C) RBR ligases use the combined mechanism of both RING and HECT ligases.E2~Ub conjugate is bound to the so-called Rcat domain (required-for-catalysis) of RBR ligase. Next, Ub is transferred from E2 to the Rcat domain with a catalytic cysteine, from which it is then transferred to the substrate protein.

Figure 13 .

 13 Figure 13. The mechanism of action of deubiquitylating enzymes, based on the example of the catalytic mechanism of cysteine proteases.

Figure 14 .

 14 Figure 14. A schematic representation of the canonical E2/E3 complexes. A) The E6-AP HECT domain with UbcH7 conjugating enzyme, which forms a U-shaped structure. The E6-AP HECT domain N lobe (consisting of 12 α-helices and six β-strands), C lobe (six α-helices and four β-strands), and UbcH7 (four α-helices and four β-strands), colored in green, red, and cyan, respectively. The two active-site loops are colored yellow. The dotted line indicates the open line of sight between the active-site cysteines of E6-AP and UbcH7 (adapted with permission from:Huang et al., Science, 1999). B) The ternary complex of c-Cbl with UbcH7, and phosphorylated ZAP-70 peptide.The TKB domain is colored green, the RING domain red, and the linker region of c-Cbl yellow. UbcH7 is colored in cyan and its active-site cysteine in orange (adapted with permission from:Zheng et al., Cell, 2000).

Figure 15 .

 15 Figure 15. Ubiquitin transfer cascade by HECT E3s. A) Left: structure of E2 (pale cyan, with active site as sphere)∼Ub (yellow) with E3 NEDD4L HECT (violet, with active site as sphere) Right: structure of E3 Rsp5 WW3-HECT (violet, with active site as sphere), Ub (yellow) and the substrate Sna3 C (green). B) Schematic views of E2-to-E3 Ub transfer and E3-to-substrate Ub ligation (adapted with permission from: Kamandurai, Elife, 2013).

Figure 16 .

 16 Figure 16. Structure of a UBC domain (here the E2, Ubc13 is shown as an example) and its interaction surfaces. In the center is the E2, Ubc13 in ribbon structure (PDB ID: 2GMI), with its interactors. The E2 in each interacting pair is shown as green. Clockwise from lower left corner: the E1/E3-binding surface as seen in complex of Ubc13 with the RING E3 Traf6 (blue) (PDB ID: 3HCT) and in Ubc12 in complex with the NEDD8-activating E1 (yellow) (PDB ID 2NVU); backside-binding surface as seen in the E2/Ub complex, UbcH5c/Ub (red) (PDB ID 2FUH); substrate-binding surface as seen in the SUMO E2 Ubc9 in complex with its substrate RANGAP1(purple) (PDB ID: 1Z5S); activated Ub/Ubl surface as seen in UbcH5b~Ub complex (PDB ID: 3A33). The active site Cys shown as yellow stick representation (adapted with permission from:Wenzel et al., Biochemical Journal, 2010).

Figure 17 .

 17 Figure 17. Crystal structure of the Ubc7:U7BR complex. A) Schematic representation of Cue1 protein showing its transmembrane (TM), ubiquitin-binding (CUE), and Ubc7-binding region (U7BR) domains. B) Ribbon diagram of the Ubc7p:U7BR structure. Helices, strands, and loops are illustrated as spirals, arrows, and tubes, respectively (adapted with permission from: Metzger et al., Molecular Cell, 2013).

  , have been used in a broad range of PPIs analyses. The two most commonly applied types of PCAs for the detection of PPIs in living cells, based on bioluminescent and fluorescent protein-fragment complementation, are referred as bimolecular luminescence complementation (BiLC) and bimolecular fluorescence complementation (BiFC), respectively.1.3.2.1 Bimolecular luminescence complementation (BiLC)

Figure 18 .

 18 Figure 18. Principle of BiLC. A pair of interacting proteins A and B is fused to the inactive fragments of a luminescent reporter protein, e.g. Renilla luciferase. When proteins A and B interact the two reporter protein fragments are brought together and the reporter activity is reconstituted. This can be detected by the emission of light since Renilla luciferase catalyzes oxidation of its substrate coelenterazine to coelenteramide emitting photon of light.

Figure 19 .

 19 Figure 19. Principle of the BiFC assay. A pair of interacting proteins A and B is fused to the inactive Nand C-terminal fragments of a fluorescent reporter protein, respectively. When proteins A and B interact the two reporter protein fragments are brought together and the reporter activity is reconstituted, reproducing fluorescence emission under excitation.

  as a bright fluorescent protein with fast and efficient maturation properties. It has been shown that Venus produces the brightest fluorescence intensity in BiFC assays, approximately ten times higher than that of EYFP-based BiFC (Shyu et al., 2006). Recently, several studies aiming to develop a Venus-based BiFC assay with low background fluorescence have been conducted (see for instance -Ohashi et al., 2012).

Figure 20 .

 20 Figure 20. A) Structure of the Venus yellow fluorescent protein (PDB ID: 1MYW) with the α-helices and β-strands visualized in rainbow colors using PyMOL software (http://www.pymol.org/) B) N-terminal fragment of Venus (VN) C) C-terminal fragment of Venus (VC).

1 KH

 1 Low fluorescence yeast nitrogen base -This yeast nitrogen base is a component of a low fluorescence yeast visualization medium. For preparation use the following reagents at the appropriate concentrations: 5 g L -1 (NH 4 ) 2 SO 4 , 1 g L -pantothenate, 2 mg/l inositol, 0.4 mg L -1 niacin, 0.2 mg L -1 PABA, 0.4 mg L -1 pyroxidine HCl, 0.4 mg L -1 thiamine;  Low fluorescence visualization medium -2% wt/vol dextrose, low fluorescence yeast nitrogen base (as aforementioned). CRITICAL: Autoclaved and cooled to 50°C before adding the essential amino acids;  Yeast extraction buffer (breaking buffer) -2% Triton X-100, 1% SDS, 100mM NaCl, 100mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0);  TCA-sample buffer -2.2 ml 70% glycerol (final: 15%), 3 ml 1.5M Tris pH 8.8 (final: 450 mM), 0.5 ml 20% SDS (final: 1%), 40 µl 0.5M EDTA (final: 2 mM), 0.1 ml 0.5% bromophenol blue (or a 'spatula tip'), 4.2 ml H 2 O.

  using either the T4 DNA (Takara) or the more recent Gibson assembly technique (NEB). Mutagenesis was carried out using a standard site-directed mutagenesis kit (QuickChange Multi Site-Directed Mutagenesis, Stratagene) or using the Gibson assembly kit (NEB). To make point mutants in the Ubc4 test constructs, phenylalanine was mutated to alanine (F63A for a single and double mutant) and alanine was mutated to aspartic acid (A97D for a double mutant). To generate double site mutation, the single site mutated plasmid was used as a PCR template. Deletion mutants were generated as shown by[START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF]. For the construction of E2 array to systematically assay E2/E3 interactions using BiFC, all E2s were tagged with the Cterminal fragment of Venus (VC). The C-terminal fragment of Venus was codonoptimized using plasmid pEB0039 with primers oEB0043 and oEB0044 and pEB0023 as a vector linearized with PacI and BsrGI restriction enzymes. Each E2 (promoter and ORF) was cloned into a plasmid construct with a codon-optimized Venus variant between the BamHI and PacI restriction sites. All plasmids constructed in this study were sequenced using a dye terminator sequencing kit (BigDye Terminator v.3.1 Cycle Sequencing Kit, Life Technologies) and analyzed in house using the Applied Biosystems 3130 XL sequencer.Polymerase chain reactionPolymerase chain reaction (PCR) was used for several applications, including: cloning, plasmid screening, and verification of genetically modified yeast strains. In general, 1 µL of DNA was subject to PCR amplification in the final reaction volume of 50 μL (final concentration <0.5 µg/50 μL). PCR was performed using Phusion High Fidelity PCR Master Mix with GC Buffer (New England Biolabs) for cloning, and long and difficult amplification s whereas GoTaq Polymerase with 5x Green GoTaq Reaction Buffer (Promega) was used for plasmid screening or verification of yeast strains. The annealing temperature for the primers was calculated using OligoAnalyzer 3.1 software (Integrated DNA Technologies). The primers used for this study are presented in the chapter Materials, Table

a 1 .

 1 5 mL microcentrifuge tube. After centrifugation (1 min at max speed in a tabletop centrifuge) the cells were resuspended in 200 μL of yeast DNA extraction buffer (breaking buffer). 200 μL of phenol-chloroform-isoamyl alcohol (PCI 50:49:1) and an equal volume of glass beads (0.5 mm diameter) were then added to the tube (under the hood). The mixture was vortexed for 1 min and placed on ice for 1 min (repeated three times) to break the cells. 200 μL of Tris-EDTA (TE) buffer (pH 8.0) was added and mixed by rapid vortexing, followed by 10 min centrifugation at 4°C. The aqueous top layer containing the chromosomal DNA was transferred to a clean 1.5 mL Eppendorf tube. 1 mL of absolute ethanol (stored at -20°C) was added to precipitate DNA, mixed by inverting the tube and centrifuged for 10 min at RT. After removal of the supernatant the pellet was resuspended in 400 μL of TE buffer (pH 8.0). To digest RNA 3 μL of RNase (10 mg/mL) was added and incubated for 5 min at 37°C. Next, 10 μL of 4M NH 4 OAc and 1 mL of absolute ethanol (stored at -20°C) were added and the sample was incubated on ice for 15 min. After a further centrifugation step at 1000 rpm for 10 min at RT, the DNA was washed with 750 μL of 70% ethanol (stored at -20°C).

  E2 and E3 genes fused to reporter proteins. To generate such strains, linear DNA fragments (PCR products or linearized plasmids) were introduced into the yeast genome and targeted at the desired locus by homologous recombination. The primers used for PCR amplification of the tagging module were designed as previously described by[START_REF] Longtine | Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae[END_REF]. The sequences of all ORFs used for the design of the primers (ORF plus the untranslated region 1000 bases upstream of the start codon ATG and 1000 bases downstream of the stop codon) were downloaded from the Saccharomyces Genome Database (SGD). The primers were purchased from Sigma. Most E2s were tagged at the C-terminal. Ubc6 was tagged N-terminally due to the presence of its C-terminal transmembrane domain. Ubc1 was tagged at its N-terminal because the Cterminal seems to be important for Ubc1 function, as the C-terminally tagged Ubc1 strain was nonviable (personal observation). Strains with E3s were obtained from the commercial collection (Bioneer), where most genes were fused C-terminally to the Nterminal fragment of Venus fluorescent protein. Additionally, several strains with E3s

Figure 21 .

 21 Figure 21. BiLC assay optimization. A) Different strains permeability to the Renilla luciferase substrate coelenterazine h, CTZ h (10 µM). B) Different CTZ h concentrations used for assay optimization (10 µM in green and 50µM in red) C) The influence of autoluminescence reducing agents on assay performance.

Figure 22 .

 22 Figure 22. The BiLC assay tested with Ubc4/Ufd4, Rad6/Ubr1 and Ubc4/Ubr1, Rad6/Ufd4. BiLC signal detected with positive control (in green). There was no signal detected for all tested E2/E3 pairs (magnification of the graph at the bottom part of this figure).

Figure 23 .

