Jagan Mohan

Thomas Wollert
email: thomas.wollert@pasteur.fr

Human Ub-like proteins as central coordinators in autophagy

Autophagy is one of the most versatile recycling systems of eukaryotic cells. It degrades diverse cytoplasmic components such as organelles, protein aggregates, ribosomes and multi-enzyme complexes. Not surprisingly, any failure of autophagy or reduced activity of the pathway contributes to the onset of various pathologies including neurodegeneration, cancer, and metabolic disorders such as diabetes or immune-diseases. Furthermore, autophagy contributes to the innate immune response and combats bacterial or viral pathogens. The hallmark of macroautophagy is the formation of a membrane sack that sequesters cytoplasmic cargo and delivers it to lysosomes for degradation. More than 40 autophagy related (ATG) proteins have so far been identified. A unique protein-conjugation system represents one of the core components of this highly elaborated machinery. It conjugates six homologous ATG8 family proteins to the autophagic membrane. In this review, we summarize the current knowledge regarding the various functions of ATG8 proteins in autophagy and briefly discuss how physical approaches and in vitro reconstitution contributed in deciphering their function.

Introduction

All eukaryotic cells need to remove superfluous or damaged components to maintain their homeostasis. Not surprisingly, failure of intracellular recycling contributes to the onset of many human diseases including cancer, neurodegeneration, diabetes, as well as diverse inflammatory disorders [START_REF] Wong | Integration of Clearance Mechanisms: The Proteasome and Autophagy[END_REF]. Two distinct mechanism work hand in hand to recycle cytosplasmic components [START_REF] Korolchuk | Mechanisms of cross-talk between the ubiquitin-proteasome and autophagy-lysosome systems[END_REF].

The ubiquitin-proteasome system takes care of folded proteins and thereby regulates protein levels and lifetimes [START_REF] Kwon | The Ubiquitin Code in the Ubiquitin-Proteasome System and Autophagy[END_REF]. The primary marker to deliver proteins to the proteasome is the small protein-tag ubiquitin (Ub), which is covalently attached to lysines of target proteins [START_REF] Swatek | Ubiquitin modifications[END_REF]. The other major recycling system, autophagy, works on an entirely different level. It transports all cytoplasmic components that cannot be degraded by the proteasome to lysosomes for degradation [START_REF] Bento | Mammalian Autophagy: How Does It Work?[END_REF]. Although various different autophagic pathways exist including chaperone mediated autophagy, microautophagy and macroautophagy, the latter is the best characterized and most versatile one.

Cargo that is targeted by macroautophagy (autophagy in the following) also acquires Ub-labels, which are, however, recognized by cargo receptors such as p62, NBR1, NDP52, OPTN, TBK1 and others [START_REF] Stolz | Cargo recognition and trafficking in selective autophagy[END_REF][START_REF] Gatica | Cargo recognition and degradation by selective autophagy[END_REF]. The major function of these receptors is tethering of cargo to autophagic membranes termed isolation membranes (IMs) or phagophores. These structures are crescentshaped double-membrane cisternae that expand around cargo to successively sequester it form the cytoplasm [START_REF] Gatica | Cargo recognition and degradation by selective autophagy[END_REF]. In order to establish tight contacts between cargo and IMs, cargo receptors need to selectively recognize and bind IMs. This is achieved through an interaction of cargo receptors with Ub-like proteins that belong to the ATG (autophagy-related) 8-family. These small proteins are covalently attached to the lipid phosphatidylethanolamine (PE) within the phagophore membrane, where they serve as interaction hubs for many binding partners [START_REF] Rogov | Interactions between Autophagy Receptors and Ubiquitin-like Proteins Form the Molecular Basis for Selective Autophagy[END_REF]. The tight interaction of cargo and phagophore is essential for the selective and exclusive uptake of distinct cargo [START_REF] Wurzer | Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy[END_REF]. Thus, much attention has been devoted to characterize these interactions to reveal how cargo is degraded by autophagy. In terms of their localization, two populations of ATG8 proteins can be distinguished.

Those that are conjugated to the inner membrane of autophagosomes interact with cargo receptors.

The second population, which is found on the outer membrane of autophagosomes, recruit downstream ATG proteins to the phagophore and coordinate expansion, maturation and completion of autophagosomes [START_REF] Kraft | Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy[END_REF][START_REF] Nguyen | Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF][START_REF] Abreu | Conserved Atg8 recognition sites mediate Atg4 association with autophagosomal membranes and Atg8 deconjugation[END_REF][START_REF] Sanchez-Wandelmer | Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation[END_REF].

Upon starvation or under cytotoxic conditions, autophagosomes capture bulk cytoplasm independently of specific cargo. In yeast, these starvation induced autophagosomes are considerably larger then selective autophagosomes and reach diameters of up to 1 µm, leading to a strongly enhanced autophagic flux [START_REF] Baba | Two distinct pathways for targeting proteins from the cytoplasm to the vacuole/lysosome[END_REF]. Moreover, usually one autophagosomes is formed at a time and at a specific site (termed phagophore assembly site, PAS) in yeast, whereas many autophagosomes form in human cells simultaneously at various locations and their number strongly increases upon starvation. As a result, the amount of cytoplasmic material that is degraded by autophagy also increases in starved human cells. Furthermore, human starvation induced autophagosomes are 1-2 µm in diameter, whereas their selective counterparts can, depending on the size of engulfed cargo, become even larger, exceeding by far the capacity of yeast autophagosomes [START_REF] Huang | Autophagy and human disease[END_REF]. The enhanced autophagic flux, either through the formation of larger autophagosomes (yeast) or through an increase in their number (human), is essential for cells to maintain vital functions during periods of starvation.

A family of autophagy specific Ubiquitin-like proteins

A variety of ubiquitin-like modifiers including Ub, SUMO, NEDD8 as well as the autophagy specific human ATG8 proteins (hATG8s) and ATG12 regulate diverse cellular function. Although all of them share no significant sequence homology, they possess a similar fold and almost identical ternary structure. Moreover, all of them are conjugated to their targets by sequential action of E1-, E2-and E3-like enzymes [START_REF] Streich | Structural and functional insights to ubiquitin-like protein conjugation[END_REF]. The autophagy conjugation system is unique as it conjugates hATG8s not to another protein, but to the phospholipid PE within phagophores [START_REF] Kabeya | GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation[END_REF].

The first step of the cascade involves proteolytic processing of hATG8s by ATG4 proteases, which cleave the C-termini of hATG8s to expose a glycine residue which subsequently conjugated to PE [START_REF] Kabeya | LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[END_REF]. The conjugation reaction itself starts with activation of hATG8s and ATG12 by the E1enzyme ATG7 (Figure 1). This step results in the formation of a thioester bond of the C-terminal glycines of hATG8s or ATG12 with a cysteine in the catalytic center of ATG7 [START_REF] Mizushima | A protein conjugation system essential for autophagy[END_REF]. In the next step hATG8s are transferred to a cysteine within the E2-like enzyme ATG3, whereas ATG12 is transferred to ATG10 [START_REF] Yamaguchi | Structural Insights into Atg10-Mediated Formation of the Autophagy-Essential Atg12-Atg5 Conjugate[END_REF][START_REF] Taherbhoy | Atg8 Transfer from Atg7 to Atg3: A Distinctive E1-E2 Architecture and Mechanism in the Autophagy Pathway[END_REF]. Finally, ATG12 is covalently attached to ATG5 and the resulting ATG12-ATG5 conjugate functions as E3-like ligase to conjugate hATG8s to PE within the phagophore [START_REF] Hanada | The Atg12-Atg5 conjugate has a novel E3-like activity for protein lipidation in autophagy[END_REF] (Figure 1). Interestingly, ATG4 can cleave hATG8s from PE of the outer membrane of completed autophagosomes to recycle them [START_REF] Sanchez-Wandelmer | Atg4 proteolytic activity can be inhibited by Atg1 phosphorylation[END_REF].

Yeast cells express only a single Atg8 variant, whereas in human cells six ATG8-homologs are expressed. These are broadly divided into the two subfamilies GABARAP (GABA type A receptor associated protein) including GABARAP, GABARAPL1 and GABARAPL2 (also known as GATE-16) as well as MAP1LC3 (microtubule associated protein light chain 3) with its members LC3A, LC3B and LC3C [START_REF] Shpilka | Atg8: an autophagy-related ubiquitin-like protein family[END_REF]. Thus, higher eukaryotes appear to have diversified Atg8-functions consistent with the observation that these hATG8s promote different steps in autophagy [START_REF] Weidberg | LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis[END_REF].