 23 Figure 23. Specific detection of Ubc4/Ufd4 interaction using BiFC. A) Assessment of the expression level of VN and VC tagged Ubc4 and Ufd4 by Western blot (immunoblots of total protein extracts). The top panels refer to the whole cell extracts treated with DTT as a reducing agent. The bottom panel refers to the whole cell extracts treated with TCEP to preserve thioester bonds, therefore see the E2 ῀ Ub conjugate. B) Ubc4 deletion mutants used in this study. Regions deleted in the Ubc4 gene (PDB ID: 1QCQ) are presented in yellow. The canonical E2/E3 interface is highlighted in black, whilst the additional so-called 'backside' E2 interacting surface is highlighted in grey. C) Immunoblots of total protein extracts from Ubc4 deletion mutants. The top panels refer to the whole cell extracts treated with DTT as a reducing agent. The bottom panel refers to the whole cell extracts treated with TCEP to preserve thioester bonds, therefore see the E2 ῀ Ub conjugate. D) Quantification of BiFC signal. To quantify the fluorescence signal in different subcellular compartments, all yeast strains express nuclear protein (here Rpn7) fused to the red fluorescent protein tdimer2. This enables to automatically segment the fluorescence images and to specifically quantify the BiFC signal in both the nucleus and the cytoplasm. E) BiFC imaging of the interaction between the Ufd4-VC and the Ubc4-VN constructs. Fluorescence images of yeast strains expressing Ubc4 and Ufd4 tagged with VN and VC at their endogenous chromosomal locus. Various strains expressing Ubc4 mutated in its interaction surface with Ufd4 were used to test the specificity of the BiFC signal.

Figure 25 .

 25 Figure 25. Yeast strains expressing 11 E2s (tagged with VC) from their own endogenous promoters. Expression assessed by Western Blot using Anti-GFP antibody (top). Rad6-VC, VC-Ubc6 and Ubc13-VC as the examples of tagged E2s versus endogenous E2s across the strains. The expression levels were compared using Rad6, Ubc6 and Ubc13-specific antibodies, respectively.

Figure 26 .

 26 Figure 26. BiFC screen results of the putative detected E2/E3 interactions. Red dot represents a very strong or strong BiFC signal, pink indicates a moderate BiFC signal and grey indicates a weak BiFC signal. Uba1, which is an E1 activating enzyme serves here as a control.

Figure 27 .

 27 Figure 27. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with Cterminally (top) and N-terminally (bottom) tagged Tfb3 ligase. N-terminally tagged Tfb3 showed a strong BiFC signal in the nucleus with several E2s such as Ubc1, Rad6, Cdc34, Ubc4, Ubc5, Ubc8 and Ubc13 contrary to C-terminally tagged Tfb3. Here, a very weak BiFC signal was only detected for Ubc1 and Ubc13.

Figure 28 .

 28 Figure 28. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with Cterminally (top) and N-terminally (bottom) tagged Prp19 ligase. N-terminally tagged Prp19 showed slightly stronger BiFC signal in the nucleus for Ubc1, Ubc13 and Ubc8 conjugating enzymes in comparison with C-terminally tagged Prp19.

Figure 29 .

 29 Figure 29. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with Cterminally (top) and N-terminally (bottom) tagged Psh1 ligase. N-terminally tagged Psh1 showed BiFC signal in the cytoplasm and nucleus when interacting with Ubc1 and in the nucleus when interacting with Ubc5 and Ubc13. No significant signal was detected for C-terminally tagged Psh1.

Figure 30 .

 30 Figure 30. BiFC imaging of the N-terminally tagged Tfb3, Prp19 and Psh1 with Ubc1, Ubc4, Ubc8 and Ubc13 as examples. Top panel for each ligase shows BiFC signal and bottom panel Rpn7 proteasome subunit fused with red fluorescent protein tdimer2 that serves as a nuclear marker. A significant BiFC signal was not detected for Tfb3 in combination with Ubc4, Prp19 with Ubc4 and Psh1 with Ubc8 and Ubc4.

Figure 31 .

 31 Figure 31. BiFC imaging of the Irc20 with Ubc1 and Rad5 with Ubc13. Top panel shows BiFC signal and bottom panel Rpn7 proteasome subunit fused with red fluorescent protein tdimer2 that serves as a nuclear marker. No BiFC signal was detected with Ubc4 conjugating enzyme (shown here as an example for a better visualization of the positive BiFC signal).

Figure 32 .

 32 Figure 32. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with a Cterminally tagged putative ubiquitin ligase Irc20 (top) and ubiquitin ligase Rad5 (bottom). The fluorescence signal was detected in the nucleus upon the interaction of Irc20 with Ubc1 conjugating enzyme and Rad5 with Ubc13.

Figure 33 .

 33 Figure 33. BiFC imaging of the Gid9 and Rmd5 (Gid2) with Ubc8. Top panel shows BiFC signal and bottom panel Rpn7 proteasome subunit fused with red fluorescent protein tdimer2 that serves as a nuclear marker. No BiFC signal was detected with Ubc4 conjugating enzyme (shown here as an example for a better visualization of the positive BiFC signal).

Figure 34 .

 34 Figure 34. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with a Cterminally tagged ubiquitin ligases Gid9 (top) and Rmd5, also called Gid2 (bottom) with Ubc8 conjugating enzyme. The fluorescence signal was detected both in the cytoplasm and in the nucleus.

Figure 35 .

 35 Figure 35. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with a Cterminally tagged ubiquitin ligase Rsp5 on a replicative plasmid. The fluorescence signal was detected nearly with all ubiquitin conjugating enzymes, except Ubc7 and Ubc11.

Figure 36 .

 36 Figure 36. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with a Cterminally tagged ubiquitin ligases Ufd2 and Ufd4. The fluorescence signal was detected in the cytoplasm and nucleus upon interaction with Ubc1, Ubc4, Ubc5, Ubc8 and Ubc13.

Figure 37 .

 37 Figure 37. Quantification of the BiFC signal in the cytoplasm and the nucleus of the yeast cells with a Cterminally tagged ubiquitin ligases Pex2 (top), Pex10 (middle) and Pex12 (bottom). The fluorescence signal was detected with ubiquitin conjugating enzymes, Ubc1, Ubc10 and Ubc13 for Pex2 and Pex10 and Ubc1 and Ubc13 for Pex12.

Figure 38 .

 38 Figure 38. BiFC imaging of the Pex2, Pex10 and Pex12. Top panel shows BiFC signal and bottom panel Rpn7 proteasome subunit fused with red fluorescent protein tdimer2 that serves as a nuclear marker.

Figure 39 .

 39 Figure 39. BiFC imaging of the cells with VN-tagged Asi1 and Asi3 together with VC-tagged Ubc6. Top panel shows BiFC signal and bottom panel Rpn7 proteasome subunit fused with red fluorescent protein tdimer2 that serves as a nuclear marker. Scale bar, 5 µm.

and

  Ubc7 were productive, we analyzed the ubiquitylation of the N-terminal sequence of Stp2, which had previously been shown to contain the minimal signal for Asidependent repression of Stp2 activity. Those experiments were performed by Audrey Brossard and showed that the N-terminal fragment of Stp2 fused to the TAP-tag was indeed ubiquitylated in an Asi3 dependent manner. Furthermore, ubiquitylation of Stp2 N-terminus was diminished in ubc6 cells and almost fully abolished in ubc7 cells.

  Khmelinskii A.*, Blaszczak E.*, Pantazopoulou M., Fischer B., Omnus D.J., Le Dez G., Brossard A., Gunnarsson A., Barry J.D., Meurer M., Kirrmaier D., Boone C., Huber W., Rabut G., Ljungdahl P.O.,[START_REF] Khmelinskii | Analysis of protein dynamics with tandem fluorescent protein timers[END_REF]. Protein quality control at the inner nuclear membrane. Nature. 516 (7531), 410-413.

Anton Khmelinskii 1

 1 *, Ewa Blaszczak 2,3 *, Marina Pantazopoulou 4 , Bernd Fischer 5,6 , Deike J. Omnus 4 , Gae ¨lle Le Dez 2,3 , Audrey Brossard 2,3 , Alexander Gunnarsson 4 , Joseph D. Barry 5 , Matthias Meurer 1 , Daniel Kirrmaier 1 , Charles Boone 7 , Wolfgang Huber 5 , Gwenae ¨l Rabut 2,3 , Per O. Ljungdahl 4 & MichaelKnop 1,8

Figure 1 |

 1 Figure 1 | The Asi complex is a Ubc6/Ubc7-dependent E3 ubiquitin ligase of the INM. a, BiFC strategy used to assay E2-E3 interactions. E2 and E3 proteins were endogenously tagged with carboxy-and amino-terminal fragments of the Venus fluorescent protein (VC and VN, respectively). Interactions between E2 and E3 proteins enable reconstitution of functional Venus that is detected with fluorescence microscopy. Rpn7 fused to the red fluorescent protein tDimer2 served as a nuclear marker. b, Quantification of BiFC signals in cells co-expressing VC-Ubc6 and VN-tagged E3s. Fluorescence microscopy examples representative of six fields of view (top). Scale bar, 5 mm. BiFC signals were measured in the cytoplasm and nucleus of individual cells (bottom, n as shown). Whiskers extend from the tenth to ninetieth percentiles. c, Microscale thermophoresis analysis of interactions between recombinant maltose binding protein (MBP)-E3 fragments and the indicated E2s. Plots show the fraction of MBP-E3 bound to the E2 at each tested E2 concentration (mean 6 s.d., n as shown). Dissociation constants (K d , mean 6 s.d.) were derived from nonlinear fits with the law of mass action (solid lines). d, Activity of b-galactosidase (b-gal) expressed from the AGP1 promoter in the indicated strains (mean 6 s.d., n 5 3 clones). a.u., arbitrary units; WT, wild type. e, Ubiquitylation of Stp2 N -TAP in strains expressing 103histidine (His)tagged ubiquitin. Total cell extracts and ubiquitin conjugates eluted after immobilized-metal affinity chromatography were separated by SDS-PAGE followed by immunoblotting with antibodies against the TAP tag, Pgk1 and ubiquitin. Representative immunoblots from three technical replicates. *P , 10 24 (b; one-way analysis of variance (ANOVA) with Bonferroni correction for multiple testing) and *P , 0.05 (d; two-tailed t-test).

Figure 3 |Figure 2 |

 32 Figure 3 | Systematic identification of substrates for Asi and ERAD E3 ubiquitin ligases. a, A tandem fluorescent protein timer (tFT) is composed of two fluorescent proteins: one more slowly maturing (for example, the red fluorescent protein mCherry, maturation rate constant m S ) and the other faster maturing (for example, the green fluorescent protein sfGFP, maturation rate constant m F ). When fused to a protein of interest, a tFT reports on the degradation kinetics of the fusion protein: whereas fusions undergoing fast turnover are degraded before mCherry maturation, resulting in a low mCherry/ sfGFP intensity ratio, the relative fraction of mature mCherry increases for proteins with slower turnover. b, Summary heat map of the screens for tFTtagged proteins with altered stability in the indicated mutants. Changes in protein stability (z-score) are colour-coded from blue (decrease) to red (increase). Only proteins with a significant change in stability in at least one mutant (1% false discovery rate and z-score . 4) are shown. Clusters of potential substrates of Asi (green), Hrd1 (red) and Doa10 (blue) E3 ubiquitin ligases are indicated. c, d, Fraction of proteins in the tFT library and in the three clusters in b with a predicted transmembrane domain or signal peptide (c) or mapped to component Gene Ontology (GO) terms (d). Each cluster is significantly enriched in proteins with a predicted transmembrane domain or signal peptide compared to the tFT library (P , 2.2 3 10 216 , Fisher's exact test). e, Quantification of sfGFP signals in strains expressing tFT-tagged proteins from the Asi cluster in b. Fluorescence microscopy examples representative of five fields of view (top). Scale bar, 5 mm. sfGFP intensities were measured in individual cells and at the nuclear rim (bottom, n as shown).For each protein, measurements were normalized to the mean of the respective wild type. Whiskers extend from minimum to maximum values. *P , 0.05 (two-tailed t-test). f, Degradation of 33HA-tagged proteins after blocking translation with cycloheximide. Whole-cell extracts were separated by SDS-PAGE followed by immunoblotting with antibodies against the HA tag and Pgk1 as loading control. Representative immunoblots from three technical replicates.