Members of the LC3 subfamily are thought to function during phagophore expansion whereas GABARABs promote later steps in autophagy, including fusion of autophagosomes with lysosomes [START_REF] Wang | GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion[END_REF][START_REF] Nguyen | Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF]. The covalent attachment of hATG8s to PE within phagophore membranes, particularly the correlation of the amount of LC3B on phagophores with autophagosome biogenesis, made LC3B the primary maker for autophagosomes in human cells [START_REF] Kabeya | GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation[END_REF][START_REF] Kabeya | LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[END_REF]. Almost all hATG8 interaction partners, in particular cargo receptors and members of the autophagy core machinery, but also canonical membrane trafficking regulators such as RAB proteins, possess a conserved motif called Atg8 interaction motif (AIM, yeast) or LC3 interacting region (LIR, human) [START_REF] Noda | Atg8-family interacting motif crucial for selective autophagy[END_REF]. The interactions of LIR containing proteins with hATG8s thus coordinate all aspects of autophagy biogenesis, including selection and recruitment of cargo [START_REF] Lamark | Regulation of selective autophagy: the p62/SQSTM1 paradigm[END_REF],

phagophore initiation [START_REF] Alemu | ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs[END_REF], expansion and maturation as well as trafficking of autophagosomes and fusion with lysosomes [START_REF] Albanesi | GABARAP-mediated targeting of PI4K2A/PI4KIIatα autophagosomes regulates PtdIns4P-dependent autophagosome-lysosome fusion[END_REF]. The signature sequence of the LIR involves an aromatic amino acid followed by two random amino acids and a branched chain amino acid (W/F/Y-X1X2-L/I/V) [START_REF] Birgisdottir | The LIR motifcrucial for selective autophagy[END_REF]. The conserved hydrophobic residues are binding two hydrophobic pockets within the LIR binding site of hATG8s (Figure 2). Moreover, flanking acidic amino acids upstream of the LIR core region provide electrostatic interactions with the N-terminal helix of hATG8s. Such electrostatic interactions are particularly important for F-type LIR motifs [START_REF] Birgisdottir | The LIR motifcrucial for selective autophagy[END_REF], which bear an inherently weaker affinity to hATG8s compared to W-type motifs [START_REF] Rozenknop | Characterization of the interaction of GABARAPL-1 with the LIR motif of NBR1[END_REF]. Furthermore, binding of certain cargo receptors such as optineurin to hATG8s is regulated through phosphorylation of serine or threonine residues that are flanking the core LIR motif which results in an enhanced interaction [START_REF] Wild | Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth[END_REF].

The Swiss army knives in authophagy: hATG8s

The Ub-like conjugation system comprises sixteen gene products which altogether coordinate the conjugation of the six hATG8s to the phagophore [START_REF] Dikic | Mechanism and medical implications of mammalian autophagy[END_REF]. Thus, from an evolutionary point of view, hATG8s and their conjugation to autophagic membranes might be considered as key step in autophagy. Furthermore, hATG8s are ubiquitously expressed in almost all tissues suggesting that they exert non-redundant functions which include many if not all steps in autophagy. Human ATG8s coordinate, for example, the initiation of autophagy through their interaction with MAP15/ERK8 [START_REF] Colecchia | MAPK15/ERK8 stimulates autophagy by interacting with LC3 and GABARAP proteins[END_REF] as well as by stimulating the assembly of the autophagy initiator complex composed of ULK1/2, ATG13 and FIP200 [START_REF] Alemu | ATG8 family proteins act as scaffolds for assembly of the ULK complex: sequence requirements for LC3-interacting region (LIR) motifs[END_REF]. The best understood function of hATG8s involves, however, tethering of cargo to autophagic membranes through their interaction with cargo receptors [START_REF] Birgisdottir | The LIR motifcrucial for selective autophagy[END_REF]. Some cargo receptors such as Nix, promotes tethering of specific cargo (here mitochondria) to phagophores, whereas others including p62 target many, if not all types of cargo to autophagosomes [START_REF] Geisler | PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1[END_REF][START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF][START_REF] Zaffagnini | p62 filaments capture and present ubiquitinated cargos for autophagy[END_REF]. Thus, whether a distinct combination of cargo receptors and hATG8s coordinate the selective degradation of cargo remains to be investigated. The unifying signal to degrade cargo by autophagy its ubiquitination. It is therefore also possible that regulation solely occurs on the level of ubiquitin E3-ligases and that all ubiquitinated cargo is recognized by cargo receptors and thus targeted to autophagic membranes [START_REF] Kuang | Emerging roles E3 ubiquitin ligases in autophagy[END_REF]. Another widely accepted function of hATG8s and GABARAPs in particular involves maturation of autophagosomes and their fusion with lysosomes [START_REF] Wang | GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion[END_REF][START_REF] Mcewan | PLEKHM1 Regulates Autophagosome-Lysosome Fusion through HOPS Complex and LC3/GABARAP Proteins[END_REF]. Although the precise mechanism of this fusion step still needs to be investigated, recent studies demonstrated that GABARAPs, but not LC3Cs, recruit the PtdIns [START_REF] Swatek | Ubiquitin modifications[END_REF] kinase IIa and the tethering factor PLEKHM1 to autophagosomes before fusion can occur [START_REF] Nguyen | Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF][START_REF] Wang | GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion[END_REF].

Consequently, LC3s cannot fully compensate for deletion of GABARAPs, whereas GABARAPs appear to be able to fully compensate for loss of LC3s [START_REF] Nguyen | Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF][START_REF] Szalai | Autophagic bulk sequestration of cytosolic cargo is independent of LC3, but requires GABARAPs[END_REF].

Despite the fact that ATG8s coordinate and regulate many steps in autophagy, they are not essential for the formation of autophagosomes since a reduced level of autophagy is observed in cells in which all hATG8s have been deleted. However, most autophagosomes in such cells do not properly elongate and seal, indicating that one of the major functions of ATG8s is to coordinate the elongation of phagophores and their closure [START_REF] Nguyen | Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF]. Interestingly, a much more severe autophagic phenotype is observed if components of the conjugation cascade such as ATG5 or ATG7 are deleted [START_REF] Tsuboyama | The ATG conjugation systems are important for degradation of the inner autophagosomal membrane[END_REF], indicating that these proteins have other functions in autophagy that are independent of the conjugation of ATG8-proteins.

LC3A -Microtubule-associated protein 1A/1B Light Chain 3A

Common to all hATG8s is their lipidation to membranes that are either direct precursors for the biogenesis of autophagosomes, phagophores or autophagosomes. This also holds true for LC3A [START_REF] Kabeya | GABARAP and GATE16 localize to autophagosomal membrane depending on form-II formation[END_REF]. Although the precise function of LC3A in autophagy is not well understood, accumulating experimental evidence suggests that it mainly functions in selective autophagy. Two studies showed that LC3A interacts with the cargo receptor p62 during aggrephagy as well as with NDP52 during autophagic clearance of Salmonella typhimurium (xenophagy) [START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF][START_REF] Verlhac | Autophagy receptor NDP52 regulates pathogen-containing autophagosome maturation[END_REF]. This indicates that LC3A might tether autophagic cargo to the phagophore to promote its selective clearance.

However, p62 and NDP52 do not only interact with LC3A but also with other LC3-family members such as LC3B (p62) or LC3C (NDP52), suggesting that LC3A, LC3B and LC3C either cooperate during xenophagy or exert partially redundant functions [START_REF] Muhlinen | LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy[END_REF]. Another xenophagy related function of LC3A involves its interaction with TRIM family proteins during innate immunity and antiviral autophagy [START_REF] Kimura | TRIM-mediated precision autophagy targets cytoplasmic regulators of innate immunity[END_REF]. The family member TRIM5 has, for example, been shown to act as cargo receptor by interacting with viral capsids as well as with LC3A [START_REF] Mandell | TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition[END_REF].

Moreover, TRIM proteins possess a RING E3-like domain, which functions as ubiquitin ligase [START_REF] Yamauchi | Ubiquitination of E3 ubiquitin ligase TRIM5 alpha and its potential role[END_REF]. It remains, however, to be investigated whether the interaction of TRIM proteins with LC3A and their ubiquitin ligase activity are functionally interdependent.

Furthermore, LC3A appears to promote autophagic clearance of mitochondria by interacting with mitochondrial cargo receptors. Mitophagy is an essential mechanism to remove damaged mitochondria to avoid release of reactive oxygen species or apoptosis inducing factors from the mitochondrial lumen. The best studied mechanisms of mitophagy involves the PINK1/Parkin system [START_REF] Youle | Mechanisms of mitophagy[END_REF]. The protein kinase PINK1 selectively accumulates on damaged mitochondria and recruits the E3 ligase Parkin which in turn ubiquitinates various proteins on damaged mitochondria [START_REF] Narendra | PINK1 is selectively stabilized on impaired mitochondria to activate Parkin[END_REF]. Ubiquitinated mitochondria are subsequently recognized by canonical cargo receptors such as p62 [START_REF] Lazarou | The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy[END_REF]. Recently, however, a number of Parkin independent autophagy receptors have been identified including BNIP3, NIX, FUNDC1 and FKBP8.