1 |

 1 Identification of Ubc6 and Ubc7 ubiquitinconjugating enzymes as functional interacting partners of Asi1 and Asi3. a, Quantification of BiFC signals in cells expressing VC-Ubc6 and all tested E3 ubiquitin ligases. BiFC signals were measured in the cytoplasm and nucleus of individual cells (n as shown). Whiskers extend from the tenth to the ninetieth percentiles. The same representation is used in c and d. b, Immunoblot showing expression levels of VC-tagged E2 ubiquitin-conjugating enzymes. Ubc11-VC could not be detected in the growth condition of the BiFC assay. c, Quantification of BiFC signals in cells co-expressing VC-tagged E2 ubiquitinconjugating enzymes and Asi1-VN or Asi3-VN (n as shown). d, Detection of a significant BiFC signal between Asi1-VN and Ubc4-VC in cells lacking UBC6 (n as shown). e, Coomassie-stained gels of recombinant proteins used in microscale thermophoresis experiments. f, mRNA levels of AGP1 and GNP1 measured with qRT-PCR in the indicated strains (mean 6 s.d., n 5 3 clones).

2 | 3 |

 23 The signal was normalized to wild type (dashed line). g, Ubiquitylation of Stp1-HA or Stp1-RI 17-33 -HA (Stp1 variant in which amino acid residues 2-64 were replaced with Stp1 residues 17-33 flanked by minimal linker sequences) (left) and Stp2-HA or Stp2D 2-13 -HA (Stp2 variant lacking amino acid residues 2-13) (right) in strains expressing 63His-ubiquitin. Stp1-RI 17-33 and Stp2D 2-13 variants exhibit compromised cytoplasmic retention and enhanced Asi-dependent degradation, whereas full-length Stp1 is degraded primarily in the cytoplasm in a SCF Grr1 -dependent manner11 . Total cell extracts (T), flow-through (F) and ubiquitin conjugates (E) eluted after immobilized-metal affinity chromatography were separated by SDS-PAGE followed by immunoblotting with antibodies against the HA-tag, Pgk1 and the His-tag. Representative immunoblots from three technical replicates. *P , 10 24 (a, c and d; one-way ANOVA with Bonferroni correction for multiple testing), and *P , 0.05, **P , 0.1 (f; two-tailed t-test). Lack of genetic interaction between ASI1 and HRD1 or DOA10 at 37 6C. Tenfold serial dilutions of strains grown on synthetic complete medium for 2 days at 30 or 37 uC. tFT screens for substrates of Asi and ERAD E3

  | Analysis of integral membrane protein substrates of the Asi E3 ubiquitin ligase. a, Differences in the log 10 mCherry/sfGFP intensity ratio between the indicated mutants and the wild type (mean 6 s.d., n 5 4) for tFT-tagged proteins from the Asi cluster in Fig. 3b. b, Quantification of BiFC signals in strains co-expressing VC-Ubc6 and Asi3-VN (top). BiFC signals were measured in the cytoplasm and nucleus of individual cells (n as shown). Whiskers extend from tenth to ninetieth percentiles. A substantial BiFC signal is retained in the asi2D mutant, despite reduced expression of Asi3 (immunoblot, bottom). c, Quantification of sfGFP signals in strains expressing tFT-tagged proteins from the Asi cluster in Fig. 3b. Fluorescence microscopy examples representative of five fields of view (top). Scale bar, 5 mm. sfGFP intensities were measured in individual cells (middle) and at the nuclear rim (bottom). For each protein, measurements were normalized to the mean of the respective wild type. Whiskers extend from minimum to maximum values. *P , 0.05 (a and c; two-tailed t-test) and *P , 10 24 (b; one-way ANOVA with Bonferroni correction for multiple testing).

TABLE OF CONTENTS 3

 OF3

	.1 Materials
	3.2 Methods
	3.2.1 Molecular biology
	3.2.2 Yeast genetics
	3.2.3 Immunoblot
	3.2.4 Luciferase assay
	3.2.5 Confocal microscopy
	3.2.6 Quantification of the efficiency of BiFC complex formation and statistical analysis
	4 RESULTS
	4.1 Bioluminescent versus fluorescent protein complementation assays to study
	E2/E3 interactions in living cells
	4.1.1 Bimolecular Luminescence Complementation (BiLC)
	4.1.2 Bimolecular Fluorescence Complementation (BiFC)
	4.2 Construction and use of an array of yeast strains to assess E2/E3
	interactions
	4.3 Detection and localization of E2/E3 pairs with BiFC
	4.4 Functional characterization of the candidate E2/E3 pair
	5 DISCUSSION
	6 CONCLUSIONS AND PERSPECTIVES
	7 REFERENCES
	8 PUBLICATION

  Furthermore, different linkages result in specific chain conformations. Chains can either be 'compact', where neighboring molecules interact with one another, for example in the canonical Lys48-linked chains or Lys6 and Lys11, or adapt 'open' conformations, where there are no interactions between ubiquitin moieties. For instance, linear Lys63 and Met1-linked chains usually adapt an 'open' conformation. It is important to note, however, that the chain conformations observed in Ub-interacting protein complexes are frequently different from those observed in isolation. This suggests either a considerable dynamic flexibility of the chains or vast chain remodeling consequent to binding (Yu[START_REF] Ye | Ubiquitin chain conformation regulates recognition and activity of interacting proteins[END_REF]. The study conducted by[START_REF] Peng | A proteomics approach to understanding protein ubiquitination[END_REF] provided first evidence of a great diversity of polyubiquitin chains in vivo. Using a multidimensional liquid chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS), they

Table 2 .

 2 Ubiquitin conjugating enzymes in yeast (Data retrieved from: Saccharomyces Genome Database) Sequence alignments and phylogenetic analysis of eleven yeast E2s. A) Protein sequence alignments performed with CLUSTALW2. Small, hydrophobic and aromatic amino acids are indicated in red, acidic amino acids in blue and basic amino acids in magenta. Green represents hydroxyl, amine, amide and basic residues. Asterisks and dots indicate identical and conserved amino acids, respectively. The differences in the sequence are marked with period (semi-conserved substitution) or colon (conserved substitutions). The sequences for the alignment were retrieved from Saccharomyces Genome Database. B) Phylogenetic tree of yeast E2s generated with CLUSTALW2. A cladogram showing branching patterns.

	Standard	Systematic	Class	Size	Molecular	Subcellular	Main functions
	gene	name		(aa)	weight	localization
	name				(~kDa)	(with GFP)
	Ubc1	YDR177W	Core	215	24	Cytoplasm	Selective degradation of short-lived and
						Nucleus	abnormal proteins,
							ERAD, vesicle biogenesis
	Ubc2	YGL058W	C-term	172	20	Cytoplasm	DNA repair, ERAD, ubiquitin-mediated
	(Rad6)		extension			Nucleus	N-end rule protein degradation
	Ubc3	YDR054C	N-and C-	295	34	Cytoplasm	Cell cycle control
	(Cdc34)		term			Nucleus
			extensions
	Ubc4	YBR082C	Core	148	16	Cytoplasm	Selective degradation of abnormal or
						Nucleus	excess proteins, involved in DNA
							replication, component of cellular stress
							response
	Ubc5	YDR059C	Core	148	16	Cytoplasm	Selective degradation of short-lived,
						Nucleus	abnormal or excess proteins, central
							component of cellular stress response
	Ubc6	YER100W	C-term	250	28	ER	ERAD
			extension
	Ubc7	YMR022W Core	165	19	ER	ERAD, proposed to be involved in
	(Qri8)						chromatin assembly
	Ubc8	YEL012W	Core	218	25	Cytoplasm	Gluconeogenesis regulation
	(Gid3)					Nucleus
	Ubc10	YGR133W	Core	183	21	Peroxisome	Peroxisomal matrix protein import and
	(Pex4)						peroxisome biogenesis
	Ubc11	YOR339C	C-term	156	18	Not visualized	Cell cycle regulation
			extension

Table 3 .

 3 Numbers of ubiquitin ligases encoded by H. sapiens and S. cerevisiae genomes based on the presence of 'catalytic' domains

	Catalytic subunit of E3s	Human	Yeast
	RING
	 RING	~300	~50
	 U-box	9	2
	HECT	28	5
	RBR	13

Table 4 .

 4 The main known or putative ubiquitin ligases and components of E3 complexes in yeast (Data retrieved from: Saccharomyces Genome Database)*

	Standard gene	Systematic	Size	Molecular	Subcellular	Main functions and/or unique features
	name	name	(aa)	weight	localization
				(~kDa)	(with GFP)
	E3s with a RING catalytic subunit:
	Air1	YIL079C	360	42	Cytoplasm	Putative E3, nuclear RNA processing and degradation
					Nucleus
	Air2	YDL175C	344	39	Nucleus	Putative E3, nuclear RNA processing and degradation
	Asi1	YMR119W	624	71	Nuclear periphery Subunit of Asi Ub ligase complex that targets misfolded
						proteins and regulators of sterol biosynthesis for
						degradation; SPS-sensor signaling of amino acids
	Asi3	YNL008C	676	78	Nuclear periphery Subunit of Asi Ub ligase complex that targets misfolded
						proteins and regulators of sterol biosynthesis for
						degradation; SPS-sensor signaling of amino acids
	Asr1	YPR093C	288	33	Not visualized	Modification and regulation of RNA Pol II, alcohol
						stress response
	Bre1	YDL074C	700	81	Nucleus	Monoubiquitylation of histone H2B on Lys123
	Cwc24	YLR323C	259	30	Nucleus	Involvement in splicing
	Dma1	YHR115C	416	46	Ambiguous	Septin dynamics control, spindle positioning
	Dma2	YNL116W	522	58	Cytoplasm	Septin dynamics control, spindle positioning
	Doa10 (Ssm4)	YIL030C	1319	151	ER	ERAD
	Etp1	YHL010C	585	67	Not visualized	Required for growth in ethanol
	Fap1	YNL023C	965	109	Not visualized	Response to rapamycin
	Far1	YJL157C	830	95	Nucleus	Cyclin dependent kinase inhibitor and nuclear anchor
	Gid9 (Fyv10 )	YIL097W	516	60	Nucleus	Subunit of GID complex, involved in ubiquitin ligation
						as a dimer with Rmd5
	Hel2	YDR266C	639	73	Cytoplasm	Ubiquitylation and degradation of excess histones
	Hrd1	YOL013C	551	64	ER	ERAD
	Hrt1	YOL133W	121	14	Not visualized	Subunit of multiple ubiquitin ligase complexes,
						involvement in cell cycle
	Irc20	YLR247C	1556	180	Nucleus	Putative E3, synthesis-dependent strand annealing-
						mediated homologous recombination, ensures precise
						end-joining, has helicase domain
	Mag2	YLR427W	670	76	Cytoplasm	Unknown function, predicted to be involved in repair of
						alkylated DNA
	Mot2 (Not4)	YER068W	587	65	Cytoplasm	Subunit of Ccr4-Not complex, involved in
						ubiquitylation of nascent polypeptide-associated
						complex (NAC) subunits and histone demethylase
						Jhd2p, transcription regulation
	Mtc5	YDR128W	1148	131	Vacuolar	Subunit of the SEA (Seh1-associated) complex, has N-
					membrane	terminal WD-40 repeats and a C-terminal RING motif
	Nam7 (Upf1)	YMR080C	971	109	Cytoplasm	Nonsense mediated mRNA decay, required for
						efficient translation termination at nonsense codons
	Pep3	YLR148W	918	107	Endosome	Vacuolar biogenesis
	Pep5	YMR231W	1029	117	Endosome	Protein trafficking and vacuole biogenesis, involved in
						ubiquitylation and degradation of excess histones
	Pex2	YJL210W	271	31	Peroxisome	Peroxisomal matrix protein import
	Pex10	YDR265W	337	39	Peroxisome	Peroxisomal matrix protein import, Ubc4-dependent
						Pex5 ubiquitylation
	Pex12	YMR026C	339	46	Peroxisome	Peroxisome biogenesis and peroxisomal matrix protein
						import
	Pib1	YDR313C	286	33	Endosome	Phosphatidylinositol(3)-phosphate binding
	Psh1	YOL054W	406	47	Nucleus	Polyubiquitylation and degradation of centromere-
						binding protein Cse4
	Rad5	YLR032W	1169	134	Cytoplasm	Postreplication repair, DNA damage tolerance, PCNA
					Nucleus	polyubiquitylation
	Rad16	YBR114W	790	91	Cytoplasm	Nucleotide excision repair
					Nucleus
	Rad18	YCR066W	487	55	Not visualized	Postreplication repair, PCNA monoubiquitylation
	Rkr1 (Lnt1)	YMR247C	1562	180	Not visualized	Ubiquitin-mediated degradation of proteins translated
						from nonstop mRNAs
	Rmd5 (Gid2)	YDR255C	421	49	Not visualized	Gluconeogenesis, polyubiquitylation and degradation of
						the gluconeogenic enzyme fructose-1,6-bisphosphatase
	Rtc1 (Sea2)	YOL138C	1341	149	Vacuole	Subunit of the SEA (Seh1-associated) complex that
						associates dynamically with the vacuole
	San1	YDR143C	610	66	Not visualized	Proteasome-dependent degradation of aberrant nuclear
						proteins
	Ssl1	YLR005W	461	52	Nucleus	Subunit of the core form of RNA polymerase
						transcription factor TFIIH, transcription and nucleotide
						excision repair
	Slx5 (Hex3)	YDL013W	619	71	Not visualized	Subunit of the Slx5-Slx8 SUMO-targeted ubiquitin
						ligase complex, genotoxic stress response

Table 5 .