Interestingly, FUNDC1 interacts with various LC3 proteins including LC3A during hypoxiainduced autophagy [START_REF] Liu | Mitochondrial outer-membrane protein FUNDC1 mediates hypoxiainduced mitophagy in mammalian cells[END_REF], whereas FKBP8 binds preferentially LC3A [START_REF] Bhujabal | FKBP8 recruits LC3A to mediate Parkinindependent mitophagy[END_REF]. Thus, LC3A might be activated in response to specific environmental cues such as hypoxia to induce Parkin-independent mitophagy.

LC3B -Microtubule-associated protein 1A/1B Light Chain 3B

LC3B is the most studied and in some aspects also best understood member of the ATG8 protein family. Although first characterized as microtubule associated protein [START_REF] Mann | Molecular characterization of light chain 3. A microtubule binding subunit of MAP1A and MAP1B[END_REF], LC3B plays an important role in autophagy and its activity is required for many different steps during the biogenesis of autophagosomes. The best characterized functions of LC3B involve tethering of cargo to autophagosomes through its interactions with cargo receptors. LC3B recruits for example the cargo receptor p62/SQSTM1 to autophagosomes [START_REF] Shvets | Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes[END_REF]. This recruitment requires LC3B to be lipidated, i.e. conjugated to PE of the phagophore, suggesting that it functions as membrane anchor for ubiquitinated protein aggregates and peroxisomes [START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF]. Moreover, p62 has a strong tendency to form larger oligomers, which are themselves substrate for selective autophagy [START_REF] Zaffagnini | p62 filaments capture and present ubiquitinated cargos for autophagy[END_REF]. The recruitment of these p62-oligomers to phagophores also depends on LC3B [START_REF] Lamark | Regulation of selective autophagy: the p62/SQSTM1 paradigm[END_REF], suggesting that LC3B coordinates delivery of different substrates to autophagosomes during aggrephagy [START_REF] Lystad | Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures[END_REF].

The accumulation of protein aggregates in neurons is a hallmark for many neurodegenerative diseases. The observation that aggrephagy counteracts neurodegeneration has therefore increased the attention devoted to the formation and clearance of a specific type of protein aggregates, termed amyloids, tremendously. The onset of Hintington's disease correlates with the appearance of Htt-amyloid fibrils that are induced by abnormal expansion of its polyQ repeat [START_REF] Ravikumar | Aggregate-prone proteins with polyglutamine and polyalanine expansions are degraded by autophagy[END_REF] [START_REF] Scherzinger | Huntingtin-encoded polyglutamine expansions form amyloidlike protein aggregates in vitro and in vivo[END_REF]. Autophagic clearance of such amyloids requires a cooperation of LC3B with the autophagy receptors p62 and NBR1 [START_REF] Kirkin | A role for NBR1 in autophagosomal degradation of ubiquitinated substrates[END_REF]. Moreover, the recently identified Ub-receptor TOLLIP contains two LIR motifs that bind to LC3B to promote aggrephagy of Htt-fibrils [START_REF] Lu | Autophagic clearance of polyQ proteins mediated by ubiquitin-Atg8 adaptors of the conserved CUET protein family[END_REF]. Thus, an increasing number of studies suggest LC3B to be an important membrane anchor that mediates intimate contact of protein aggregates and amyloids to phagophores. The tethering function of LC3B requires interaction with various cargo receptors, indicating that such receptors have redundant functions. The complexity of interactions that coordinate tethering of protein aggregates and amyloids to autophagic membranes is further increased by the fact that other ATG8s are involved in this process. LC3C, for example, also interacts with NBR1 and p62 in aggrephagy. It remains to be investigated whether LC3B and LC3C are activated by different environmental or cellular cues and can partially replace each other, or whether both proteins cooperate by promoting different steps in aggrephagy.

Interestingly, both LC3B and LC3C also play an important role in xenopahgy of intracellular pathogens such as Salmonella spp. Antibacterial autophagy can be regarded as innate immune response and requires intracellular pathogens to be exposed to the cytoplasm. Damaged or ruptured pathogen-containing phagosomes expose for example otherwise inaccessible glycans to cytoplasmic proteins. Beta-galactoside moieties of such glycans are recognized by cytoplasmic galactin 8, which induces ubiquitination of bacteria and recruitment of the cargo receptor NDP52 [START_REF] Thurston | Galectin 8 targets damaged vesicles for autophagy to defend cells against bacterial invasion[END_REF]. Autophagic clearance of such pathogens is promoted by an interaction of the autophagy receptors NDP52 and Optineurin with not only LC3B, but also with LC3C. While the interaction of NDP52 with LC3C tethers bacteria to the phagophore [START_REF] Muhlinen | LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy[END_REF], the maturation of these phagophores into autophagosomes appears to depend on the interaction of NDP52 with LC3B [START_REF] Verlhac | Autophagy receptor NDP52 regulates pathogen-containing autophagosome maturation[END_REF].

Parkin-mediated mitophagy is another selective autophagy pathway that depends on LC3B.

During this process, LC3B has been shown to promote the expansion of phagophores around damaged mitochondria through its interaction with the autophagy receptors Optineurin and NDP52 [START_REF] Wong | Optineurin is an autophagy receptor for damaged mitochondria in parkin-mediated mitophagy that is disrupted by an ALS-linked mutation[END_REF]. Interestingly, the interaction of LC3B and Optineurin is phosphoregulated with phosphorylation of Optineurin strongly enhancing its affinity to LC3B [START_REF] Wild | Phosphorylation of the autophagy receptor optineurin restricts Salmonella growth[END_REF].

The function of LC3B in autophagy is, however, not only limited to cargo selection. LC3B is also involved in coordinating phagophore expansion and their maturation into autophagosomes [START_REF] Weidberg | LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis[END_REF][START_REF] Mcewan | PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins[END_REF]. The observation that LC3B accumulates on phagophores is the presumably strongest indication that LC3B coordinates autophagosome maturation. The correlation of phagophore maturation with the amount of LC3B on such membranes rendered GFP-LC3B to be one of the most widely used makers for autophagosomes [START_REF] Kabeya | LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing[END_REF][START_REF] Mizushima | In vivo analysis of autophagy in response to nutrient starvation using transgenic mice expressing a fluorescent autophagosome marker[END_REF]. GFP-LC3B is also often used to follow dynamics of autophagosome formation whereas the RFP-GFP-LC3B tandem protein is a widely used marker to track fusion of autophagosomes and lysosomes and to perform semiquantitative autophagy flux measurements [START_REF] Mizushima | Methods for monitoring autophagy[END_REF][START_REF] Mizushima | Methods in mammalian autophagy research[END_REF].

As already mentioned, knockout of all LC3 proteins does not inhibit autophagy. However, recent studies have shown that smaller autophagosomes are formed in such cells that possess in addition slower maturation rates. Thus, LC3 proteins are not essential for autophagy, but regulate autophagosome size presumably by coordinating their expansion [START_REF] Nguyen | Atg8 family LC3/GAB ARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF]. Moreover, LC3B and GABARAPL2 can promote membrane fusion in vitro. This effect depends on the N-terminal positively charged helix in both proteins, implying that LC3B and or GABARAPL2 are involved in the closure of autophagosomes or their fusion with lysosomes [START_REF] Weidberg | LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis[END_REF].

How all these macroscopic observations relate to specific molecular functions of LC3B on lipid bilayers during the initiation, formation, maturation and degradation of autophagosomes remains, however, to be elucidated. The lack of a detailed molecular characterization of LC3B in such processes is partially the result of the high degree of redundancy of LC3 proteins. This, however, not necessarily reflect physiologically relevant redundant functions, but rather the high degree of homology of ATG8s in general and LC3 or GABARAP subfamilies in particular.

LC3C -Microtubule-associated protein 1A/1B Light Chain 3C

While LC3A and LC3B are very homologous in terms of their sequence identity, LC3C is the most distant member of the LC3 subfamily and contains N-and C-terminal extensions that are not present in LC3A or LC3B. The C-terminus is, as that of the other ATG8 proteins, cleaved by ATG4 to expose the glycine residue that is subsequently conjugated to PE. Both, activation of LC3C as well as its cleavage from membranes is catalyzed by ATG4B, which is the principle ATG8-protease that processes the other hATG8s as well [START_REF] Li | Kinetics comparisons of mammalian Atg4 homologues indicate selective preferences toward diverse Atg8 substrates[END_REF][START_REF] Li | A high-throughput FRET-based assay for determination of Atg4 activity[END_REF][START_REF] Kauffman | Delipidation of mammalian Atg8-family proteins by each of the four ATG4 proteases[END_REF]. The efficiently of ATG4B towards LC3C is, however, low suggesting that LC3C might be activated by another ATG4 homolog and thus differentially regulated [START_REF] Li | A high-throughput FRET-based assay for determination of Atg4 activity[END_REF]. Given that LC3C appears to be involved in many steps in autophagy including initiation, mobilization of membranes for phagophore expansion and recognition of specific cargo during selective types of autophagy [START_REF] Weidberg | LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis[END_REF][START_REF] Stadel | TECPR2 Cooperates with LC3C to Regulate COPII-Dependent ER Export[END_REF][START_REF] Suzuki | Structural basis of the autophagy-related LC3/Atg13 LIR complex: recognition and interaction mechanism[END_REF], its independent regulation would allow cells to modulate distinct aspects of autophagy.