 5 The

	Ubiquitin ligase (E3)	Ubiquitin (E2) conjugating enzyme	Method/assay	Reference
	Apc11	Ubc1	Reconstituted Complex	Kimata (2011)
	Apc11	Ubc4	Reconstituted Complex	Enquist-Newman et al. (2008); Foster and Morgan
				(2012); Zhang et al. (2013)
	Asr1	Ubc1	Two-hybrid	Yu (2008)
	Asr1	Ubc5	Two-hybrid	Yu (2008)
	Bre1	Rad6	Affinity Capture-Western	Wood et al. (2003); Krogan et al. (2006)
			Reconstituted Complex	Kim and Roeder (2009)
			Biochemical Activity
	Cdc53	Cdc34	Affinity Capture-Western	Patton et al. (1998); Skowyra et al. (1999)
	Cul3	Cdc34	Reconstituted Complex	Michel et al. (2003)
	Cul3	Ubc4	Affinity Capture-MS	Geyer et al. (2003)
	Cul3	Ubc5	Reconstituted Complex	Harreman et al. (2009)
	Dma1	Ubc4	Reconstituted Complex	Loring et al. (2008)
	Dma1	Ubc13	Reconstituted Complex	Loring et al. (2008)
	Dma2	Ubc4	Reconstituted Complex	Loring et al. (2008)
	Dma2	Ubc5	Two-hybrid	Chahwan (2013)
	Dma2	Ubc13	Two-hybrid	Chahwan (2013)
			Reconstituted Complex	Loring et al. (2008)
	Doa10	Ubc6	Affinity Capture-Western	Neuber et al. (2005)
	Doa10	Ubc7	Affinity Capture-Western	Neuber et al. (2005); Bagola et al. (2013); Kreft and
				Hochstrasser (2011)
			Reconstituted Complex	Bagola et al. (2013)
	Doa10	Ubc13	Two-hybrid	Chahwan (2013)
	Gid2	Ubc8	Biochemical activity	Santt et al. (2008)
	Gid9	Ubc8	Biochemical activit	Santt et al. (2008)
	Hel1	Ubc4	Affinity Capture-Western
			Reconstituted Complex

known physical E2/E3 interactions in S. cerevisiae (Data retrieved from: BioGRID database of protein and genetic interactions)* Bailly et al. (1997); Ulrich and Jentsch (2000) Ho et al. (2002) Ulrich and Jentsch (2000); Uetz et al. (2000); Fu et al. Krogan et al. (2006); Collins et al. (2007); Michel et al. (2003); Zaidi et al. (2008); Han et al. IP), chip arrays, surface plasmon resonance (SPR), mass spectrometry or X-ray crystallography, and NMR spectroscopy

  1.3.1 Methods to assay protein-protein interactions under near physiological conditions 1.3.1.1 Förster resonance energy transfer (FRET)

  chemicals and reagents used in this study were of molecular biology and analytical grade. Plasmid DNA extraction kits as well as PCR products clean-up and gel extraction kits were purchased from Macherey-Nagel. Restriction enzymes and Gibson Assembly Master Mix used for molecular cloning were purchased from New England Biolabs. Other reagents, bacteria and yeast media, and all necessary equipment are listed below. Tetracycline (10 mg/mL) -dissolved in dH 2 O, filter sterilized. CRITICAL:

	 S-(2-Aminoethyl)-L-cysteine hydrochloride (Thialysine, MW = 200.69 g mol -1 ,  Zinc sulfate monohydrate (ZnSO4. H 2 O, MW = 179.47 g mol -1 , Sigma-Aldrich, Equipment
	Sigma-Aldrich, cat. no. A2636) (see REAGENT SETUP); cat. no. 96495);  Electronic 96-channel handheld pipette VIAFLO 96 (Integra Bosciences, cat. no.
	 Tetracycline hydrochloride (MW = 480.90 g mol -1 , Sigma-Aldrich, cat. no.  Biotin (Vitamin H, MW = 244.31 g mol -1 , Sigma-Aldrich, cat. no. B4501); 6000);
	T3383) (see REAGENT SETUP);  Calcium pantothenate (MW = 244.31 g mol -1 , Sigma-Aldrich, cat. no. C8731);  Refrigerated incubator shaker-incubator to grow yeast cultures at 20°C.
	 Uracil (Ura, MW = 112.09 g mol -1 Sigma-Aldrich, cat. no. U1128);  Myo-inositol (MW = 180.16 g mol -1 , Sigma-Aldrich, cat. no. 17508); Alternatively, cultures can be grown at 25°C or 30°C in a standard incubator;
	 L-histidine (His, MW = 155.15 g mol -1 Sigma-Aldrich, cat. no. H8000);  Niacin (Vitamin B3, nicotinic acid, Sigma-Aldrich, cat. no. 72309);  Inverted fluorescence microscope with 63x water immersion objective and 96-
	 L-lysine (Lys, MW = 146.19 g mol -1 Sigma-Aldrich, cat. no. L5501);  L-Leucine (Leu, MW = 131.17 g mol -1 Sigma-Aldrich, cat. no. L8000);  L-methionine (Met, MW = 149.21 g mol -1 Sigma-Aldrich, cat. no. M9625);  Amino acid stock solution (see REAGENT SETUP);  Ammonium sulfate ((NH 4 ) 2 SO 4 , MW = 132.14 g mol -1 , Sigma-Aldrich, cat. no. well plate adaptor. In our laboratory we were using Leica SP8 confocal  PABA (4-amniobenzoic acid, MW = 137.14 g mol -1 , Sigma-Aldrich, cat. no. microscope; A9878);  Luminometer -after testing different luminometer, we were using Centro XS3  Pyridoxine hydrochloride (MW = 205.64 g mol -1 , Sigma-Aldrich, cat. no. LB960 Microplate Luminometer (DLReady, Berthold Technologies) for the P9755); final measurements.
	A6387);
	449709);
	 clonNAT (Nourseothricin-dihydrogen sulfate, MW = 1359.47 g mol -1 , Werner BioAgents, cat. no. 5.001.000) (see REAGENT SETUP);  Hygromycin B (MW = 527.52 g mol -1 , TOKU-E, cat. no. H007) (see REAGENT SETUP); Reagent setup  Boric acid (H 3 BO 3 , MW = 61.83 g mol -1 , Merck Millipore, cat. no.  Zinc acetate (5 mg/mL) -dissolved in dH 2 O, filter sterilized; 1007650500);  clonNAT (100 mg/mL) -dissolved in dH 2 O, filter sterilized. This stock solution  Copper (II) sulfate pentahydrate (CuSO 4 .5H 2 0, MW = 249.69 g mol -1 , Sigma-Aldrich, cat. no. 209198);  Potassium iodide (KI, MW = 160.00 g mol -1 , Sigma-Aldrich, cat. no. P4286);  Iron (III) chloride (FeCl 3 , MW = 162.20 g mol -1 , Sigma-Aldrich, cat. no. F7134);  Manganese (II) sulfate monohydrate (MnSO 4 .H 2 O, MW = 169.02 g mol -1 , Sigma-Aldrich, cat. no. 221287); is stable for 4 weeks at 2-8°C. For longer storage the solution must be frozen at - Low fluorescence visualization medium (Yeast nitrogen base without riboflavin 20°C or deeper; and folic acid) was prepared according to the protocol of Sheff and Thorn,  Hygromycin B (300 mg/ mL) -dissolved in dH 2 O, filter sterilized. This stock (2004); solution is stable for several months at 2-8°C. CRITICAL: Do not freeze  Coelenterazine native (CTZ, Interchim, cat no. FP-BV0731); hygromycin stock solutions;  Coelenterazine h (CTZ h, Euromedex, cat. no. 21159-AAT);  Canavanine (50 mg/mL) -dissolved in dH 2 O, filter sterilized. This stock  Coelenterazine hcp (CTZ hcp, Euromedex, cat. no 21154-AAT); solution is stable for several weeks at 2-8°C. For longer storage needs freezing
	at -20°C;
	243655);

Reagents

 Bacto yeast extract (BD Biosciences, cat. no. 288620);

 Bacto peptone (BD Biosciences, cat. no. 211677);

 Difco dextrose (Glucose, BD Biosciences, cat. no. 215530). CRITICAL:

Dextrose should be autoclaved separately from amino acids or protein containing reagents present in media components due to the Maillard's reaction that leads to the formation of brown pigments;  Difco nutrient broth (BD Biosciences, cat. no. 234000);  Difco yeast extract (BD Biosciences, cat. no. 210934);  Difco yeast nitrogen base w/o amino acids and ammonium sulfate (BD Biosciences, cat. no. 233520);  Zinc acetate (MW = 183.48 g mol -1 , Sigma-Aldrich, cat. no. 383317) (see REAGENT SETUP);  Potassium acetate (MW = 98.14 g mol -1 , Sigma-Aldrich, cat. no. P1190);  α-Glutamic acid (MW = 187.13 g mol -1 , Euromedex, cat. no. 2030);  L-Canavanine sulfate salt (Canavanine, MW = 274.25 g mol -1 , Sigma-Aldrich, cat. no. C9758) (see REAGENT SETUP);  Glutamic acid, monosodium salt (MW = 167.15 g mol -1 , Euromedex, cat. no. 2030);  Potassium phosphate monobasic (KH 2 PO 4 , MW = 136.09 g mol -1 , Sigma-Aldrich, cat. no. P0662);  Magnesium sulfate (MgSO 4 , MW = 120.37 g mol -1 , Sigma-Aldrich, cat. no. 246972);  Sodium chloride (NaCl, MW = 58.44 g mol -1 , Sigma-Aldrich, cat. no. S9888);  Calcium chloride (CaCl 2 , MW = 110.98 g mol -1 , Sigma-Aldrich, cat. no.  Sodium molybdate (Na 2 MoO 4 , MW = 205.92 g mol -1 , Sigma-Aldrich, cat. no.  Thiamine hydrochloride (Vitamin B1, MW = 337.27 g mol -1 , cat. no. T4625);  Low fluorescence yeast nitrogen base (yeast nitrogen base without riboflavin and folic acid), was prepared according to the protocol of Sheff and Thorn, 2004) (see REAGENT SETUP);  YPD (Yeast extract/peptone/dextrose) medium (see REAGENT SETUP);  GNA pre-sporulation medium (see REAGENT SETUP);  Zinc acetate sporulation medium (ZnSPO) (see REAGENT SETUP);  Diploid selection medium (Synthetic minimal medium based on glutamate (SED), here lacking leu and ura with an addition of clonNAT) (see REAGENT SETUP);  Haploid selection medium (Synthetic minimal medium based on glutamate (SED), here lacking leu/lys/ura with an addition of clonNAT, hygromycin B, canavanine and thialysine for MATα haploid selection) (see REAGENT SETUP);  Yeast extraction buffer (breaking buffer) (see REAGENT SETUP);  TCA-sample buffer (see REAGENT SETUP). Consumables  Pipet tips for VIAFLO electronic handheld pipettes (Griptips 125 µL, Integra Biosciences, cat. no. 4424);  96-well culture plates (ScreenMates, Matrix Technologies, cat. no. 4912). These plates were used to grow yeast culture, sporulate and select the haploids;  Sealing films for yeast 96-well plate culture (Excel Scientific, cat. no. BS-25);  96-well glass imaging plates (Imaging plates CG (96-well), Zell-kontakt GMBH);

 96-well white solid plates for bioluminescent assays (Fisher Scientific, cat. no.