The first cargo receptor that was found to distinguish between different ATG8s by selectively recognizing LC3C was NDP52. Both proteins function in xenophagy of the intracellular pathogen Salmonella enterocolitica. Interestingly, LC3C binds an unusual LIR motif in NDP52, which consists of three consecutive leucine residues instead of the canonical WxxL sequence [START_REF] Muhlinen | LC3C, bound selectively by a noncanonical LIR motif in NDP52, is required for antibacterial autophagy[END_REF]. As a result of this selective interaction, LC3C recruits downstream ATGs to the phagophore to promote antibacterial xenophagy. Other proteins such as WDR81 also selectively bind LC3C using, however, a canonical LIR motif [START_REF] Liu | The BEACH-containing protein WDR81 coordinates p62 and LC3C to promote aggrephagy[END_REF]. Similarly, LC3C promotes mitophagy in a piecemeal fashion through a canonical LIR-mediated interaction with p62 [START_REF] Guerroué | Autophagosomal Content Profiling Reveals an LC3C-Dependent Piecemeal Mitophagy Pathway[END_REF]. Taken together, more and more evidence suggest that the selective interaction of LC3C with autophagy proteins is important for selective autophagy. Whether specific LC3C-receptor pairs promote different types of selective autophagy remains, however, to be investigated.

Another exciting function of LC3C is related to autophagy initiation upon starvation that occurs at the endoplasmic reticulum at special PtdIns(3)P rich domains referred to as omegasomes [START_REF] Hayashi-Nishino | A subdomain of the endoplasmic reticulum forms a cradle for autophagosome formation[END_REF]. LC3C has been found to be conjugated to ER-membranes at ER-exit sites, at which COPII vesicles usually bud off to transport proteins from the ER to the Golgi [START_REF] Barlowe | COPII: a membrane coat formed by Sec that drive vesicle budding from the endoplasmic reticulum[END_REF]. Two different functions of the ER-associated pool of LC3C have been described. One concerns efficient export of cargo from the ER by facilitating COPII coat formation on ER-exit sites. The second, autophagy related function involves an interaction of LC3C with TECPR2 that links the secretory pathway to autophagosome formation. Interestingly, COPII vesicles are involved in the formation of autophagosomes probably by delivering membranes to the phagophore [START_REF] Stadel | TECPR2 Cooperates with LC3C to Regulate COPII-Dependent ER Export[END_REF]. Whether LC3C modulates COPII budding at the ER to promote phagophore expansion remains to be shown.

GABARAP -Gamma-aminobutyric acid receptor-associated protein

Initially identified as GABA-receptor associated protein involved in the intracellular trafficking of the GABA-receptor [START_REF] Wang | GABA(A)receptor-associated protein links GABA(A) receptors and the cytoskeleton[END_REF], GABARAP regulates various steps in autophagy [START_REF] Joachim | Control of GABARAP-mediated autophagy by the Golgi complex, centrosome and centriolar satellites[END_REF]. In selective autophagy, GABARAP coordinates together with the PtdIns(3)P-binding protein ALFY the deliver p62-oligomers to phagophores. During this process, ALFY induces on the one hand the formation of p62-oligomers which in turn become substrates for autophagy. On the other hand ALFY tethers through its interaction with GABARAP p62-oligomers to phagophore membranes [START_REF] Lystad | Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures[END_REF]. But not only protein aggregates are tethered by GABARAP to phagophores. Pattern recognition proteins of the TRIM family recognize intracellular pathogens and are involved in delivering them to autophagosomes through their interaction with GABARAP. TRIM5α, for example, bind viral capsids and GABARAP to promote anti-viral xenophagy [START_REF] Mandell | TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition[END_REF].

Upon induction of nonselective autophagy, GABARAP translocate from centriolar satellites that have been identified to be intracellular GABARAP-reservoirs, to autophagosomes [START_REF] Joachim | Centriolar Satellites Control GABARAP Ubiquitination and GABARAP-Mediated Autophagy[END_REF]. One of serval functions of GABARAP in non-selective autophagy involves activation of ULK1 which in turn promotes expansion of the phagophore [START_REF] Kraft | Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy[END_REF]. GABARAP also coordinates fusion of autophagosomes and lysosomes. Such fusion reactions are driven by membrane anchored SNARE proteins. Although most SNAREs possess a C-terminal transmembrane domain that constitutively anchor them into membranes, the autophagic SNARE syntaxin (STX)17 is recruited to completed autophagosomes by inserting its hairpin-tail into the out leaflet of autophagosomes.