3789).

 Thialysine (50 mg/mL) -dissolved in dH 2 O, filter sterilized. This stock solution is stable for several weeks at 2-8°C. For longer storage needs freezing at -20°C; 

  2 g L -1 L-methionine. To select auxotrophic yeast strains appropriate amino acids were dropped out. Autoclaved using Haploid selection medium -0.17% wt/vol Difco yeast nitrogen base w/o amino acids and ammonium sulfate, 0.1% wt/vol glutamic acid, 2% wt/vol dextrose. As for diploid selection medium, yeast strains contain resistance cassettes with the NatMX6 genes that enable the cells to grow in the presence of the eukaryotic antibiotic clonNAT and URA3 marker for the selection in the media lacking uracil. Additionally, yeasts have a selection module conferring hygromycin

a short 15' cycle;  YPD medium -1% wt/vol Bacto yeast extract; 2% wt/vol Bacto peptone, 2% wt/vol dextrose in dH 2 O, sterilized by autoclaving;  GNA pre-sporulation medium -3% wt/vol Difco nutrient broth, 1% wt/vol Difco yeast extract, 5% wt/vol dextrose in dH 2 O, sterilized by autoclaving;  ZnSPO medium -1% vol/vol zinc acetate 2% wt/vol potassium acetate in dH 2 O, add 10x complete amino acid stock sterilized by autoclaving;  Diploid selection medium -0.17% wt/vol Difco yeast nitrogen base w/o amino acids and ammonium sulfate, 0.1% wt/vol glutamic acid, 2% wt/vol dextrose.

Yeast strains contain NatMX6 resistance cassette that enable the cells to grow in the presence of the eukaryotic antibiotic clonNAT (nourseothricin) and a URA3 auxotrophic marker for the selection in synthetic media lacking uracil.

CRITICAL: Autoclaved and cooled to 50°C before adding antibiotics and the desired amino acids;  resistance gene that in the presence of antibiotic hygromycin B enables to grow only MATα cells. Canavanine and thialysine needs to be added to this media.

Table 6 .

 6 Antibodies used in this study Primers were named according to the name of the modified gene or other amplified element. The forward (sense) primer is indicated by the letter F. The reverse (antisense) primer is indicated by the letter R.

	Antibody	Type	Specificity	Company/	Dilution
				Reference
	Anti-GFP (9F6)	Mouse monoclonal	N-terminal part of GFP	Selleckchem	1:5000
	Anti-GFP (clones 7.1	Mixture of mouse	C-terminal part of GFP	Roche	1:1000
	and 13.1)	monoclonal antibodies
		7.1 and 13.1
	Anti-GFP	Rabbit polyclonal	GFP	Clontech	1:1000
	Anti-yRad6	Rabbit polyclonal	Rad6 from S. cerevisiae	Davies et al., 2010	1:5000
	Anti-yUbc6	Rabbit polyclonal	Recombinant His-UBC6 (without the	Walter et al., 2001	1:5000
			transmembrane domain) from S.
			cerevisiae
	Anti-yUbc13	Rabbit polyclonal	Recombinant His6-Ubc13 from S.	Ulrich, 2003	1:10000
			cerevisiae

Table 9 .

 9 Yeast strains used in this study*

	HIS3	Benoit Palancade
	HIS3	Benoit Palancade
	KAN	Taddei lab
	KAN	Taddei lab
	HPH	Michnick lab
	NAT	Michnick lab
	NAT	Michnick lab
	HPH
	KAN
	KAN
	KAN
	KAN
	KAN
	KAN
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT
	NAT

Table 10 .

 10 Summary table of BiFC screen resultsComparison of the BiFC signal produced with N-terminally and C-terminally tagged

	BiFC signal	Number of positive hits
	Overall signal detected	128
	Very strong/strong signal (red dots)
	Moderate signal (pink dots)
	Weak signal (grey dots)
	Interactions previously reported
	New putative E2/E3 pairs

��������������������������

  ����� ��� ��������� ���� ������������� ��� �� ������������ �������� ��� �������������� �������� ����� ���������������������������������������������������������������������������������������� ��� ����� ���������� ���� ��� ��������� ������ ���������� ���������� � �� �������� ���������� ���� ���������� ������������� ��� ����� ����������� ��������� ����������� ��� �������� ��� ���������� ���� ���� �������� ��� �� ������� ����������� ����������� ����� ����� ��� ����������� ���� �������� ��������� ��������� ���� ��������� �������� �������� ����� ���� ���� ������ ������� ����� � �� ����������� ���� �������������������������������������������������������������� ������� ��� ��������� ���� ����� �������� ������� ������� ��� ��� ������� ����� ���� ����� ��� ������� ��� ������� ���������� ���������� ������ �������� ����������������� ������� ��� �������� ���� ������ �������� ����������������������������������������������������������������������������������������������������� �������� ��� �������� ��������� ��������� ��� ��� ���������� ������ ��������� ������ ������� ������������ ������ ������������ ����� ��������� ����������� �������� �������� ����������� ���� � ���� ������������ �������������� �������� ����� ����������� ��������� ��� ����� ��������� ���� ������� ��� ������� �������� ����������� �������� �������� ����� �� �������� � ������� ��� ������� ��� ���� ����� ��� ��� ������������������������������������������������������������������������������������������������� ���� ����������� ��������� ���� ���� �������� ���� ������������ ������ ����������� ��������� ��� �������� �� ��������� �������� ��������� ����� �������� ���� ������� ��� ������������ �������� ���������� ����� ���� ������ ������� ���� ������� ����������� ��� �������� �������� ���� ������ ����������� ������������ ����� ������ ���� �������� ��� ����� ��������� ���� ����������� ����� ����������� �������� �� �� ��� ��������� �� ������� ���� ��������� �������� �������� ����� ���� �������������� ������ ����� ��������� ���� ���������� �������������������������������������������������������������������������������������������������� ���� ����� ����� �������������� ����������� ����� ����� ����� ����������� ��������� ���� �������������������������������������������������������������� Δ� ������������������������������� ��������������������������������������������������������������������������������������������� �������������� ��������� ��������� ����� ���������� ������ ������������ ��� ����� ������� ����� �� ������ ��� ��������� ������ ������������� ���� ����������� ��� ��������������� �������� ��� ����������� ���� ���������� ������������������������������������������������������������������������������������������������ ����������� ��� ���� ������� �������������� �������� ��� ���� ����������� ��� ��� ��������������������� �������� ���������� ����� ���������� ���� ��������� ��������� �������� ��� ������������ ������ ����� ���� ���� ������������������������������������������������������������ �� �� ��� ������� ��� ������� ���������� ���������� ��� ���� ���� �������� ���� ��� ������������ ���� ������ ��������� ���� �������������������������������������������������������������������������������������������������� ��� ����������� ����������� ����� ��� �� �������� ���������� ������ ���� �� ���� ������������� ������� ��������� �������������������������������������������������������������������������������������������������� ���������� ���������� �� ��������� ����� �������� �������� ������ ���� ��� ��������� ��� ��������� �������� ���� ����������� ��� ���������� ���� �������� �������� ���� �� ��� ������� ��� ������ �������� ����� ��������� ����� ���������� ���������� ���� ����������� ��� ���� �������������� ������ ������� ��� ���� �������������� ����������� �� ���������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������� ����������� ��� ��������� ���� ��������� ��� ���������������� ����� ����� ���� �������������� ������� ��������� ��������� ��� ���� ���������� ����� ��������� ����� ������� ����������� ����������� ��������������������������������������������������������������������������������������� ���������� ��� ���������� ���� ��� ������������ ���� ����������� ���������������� ���� ��� ������������� ����� ����� ���� �������������� ������� ���� ���� ��� �������� ����� ���� ���������� ���� ����� �������� ������������� �������� �������� ��� �� ����������� ������������� ������ ��� ���� �������� �������� ��� ����� � �� ����� ������� ����� ���� ����� ����� ��������� ���������� ���� ��������� ����������� ����� ��������� ���� ����������������������������������������������������������������� ������ �������� �� ���� �������� ����� ������ �������� ����������� ������ ���� �������� ����� �������������������� ���� �������� �������� ����� ��������� ���� ���� ����� ���� ������ ��� ��������� ������� ����� ����������� ��� ����������� ���� ��� ���� ������ ������ ��� ����������������� ���� ����� ����� ���� ���� ��� ������� ���� ��������� ��� ��� ������������ ������� ���� ����� ������ ����������� ������ ��������� ��� ������������������� ���� ���� ���� ��� ������� ���� ��������� ��� ���� �������� ����������� ��� ��� ������������������������������������������������������������������������������������������� ����������������������������������������� ���� ������� ���� ��������� �������� �������� ����� �������������� ���� ���� ���������� ��� �������� �������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������������µ�� ���������������������������������������������µ���������������������������������������������������� ����µ�������������������������������������������������������������������µ������������������ � �� ��µ�� ��� ��� ������ ��� �������� ���������������� �������� ����µ�� ��� ��������� ���� ��������µ�� �������� �������µ�� ��� � � �� ���� �����µ�� ��� ���������� ��� ������� ���� ����������� ��������� �������������� ��������� ������������������� ��� ������� ��������� ���������������� �������� �������� �� �������� ��� ���� ��������� ������ �������� ���µ�� ��� �µ�� ������� ���� ������ ��� ����� ����� ����� �������� ������� ������� ������� ����� ������������� ������ ���� ������� ��������� ���������� ������� ���� ������� ����� ������� ����� ���������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������ ������� ��� ����� ��� ���������� ��� ���������������� ��� ������ ������ ��� ����� ���� ����������� ��� ����� �������� ���������� ��� ������ ������� ��� ����� �������� ������ ����� ��������� ��� �������� ���� �������������������������������������������������������������������������� ��� ������������ ������� ������������ ����� ���������� ���µ�� ��� ���������� �������� ������ ����� ��������� ����� ����� ����� ������ �� ���������� ����������� ������� ��������� ������ �������� �������� ��µ�� ��� ���������� �������� ������� ����� ������������ ����� ����� ���� ������ ����� ������������� ����� ���� �������������� ��������������� ������� ����µ�� ��� ������ ������� �������� �������� �������� �������� ����� ��������� ������ ���� ���������� ������ ���� ������ ����������������������������������������������������������������������������������������������� ���� ������� ����� ������� ����� ���� ���������� ��������� ������ ���������� ������� ������������ ���� ���������� ���� ������� ��� �� ����� ������ ������ ������ �� ��������������� ����� ������� ��� �������� ���� ������������ ���� �������� ���� ���� ����� ������� ���� ������������ ��� ����µ�� ���� ����� ��� ���������� ������������������������������������������������������������������������������������������������� ����� ����� ����� ���� ��������� ������� ����� �� ��������� ����� ������ ����� ������� ����� �������� ���� ������� ����� ���������� ��� ����� ���� ������� ������ ������ ��������� ����� �������� ��� ���� �������� ������������������������������������������������������������������������������ ������ �������� ����� ����� ��� ���� ������ ������ ��� ����������� ����� ���� ���� ����� ����� ���������� ��������� ���� ����������� ���� ���� ��������� ����� ���� ������������� ��������� ��������� ������ ���� ������������ ���������� ������ ���� �������� ���� �������� ������� ��� ���� ���� ����� �������� ���� �������� ������������ ��� ���� �������� ������� ����� ����� �������� ������ ���� ������� ��� ����� ���� ��������� ����������������������������������������������������������������������������������������������� ���� ���� ���� �� �������� �������� ������� ���������� ������� ���� ���� �������������������������� �������� ���������������������������������������������������µ����������������������������������������������µ�� ��� ���� ����� ����������� ������� ������� ����� ������� ������ ��� � � � �� � �� ������ ������ �����µ�� ��� ������������������������������������������������������������µ������������������ � ����µ�������� ���������������������������������������������µ��������µ������������������������������������������� ��������������������µ������ � ����������µ�����������������������������������������µ������������������ ������ ����������� ��� ������� ��������� ���������� ������� �� ��������� �������� ������������� ����������� ������� ���µ�� ��� �µ�� ������� ���� ������ ��� ����� ����� ����� �������� ����������� ������� ������� ������� ����� �������������� �� ������ �������� ��� ����� ������� �������� ��� ������ ��� ��������� ������� ���� �����������������������������������µ������������������������������������������������������������ �������� �� �������� ���� ���� ���� ��� ����� ���� �������� ����������� ������� �������� ��� �� ������� ��� ��������� ����� ���� ������� ����� ������� ����� ��������� ��������� ����� ������ �� ����������� ��������� �������� ���� ���� ����� �������� ���� ��� ��� ��������� �������� ���� �������� ������ ���� ���������� ��������� ������ ��� ������ ��� ������� ��� ����� ��� ���������� ��� ���������� ��� ������ ������ ��� ����� ���� ������������������� ����������������������������������������������������������������������������������������������� ����� ��� ����� ������������ ������������ ���� �� ���������� ���� ������� ����������� ���� ������ ���� ����������������������������������������������������������������������������������������������� ����� ����������� ��� ���� ��������� ��� �� ���� �������� ��� ��������� ����� ��� ���� ������� ���� ���� ��� ���� ������������� �������������������������� �������������������������������������������������������������������������������������������������� ������ ����� ��������� ��� ������������ ������� ����� �� ���������� ����������� ��� ����� ������� ���� �������� ���������� ��������� ������� ��������� ��� ����������� ������� ��� ���������� ����������� ��������� ������� ����������� ��� ���������� ���� ��� ���������� �������� ��� ��������� ���� ������ ������� ����������� ������� ����������� ��������������� ����� �� �� ������������� ������������ ��� ���� ��������� ����� ��������� ������ ������ ��� ������� ��� �������� ������� ����������� ��������� ������� ����������� ��� ���������������������������������������������������������������� ��� ����� ����������� ������ ����� ������������� ��� �������� ��������� ����� ���� �������������� ������ ������� �������������� ��� ������������ ������������ ��� ����� �������� ������ ���� ������������� ���� ���������� ���� ��������� ��� ��� ���������� ���������� ��� ���� ����������� ��������� ������ ����� ����������� ����������� ����� ����� ���������� ������� ������ ��� ������������ ����������� ����� �� ������ ��� ��������� ��������� ����� ���� ����� �������� ����� ����� ��� ���� ��� ���������� ���������� �� ��������� ��������� ������������������������������� � ��� ���������������������������������������������������������������������������������������������������� ���������� �� ������������ ��������� �� �������� ���� ����� ��������� � ��������� �� �� � �� ������ �������� ��������������������������������������������������������������� ������������������������������������� �������� ��� ���������� ������������� ���� ��� ���� ����������� ��������� ��� ���� ����� ������������ ���������� ������������ ��� ��������� ����� ����������� �������� �������� ���� ����� ���� ��� ��������� ����� ���� �������������� ����� � �� ������ ��������� ����������� ������ ������ ����� ���� �������� ������� ������� ������� ��� �������������� ������ ������������ ������ ���� �������� ����������� ������ ������ ����� ���� ������������������������������������������������������������������������������������ ������������ ������������������������������∆��������������������������������� ������������������������������������������������������������������������������������������������� ������ ��� ������ ������� ������������� ������������� ���� ���� ��������� ����������� ����� �������������� ������������ ����� ���� ��� ������������ ��� ������� ������������ �������������� ������ ����������������������������������������������������������������������������������������������� ��� ������� ������������� ������� ���� ������� ������� ����� ���� ������� ��������� ������ ��������� ������� ��� ����������������������������� �� ��������������������������������������������������������������������� ������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������� �������������������������� ����������������������� �� �� ������������ ���������� ��� �� ������� �������� ��������� ���������� ��� ������������ ��������� ������ ��� �������� ��� �������� ��������� ����� �� ��������� ������ �������� ������� ������������ �������� �������� ��� ���������� ����� ������� ����� �������� �������� ���������� ���� ������ ������� ��� �������� ��� �������� ������������ ���� ���� ��������� ��������� ����������� ��������� ����� �������� ����������� ��������� ��� ��������� ��� ����������� ������ ��� ����� �������������� ��� ���� ���� ������������� ����� ������� ��� ���� ���� ������ ����������� �������� �������� �� � ������������� ��� �������� ����������� ������ ������ ���� ����������� ��� ���� ���� ���������� �������� ��� ���������� ����� ���� ������� ����� ��� �������� ��� ��� �� � ������ �������� ���� ���������� ����������� ��� �������� ��� ����� ��� �� ������ ��������������������������������������������������������������������������������������������������� ������������

	�	������������������������������������������������ ������� ��� ��������� �� ���� ��������� ���� �������� ��� �������� ��������� ��� ����� ���������� ���� ����� ���� �������������������������������������������������������������������������������������������������� ������������ ������ �������� ����������� ��������� ������ ��� ���� ���� ��� � �� �� ���� ��������� ��� ���� ����������������������������������������������� � ��������������������������������������������������� ����� � ������������������������������������������������������������������������������������������ � � ����� ����� �������������� ��������� ���� ��������� ��� ����� ������ ��������� ������ ����� ��� ��������� ��������� �������������������������������������������������������������������������������������� ���������������������������������������� ��������������������������������������������������������������������������������������������������� ������ �������� ��������� ������� �������������� ����� ��� ����� ���������� ��� ������� ����� ����� ����� ��������������������������������������� ��� ����������������������������������������������������������� ������� �������������� �������� ������������������������������������������������������������������������������������������� ����������������������������������������������� ������������������������������������������������������������������������������������������� �������������� ������� ������� �������������� ����� ���������� ���� ���������� ��� �������� ������� ��������� ���������� ����� ���� ���� ��������� ������� ������ ���� ����� ������������ ��� ����������� ���� �������������������������������������������������������������������������������������������������� ��� ���� ���������� ����������� ��� ������������� ����� ��������� ����� ���� ������������ ���� ������������������������������������������������������������������������������������������������ ������ ���� ����������� ����� ����� ��������� �������� ������ ������ ������������� ���� ��������� �� � ���� ��������������������������������������� �� ������������������������������������������������������������ ��� ��� ���� ���������� �������� ������ ��� ������������ ��� ���������� ��������� ���� �������� ������� ���� ���� ���������� ����� ������������� �������� ����������� ������ ���������� ����������� ��� ����� �������� ������ ����������� ��� �������������� ���������� �������� ������� ��������� ����� �� ������� �������� ����� ������������ ����� ����� ���� �������� ����������� ��� �������������� ����� ��������������� �������� �������������������������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������������������� ����� ��������� ��������� ���� ������� ��������� ���� ����� ��������� ������� ����� ������������� ����������� ����� ����� ��������� �������� ������� ���������� ��� ���� ������������ ��������� ��� ����� ����� ����������� ��������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������� ����� ������� ������ ���� ����� ����������� ������� ����������� ��� ������������� ���� ����� ��� ����� ���� �������� ������������ ��� ���� �������� ������� ����� ����� ������ ��� ����������� ���� ����������� ��� ������������������������������������������������������������������������������������������������ ��������� ������ ���� ����������� �������� ����������� ��� �������������� ����� ���� ������ �������� ���������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������� ����� �������� ������ ����������� ��� �������������� ����� ���� ������ �������� ������������ ��� ���� �������� �������������������������������������������������������������������������������������������������� �������� �� ������ ���� ������ ������ ��� ������� ����������������� ��������� ������������� ������������ ��� ���� �������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������������� ���� ����� ������ �� ���������� ������ ���� ��� ����� ���� ��� ���������� ������� ���� ��� ����� ������������������������� ���������������������� �������� ��� ������������������������������� �������������������������������������������������������������������� � �����������������������α� ����Δ��������������������Δ������������������Δ������Δ������Δ������������������������������� ��� ���� ��������� ������� ����� ��������� ������� �������������� ����� ��� ���������� ��� �������������� ����� �� �� ���������������������� ����� ������� ����� � ��� ���������� ���������� ��� ����� ������������� ������� ������� �� ��� ���������� ������� ������������� ����� ������� ��������� ���� �������� ��������� �����Δ���������������� ����Δ��������������� ���������� ���� ���������� ��� �������� ������� ������� ���������������������������������� ���������������������������������������������������������������������������������������������������� ����������������������� �� ���������������������������������������������������������������������� ������������������������������������������������������������������������������������������������ ������ ���� ���� ��������������� ����������� ����� ������ �� � ��������������� ���������� ���� ��������� ������������ ��������� ���� ����� ����������� ����� ���� ����Δ�� ������ ��� ���� ������ ������� ��� ���� ���������� ����� �������� ������������ ���������������������������������������������������������������� ���������� ���� ������������ ������������������������������������������������������������������������ ���� ���� �������� ��� ���������� �������� ������������ ���� ��������� ��� ����� �������� ��� ���� ��������� ����� ����� ���� �������������� ������ ���� ���������� ��� ���� ���������� ������� �������� ������������������������� ����������������������� �� ����� ����� ��������� ���������� �������� ����� ��� ����� ��������� ��� ���� ��� ������ ����������� ������������������������������������������������������������������������ �� ���������������������������������������������������� �� �� �� ���������������������� ����� �� �� ������������������������������������������������������������������������������������������ ���� ���� ������ ������ ���� ������� ���� ��� ���������� ������ ������ ��� ���� ����� ������������ ���������� ������ ���������� ������ ���� ������ ������� �������� ��������� ����� ��� ���������������������������������������������������������������������������� �� ����������������������������������������������������������������������������������������������� ����������������������������������������������������������������� ���� ���������� ����� ���� �������� �������� ������ ������������ ����� ��������� ������������ �������� ������� ���� �������������������������������������������� �� �������������������������������������� ��������������������������������������������������������������������������������������������� �� �� �� ����� ��������� ��������� ����� ����� ���� ��������� ��� ����� ������������ �� � ���� ������� ����� ���� ���������������������������������������������������������� � ������������������������������������� ��� ������� ��������� ���� ���������� ��� ���� ������������ �������� ��� �������� ������� ���� � ����� ����� ������������������������������������������������������������������������ ����������������������������������������������������� ������ �������� ���� ���� ������������� ��� ���� �������� ������� ����� ��������� ��� ����������� ��������� �� �� ��������� ��� ��� ���� ���� ������������ ������� ���� ����� �������� ��� ��� ������� ��������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������� ���� ����� ����� ��� ��� ������� ���� ��������� ��� ��� ������������ ������� ���� ����� ������ ����������� ��������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������ ���������������������������������µ������������������������������������������������������������������ ����������������������������������������������������������������������������������������������������� �������������������������������������������������������������������������������������������������� ����� ���� ���������� �� �������� ��� ������ �������� ���� �������� ������ ���������� ��������� ��� ���� ������������������������������������������������������������������������������������������������� ���� �������� �������������������� ������������ ��� ������ ���������� ������ ���� ���������������� ����� �������� ���� ������������ ��� �������������������� �� ������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������������� ������������������ ������ ��� ����� ����������������������������� ����� ���������������������������������� ���������������������������������������������������������������������������������������������������� �������� ����� ���� ��� ������� ������� �������� �������� �������� �������� ��� ��������� ��������������� ��������������������������������������������������������������������������������������������� ������������������������������������������� ������������������������������������������������������ ��������������������������������������������������������������������������������������������� ������������������ ���������������� ����� ���������� ���� ������� ����� ����� ����� ������ ����� ��������� ��������� ���� ������� ��������� ��� ������� ����� ����������������������������������������������������������������������������������������������� ����������������������������������������µ���������������������������������������������������������� ������ ��� ���� ����� ����� ��������� ��� �� ��������� �������� ������� ���� ������� ����� ������� ����� ���� ���������������������������������������������������������������������������� �������������������������� ������������� ������������� �������� ����� ������� ������� ����� ���� ����� ��������� ������ ����������������� �� � ����������������������������������������������������������� ��� ��� ������������� ����������������������������������������������� �� �������������������������������������������� ����������������������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������������������� �������� ������� ���� ������������ ������ ����� ����� �������� ����� �� �������� �������� ������� ������������������������ ����� �������� ��� ������������ ����������� ��� ���� ������ ��� ���� ����� ���� ��������� ���� ����� ����� ����� ��� ������������� ������� ������ ��� ������� ���� ���� ���� ��� �������� ���������������������������������������������� � ������������������������������������������������ � �������� �������� �������� ������������������� �������������� ������������ ������������ ������������ �������������� �������������� ��������� ��������� ��������� ������� ����������� ����� ��������� ��������� ��������� ����������� ���� ��������� ��������� ��������� ��������������������� ����� ���� ��� � ����������������������������������������������� �������������������������� ������������������������������������������������� � �������� �������� �������� ������������������� �������������� ���������� ����������� ����������� �������������� �������������� ��������� ��������� ��������� �������������������� ����� ��������� ��������� ��������� �� ������� ���� ��������� ��������� ��������� ��������������������� ���� ���� �� � �������������������� � � � ��� �������������������� � � ������������������������������������������������������������� ������� ����������� ��������� ������� ������� ������ �����������������Δ���� �������������� ������� ������ �����������������Δ�������������������Δ������ ���� �� � ������� ������ �����������������Δ�������������������Δ������� ���� �� � �������� ������ �����������������Δ�������������������Δ�����������Δ��������� ���� �� � �������� ������ �����������������Δ��������Δ���������� �������������� �������� ������ �����������������Δ��������Δ�������� �������������� ������� ������ ���������Δ������Δ�������Δ������Δ�� ���� �� � �������� ������� ����Δ�������� ����������������� �������� ������� ����Δ������� ���� �� � �������� ������� �����Δ�������� ���� �� � ������ ������ �������������Δ������Δ�������Δ������Δ������Δ��������������������Δ� ���� �� � ������ ������ �������������Δ������Δ�������Δ������Δ�� ���� �� � � � ����Δ���������������������Δ�������������� �������� ������ �������������Δ��������������������Δ������������� ���������� � � ����Δ������������������������ �������� �������� ����Δ���������������� ���������� �������� �������� ��������������� ���������� �������� �������� ���������������� ���������� �������� �������� ��������������� ���������� �������� �������� ��������������� ���������� �������� �������� ����Δ���������������� ���������� ��������� �������� ����Δ���������������� ���������� �������� �������� ��������������� ���������� �������� �������� ��������������� ���������� �������� �������� ���������������� ���������� �������� �������� ���������������� ���������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� ��������� ������� ������������������������ ���������� �������� ������� ���������������� �������� �������� ������� ��������������� ������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� ��������� ������� ����Δ��������������� ���������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ���������������� �������� ��������� ������� ����Δ��������������� ���������� �������� ������� ���������������� �������� ��������� ������� ����Δ��������������� ���������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ����������������� �������� ��������� ������� ����Δ����������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ����������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� ��������� ������� ����Δ��������������� ���������� �������� ������� ���������������� �������� ��������� ������� ����Δ��������������� ���������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ������������������� �������� ������� ����������� ��������� ������� ��������� ������� �����Δ���������������� ���������� ������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ���������������� �������� �������� ������� ����������������� �������� �������� ������� ���������������� �������� �������� ������� ������������������ �������� �������� �������� ��������������������Δ���������������� ���������� �������� �������� ������������������������������� ���������� �������� �������� �����������������������������������Δ�������� ���������� �������� �������� ��������������������Δ���������������� ���������� �������� �������� ��������������������Δ��������������������Δ�������� ���������� �������� �������� ��������������������Δ��������������������Δ�������� ���������� ��������� ������� ����������������� ���������������� ��������� ������� ���������������������Δ�������� ���������� ��������� ������� ���������������������Δ�������� ���������� ������� ����� ��������������������������������������� ���������� � � ����������������������������������������� � ������� ������� ����Δ������ ���������� ������� ������� ����Δ������ ���������� ������� ������� ����Δ������ ���������� ������� �������� ����Δ��������� ��������� �������� ������� ����Δ�������������Δ��������� ��������� �������� ������� ����Δ�������������Δ��������� ��������� �������� ������� ����Δ�������������Δ�������������Δ��������� ��������� �������� ������� ����Δ�������������Δ�������������Δ��������� ��������� ������� ������� ����Δ��������� ��������� ������� ������� �����Δ��������� ��������� �������� ������� ����Δ�������������Δ��������� ��������� �������� ������� �����Δ�������������Δ��������� ��������� �������� ������ ����Δ����������������������� ��������� �������� �������� ����Δ����������Δ�����������������������������������������������Δ�� ��������� ������� �������� ����Δ��������� ��������� ������� �������� ����Δ��������� ��������� ������� �������� ����Δ��������� ��������� ������� �������� ����Δ��������� ��������� ������� �������� ����Δ��������� ��������� �������� �������� �������������������� ��������� �������� �������� ��������������������� ��������� �������� �������� �������������������� ��������� �������� �������� �������������������� ��������� �������� �������� �������������������� ��������� �������� �������� �������������������� ��������� �������� �������� �������������������� ��������� �������� �������� ����������������������� ��������� �������� �������� �������������������� ��������� �������� �������� �������������������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� �������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ���������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� ������� ������� ������������������� ����Δ�������� ���� ��� �������� �������� �������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������� ����Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ���������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� �������� ������������������������Δ��������� ��������� �������� ������� ����������������� ��������� �������� ������� ������������������ ��������� �������� ������� ����������������� ��������� �������� ������� ����������������� ��������� �������� ������� ���������������������Δ��������� ��������� �������� ������� ����������������������Δ��������� ��������� �������� ������� ���������������������Δ��������� ��������� �������� ������� ���������������������Δ��������� ��������� ������� ����������� ��������� ������� �������� ������� ���������������������Δ��������� ��������� �������� ������� ����������������������Δ��������� ��������� �������� ������� ���������������������Δ��������� ��������� �������� ������� ���������������������Δ��������� ��������� ������� ������� ���������������������Δ�������������Δ��������������Δ��������� ��������� ������� ������� ����������������������Δ�������������Δ��������������Δ��������� ��������� ������� ������� ���������������������Δ�������������Δ��������������Δ��������� ��������� ������� ������� ���������������������Δ�������������Δ��������������Δ��������� ��������� ������� ������� ������������������� ��������� ������� ������� ������������������� ��������� ������� �������� �����Δ��������������������������������������������������������������� ��������� ������� �������� �����Δ��������������������������������������������������������������� ��������� ������� �������� ������������ ��������� ������� �������� ������������ ��������� � � � � �������������������������������������������������������� �������� ������������ ������� ������� ���������������������������������������������������� ���� �� � ������� ����������������������������������������������������� ���� �� � ������� ���������������������������������� ���� �� � ������� ���������������������������������� ���� �� � ������ ������������������������������������������ ���� �� � ������ �����������������������������Δ��������� �������������� ������������� ������������������������ ���� �� � ������ ��������������������������������������������������������������� ���� �� �� ������������� ������������� ���� �� � ������� ������������� ���� �� � ������� ������������� ���� �� � ������ ����������������� ���� �� �������� �������������������������������������������������������������Δ�������� ��������� �������� ���������������������� ���� �� � �������� �����������Δ��������������������������������������������������������� ��������� ��������������� ��������������������������������������� ��������� ������� ��������������������������� Δ�� � ���������� ������� ���������������������������� ���������� ������� ������������������������������������ ���� � ���������� ������� ��������������������������� �� � ���������� ������� ��������������������������� ���� � ���������� ������� ��������������������������� ���� � ���������� ������� ��������������������������������� ���������� ������� �������������������������������������������� ���������� � � � ��� ����������� ��� ���������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������ �������� ��� ������������������������������������������������������������������������������������ ������������������������������������������������������������������������������������������� ��� �������������������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������� �������� ��� ����������������������������������������������������������������������������������������� ����������������������������������������������� ��� ����������������������������������������������������������������������������������������� �������� ��������������������� ��� ������������������������������������������������������������������������������������� ������������������������������ ��� ���������������������������������������������������������������������������������������������� ������������������������������� ��� ����������������������������������������������������������������������������������������� ���������������������������������������� ��� �������������������������������������������������������������������������������������������� ������������ ���� ���������������������������������������������������������������������������������������� ������������ ���� ������������������������������������������������������������������������������������������� ��������������������� ���� ��������� ��������������������������������������������������������������������������������� �������������������������������������������������������������������������������������� ���� ����������������������������������������������������������������������������������������� ������������������������������������������������������� ���� ���������������������������������������������������������������������������������������� ������������������������������������������������������������������� ���� ����������������������������������������������������������������������������������������������� ��������������������� ���� ��������������������������������������������������������������������������������� ������������������������������������������������������������������������������������ ���� ��������������������������������������������������������������������������������������� ������������������������������������������������������������� ���� ������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������� ���� ��������������������������������������������������������������������������������������� �������������������������������������������������������� ���� ������������������������������������������������������������������������������������������������ �������������������������������������������������������������������������� ���� ������������������������������������������������������������������������������������ �������������������������������������������������������������� ���� ���������������������������������������������������������������������������������������� ��������������������������������������������������������������� ����������������������������������������������������������������������������������������� ���� ������������������������������������������������������������������������������������������� ������������������������������������ ���� ���������������������������������������������������������������������������������������������� ������������������������������������������������� ���� ���������������������������������������������������������������������������������������� ����������������������������������� ���� ����������������������������������������������������������������������������������������� ������������������������������������������������� ���� ��������������������������������������������������������������������������������� ����������������������������������������������������� ���� ���������������������������������������������������������������������������������������������� ����������������������������������������������������� ���� ������������������������������������������������������� �������������������������������� ������������������ ���� �������������������������������������������������������������������������������������������� ����������������������������������������������������������������� ���� ��������������������������������������������������������������������������������������������� ���� ��������������������������������������������������������������������������������������������� ���������������������������������������������������������������������������������������� ���� ��������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������� ���� ����������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������� ������������� ���� ������������ ������������������������������������������������������������������������ ��������������������������������������������������������������������������� ���������� ������������������������������������������� ���� ��������������������������� �������������������������������������������������������������������� ������������������������������������������������������������������������������������������ �������� ���� ���������������������������������������������������������������������������������������������� ������������������������������������������������������������������� ���� ���������������������������������������������������������������������������������� ���� ������������������������������������������ ���������������������������������������������� ������������������������������������������������������������������������������������ ���� ���������������������������������������������������������������������������������������� ����������������������������������������������������������������������������������� ���� �������������������������������������������������������������������������������������������� ������������������������������������������������������������������������������������������������ ������������������������������������ ���� ��������������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������� ���� �������������������������������������������������������������������������������������������� ���������������������������������������� ���� ������������������������������������������������������������������������������������������ ����������������������������������������������������������������������������������������� �������������������������������������� ���� ������������������������������������������������������������������������������������������ �������������������������������������������������������������������������������������� ���������������������������������������������������������������������� ���� ���������������������������������������������������������������������������������������� ��������������������������������������������������������������������������������������� ���������������������������������������� �

8 /

5 D E C E M B E R 2 0 1 4 | V O L 5 1 6 | N A T U R E | 4 1 1Macmillan Publishers Limited. All rights reserved ©2014

1 2 | N A T U R E | V O L

[START_REF] Mekhail | The nuclear envelope in genome organization, expression and stability[END_REF]

| 1 8 / 2 5 D E C E M B E R2 0 1 4Macmillan Publishers Limited. All rights reserved ©2014

Macmillan Publishers Limited. All rights reserved ©2014

To delete RING of Asi3-F To delete RING of Asi3-R Delete Asi1 gene and amp KAN Delete Asi1 gene and amp KAN Integration -for Asi1 deletion To clone 5'UTR of Ubc7-F To clone 5'UTR of Ubc7-R To clone 5'UTR of Rad6-F ORF and 5'UTR of Ubc7-F ORF and 5'UTR of Ubc7-R ORF and 5'UTR of Rad6-F ORF and 5'UTR of Rad6-R To clone 5'UTR of Stp1-F To clone 5'UTR of Stp1-R To clone 5'UTR of Stp2-F To clone 5'UTR of Stp2-R ORF and 3'UTR of Stp1-F ORF and 3'UTR of Stp1-R ORF and 3'UTR of Stp2-F ORF and 3'UTR of Stp2-R To clone Asi1-F To clone Asi1-R To clone Asi2-F To clone Asi2-R Acknowledgements We thank M. Lemberg, E. Schiebel and B. Bukau for support and discussions, A. Kaufmann, C.-T. Ho, A. Bartosik and B. Besenbeck for help with tFT library construction, K. Ryman for the qRT-PCR analysis of gene expression, M. Hochstrasser for strains, the GeneCore and the media kitchen facilities of the European Molecular Biology Laboratory (EMBL) and Donnelly Centre for support with infrastructure and media. This work was supported by the Sonderforschungsbereich 1036 (SFB1036, TP10) from the Deutsche Forschungsgemeinschaft (DFG) (M.K.), the Swedish Research Council grant VR2011-5925 (P.O.L.), INSERM and grants from ANR (ANR-12-JSV8-0003-001) and Biosit (G.R.), fellowships from the European Molecular Biology Organization (EMBO ALTF 1124-2010 and EMBO ASTF 546-2012) (A.K.) and fellowships from the Ministe `re de la Recherche et de l'Enseignement Supe ´rieur and La Ligue Contre le Cancer (E.B.). M.K. received funds from the CellNetworks Cluster of Excellence (DFG) for support with tFT library construction. W.H. acknowledges funding from the EC Network of Excellence Systems Microscopy. C.B. was supported by funds from the Canadian Institute for Advanced Research (GNE-BOON-141871), National Institutes of Health (R01HG005853-01), Canadian Institute for Health Research (MOP-102629) and the National Science and Engineering Research Council (RGPIN 204899-6).

Oligonucleotides

Table 7. Primers used in this study Primer name Description* Sequence (5'3') oEB0001 oEB0002 oEB0003 oEB0004 oEB0005 oEB0006 oEB0007 oEB0008 oEB0009 oEB0010 oEB0011 oEB0012 oEB0013 oEB0014 oEB0015 oEB0016 oEB0017 oEB0018 oEB0019 oEB0020 oEB0021 oEB0022 oEB0023 oEB0024 oEB0025 oEB0026 oEB0027 oEB0028 oEB0029 oEB0030 oEB0031 oEB0032 oEB0033 oEB0034 oEB0035 oEB0036 oEB0037 oEB0038 oEB0039 oEB0040 oEB0043 oEB0044 oEB0045 oEB0046 oEB0047 oEB0048 oEB0049 oEB0050 oEB0051 oEB0052 oEB0053 oEB0054 oEB0055 oEB0056 oEB0057 oEB0058 oEB0059 oEB0060 oEB0061 oEB0062 oEB0063 oEB0064 oEB0065 oEB0066 oEB0067 oEB0068 oEB0069 oEB0070 oEB0073 oEB0074 oEB0075 oEB0076 oEB0077 oEB0078 oEB0079 oEB0080 oEB0081 oEB0082 oEB0083 oEB0084 oEB0085 oEB0086 oEB0087 oEB0088 oEB0090 oEB0091 oEB0092 oEB0093 oEB0094 oEB0095 oEB0096 oEB0097 oEB0098 oEB0099 oEB0100 oEB0101 oEB0119 oEB0120 oEB0121 oEB0122 oEB0123 oEB0124 oEB0125 oEB0126 oEB0127 oEB0128 oEB0129 oEB0130 oEB0131 oEB0133 oEB0134 oEB0135 oEB0136 oEB0137 oEB0138 oEB0139 oEB0140 oEB0141 oEB0142 oEB0143 Plasmids Table 8. Plasmids used in this study Plasmid name Features Bacterial marker Eukaryotic marker Source pEB0001 pEB0002 pEB0003 pEB0004 pEB0005 pEB0006 pEB0007 pEB0008 pEB0013 pEB0014 pEB0015 pEB0016 pEB0017 pEB0018 pEB0022 pEB0023 pEB0024 pEB0025 pEB0026 pEB0028 pEB0029 pEB0030 pEB0031 pEB0032 pEB0033 pEB0034 pEB0035 pEB0036 pEB0037 pEB0038 pEB0039 pEB0040 pEB0041 pEB0042 pEB0043 pEB0044 pEB0045 pEB0046 pEB0047 pEB0048 pEB0049 pEB0050 pEB0051 pEB0052 pEB0054 pEB0055 pEB0056 pEB0057 pEB0058 pEB0059 pEB0060 pEB0073 pEB0074 pEB0075 pEB0076 pEB0080 pEB0085 pEB0086 pFA6a

ssy5::HIS asi3(ΔRING)-VN::KAN BY4741 ssy5::HIS ubc6::NAT asi3(ΔRING)-VN::KAN scEB0115 asi1::ASI1-VN-ura3 ubc6::VC-UBC6-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 rad6::RAD6-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 cdc34::CDC34-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc1::VC-UBC1-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc5::UBC5-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc8::UBC8-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc13::UBC13-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc10::UBC10-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc7::UBC7-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc11::UBC11-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc9::UBC9-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc4::UBC4-VC-natMX4 ubc6::KAN scEB0115 asi3::ASI3-VN-ura3 ubc12::UBC12-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 rad6::RAD6-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 cdc34::CDC34-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc1::VC-UBC1-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc5::UBC5-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc8::UBC8-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc13::UBC13-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc10::UBC10-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc7::UBC7-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc11::UBC11-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc9::UBC9-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc4::UBC4-VC-natMX4 ubc6::KAN scEB0115 asi1::ASI1-VN-ura3 ubc12::UBC12-VC-natMX4 BY4741 asi3::asi3(ΔRING)-VN-NAT

limitations of the Y2H method have been extensively reviewed by Semple and colleagues (2002).

Various methodologies exist for PPIs visualization in living cells, which minimalize perturbation of the natural cellular environment. The most prominent are Förster resonance energy transfer (FRET) [START_REF] Förster | Transfer mechanisms of electronic excitation[END_REF], bioluminescence resonance energy transfer (BRET) [START_REF] Xu | A bioluminescence resonance energy transfer (BRET) system: application to interacting circadian clock proteins[END_REF][START_REF] Pfleger | Bioluminescence resonance energy transfer (BRET) for the real-time detection of protein-protein interactions[END_REF][START_REF] Pfleger | Analysis of protein-protein interactions using Bioluminescence Resonance Energy Transfer[END_REF] fluorescence cross-correlation spectroscopy (FCCS) [START_REF] Bacia | Fluorescence cross-correlation spectroscopy in living cells[END_REF] and protein fragment complementation assays (PCAs), including the approach based on the use of fluorescent proteins such as the bimolecular fluorescence complementation (BiFC) (Hu et al., 2002), or luminescent reporter proteins for example luciferases [START_REF] Paulmurugan | Noninvasive imaging of protein-protein interactions in living subjects by using reporter protein complementation and reconstitution strategies[END_REF][START_REF] Stefan | Quantification of dynamic protein complexes using Renilla luciferase fragment complementation applied to protein kinase A activities in vivo[END_REF].

for which we did not detect any interaction is Ubc11, which is also the sole E2 that we could not detect by Western blot. Still, this protein must be slightly expressed since we could detect a weak BiFC interaction with Uba1. Finally, this that we found more interactions with highly expressed E2s could also be due to the fact that some E2s such as Ubc13 are expressed at a level that tends to produce non-specific interactions. We therefore need to be very careful in the way we interpret our data.

In addition to the sensitive detection of E2/E3 interactions, BiFC enables us to visualize individual cell compartments, therefore providing information on interaction localization. However it does not enable a real-time detection of BiFC complex formation due to the slow fluorophore maturation (Hu et al., 2002). The localization of the BiFC signal may therefore not always reflect the initial subcellular interaction localization, as this may develop later in a different region, especially in the case of dynamic protein complexes [START_REF] Sung | Genomewide bimolecular fluorescence complementation analysis of SUMO interactome in yeast[END_REF]. It cannot be excluded that similarly to all other recombinant protein tagging approaches, BiFC fragments may alter the localization and/or the function of the fused partner proteins. It therefore is important to compare the subcellular localization of tagged versus untagged proteins [START_REF] Vidi | Fluorescent and bioluminescent protein-fragment complementation assays in the study of G protein-coupled receptor oligomerization and signaling[END_REF]. Despite these limitations the fact that BiFC enables to detect subcellular localization of proteins can help to select interesting E2/E3 pairs for further investigation and can give some clues on their function. The prominent nuclear rim localization of the Ubc6/Asi3 BiFC signal caught our attention and is one of the observations that prompted us to further investigate this interaction. Similarly, the fact that Pex10, but not its partners Pex2 and Pex12, produces a strong BiFC signal with Ubc13 at the vacuole is intriguing and suggests that the Ubc13/Pex10 pair could have a specific function in this compartment.

The BiFC screen for E2/E3 interactions enabled us to identify numerous new putative E2/E3 pairs. Among those interactions we could demonstrate, thanks to collaboration with the Knop and Ljungdahl laboratories, that the Asi-complex proteins Asi1 and Asi3 functionally interact with Ubc6 and Ubc7 and this led to the identification of a new quality control pathway at the inner nuclear membrane. Because BiFC can give false positive results, it is however very difficult to say how many of the other newly E2/E3 interaction are really active in the cell. One way to address this question systematically would be to perform subscreens with E2s mutants designed to abolish E3 interactions. However such mutants are difficult to design since there is more

We analysed this novel function of the Asi ubiquitin ligase with ten tFT-tagged substrates. Genetic crosses with additional deletion mutants revealed the involvement of Cue1 in Asi-dependent degradation (Extended Data Fig. 4a), in agreement with our biochemical analysis (Fig. 1c).

Several Asi substrates that were reproducibly stabilized in asi1D and asi3D mutants were not stabilized in strains lacking ASI2 (Extended Data Fig. 4a), suggesting that Asi2 might function as a substrate-specific recognition factor. The Asi2-independent nature of the interaction between Asi3 and Ubc6 further supports this notion (Extended Data Fig. 4b). With the exception of Aqy2, which was not expressed during exponential growth in liquid medium, all tFT-tagged substrates localized to the endoplasmic reticulum in wild-type cells and eight of them accumulated at the nuclear rim specifically in the asi1D mutant (Fig. 3e and Extended Data Fig. 4c). This result is consistent with protein stabilization at the INM where the Asi proteins reside. Cycloheximide chase experiments with haemagglutinin epitope (HA)-tagged variants revealed substantial turnover of Vtc1, Erg11, Vcx1 and Vtc4 in wildtype cells. All four proteins were stabilized specifically in the absence of ASI1 (Fig. 3f and Extended Data Fig. 5), further validating our screening approach (Supplementary Note 1). Interestingly, Vtc1 and Vtc4 were previously shown to localize to the vacuolar membrane 22 . Both