The fusion reaction itself is driven by an interaction of STX17 with two lysosomal SNAREs [START_REF] Tsuboyama | The ATG conjugation systems are important for degradation of the inner autophagosomal membrane[END_REF].

Before SNAREs are able to execute the fusion reaction, the two membrane compartments need to be tethered which involves tethering factors that can bridge distances of several nanometers between the opposing membranes. During autophagy, the first intimate contact of autophagosomes and lysosomes is established by the HOPS complex [START_REF] Jiang | The HOPS complex mediates autophagosome-lysosome fusion through interaction with syntaxin 17[END_REF], which itself is target of intense regulation through the small GTPase RAB7 and its effector PLEKHM1 [START_REF] Mcewan | PLEKHM1 regulates autophagosome-lysosome fusion through HOPS complex and LC3/GABARAP proteins[END_REF][START_REF] Ganley | Distinct autophagosomallysosomal fusion mechanism revealed by thapsigargin-induced autophagy arrest[END_REF]. Apart from the tethering and fusion machinery, the lipid PtdIns(4)P is involved in regulating the fusion of autophagosomes and lysosomes. PtdInsPs are selectively recognized by membrane binding domains of proteins and provide identity to membranes. Although most PtdIns(4)P is found in membranes of the Golgi, recent studies identified significant amounts of this lipid in membranes of autophagosomes and lysosomes. The autophagosomal pool of PtdIns(4)P is generated by the PtdIns(4)-kinase IIa, which is recruited to phagophores by GABARAP [START_REF] Wang | GABARAPs regulate PI4P-dependent autophagosome:lysosome fusion[END_REF]. How PtdIns(4)P regulates fusion of autophagosomes and lysosomes remains, however, to be identified.

GABARAPL1 -Gamma-aminobutyric acid receptor-associated protein like 1

The high degree of redundancy between GABARAP, GABARAPL1 and GABARAP-L2 made it difficult to correlate a specific autophagic activity with one particular member of the GABARAP subfamily. Thus, most GABARAP interaction partners have been found to bind GABARAP-L1 and -L2 as well. For example, all three GABARAPs interact with ALFY in aggrephagy, with TRIM5α in antiviral xenophagy and with HIV-1 Nef in viral budding [START_REF] Mandell | TRIM proteins regulate autophagy and can target autophagic substrates by direct recognition[END_REF][START_REF] Lystad | Structural determinants in GABARAP required for the selective binding and recruitment of ALFY to LC3B-positive structures[END_REF][START_REF] Boeske | Direct binding to GABARAP family members is essential for HIV-1 Nef plasma membrane localization[END_REF]. Whether one of the three proteins is the major interaction partner remained, however, unclear. From a structural perspective, the redundancy of GABARAPs towards their interaction partners has been traced back to a selective GABARAP interaction motif, which possesses high affinity towards all three GABARAP proteins opposed to canonical LIR motifs that bind LC3s and GABARAPs equally well [START_REF] Rogov | Structural and functional analysis of the GABARAP interaction motif (GIM)[END_REF]. Interestingly, although binding studies in vitro or in vivo were often not able to distinguish between different GABARAPs, the action of one member is under certain conditions favored over that of the others. GABARAPL1, for example, plays important roles in mitophagy and xenophagy depending on the physiological state of the cell.

The mitochondrial autophagy receptor Nix possesses a LIR motif that binds both, LC3s and GABARAPs. Mutations within this motif that abrogate LC3/GABARAP binding let, however, to a remarkable depletion of GABARAPL1 from depolarized suggesting that GABARAPL1 plays an important role in mitophagy [START_REF] Novak | Nix is a selective autophagy receptor for mitochondrial clearance[END_REF]. Moreover, damaged mitochondria release cytochrome c, which activates pro-apoptotic caspases. The proteolytic cleavage of ATG4D by caspase 3 changes the substrate specificity such that GABARAPL1 is preferentially processed.

Inhibiting the proteolytic processing of ATG4D leads to an accumulation of damaged mitochondria, thus promoting apoptosis [START_REF] Betin | Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis[END_REF]. Thus, although these data strongly suggest that GABARAPL1 possesses an anti-apoptotic function by promoting clearance of damaged mitochondria, the precise role of GABARAPL1 in this process remains to be elucidated.

GABARAPL2 -Gamma-aminobutyric acid receptor-associated protein like 2

GABARAPL2, also known as GATE-16, has previously been found to preferentially localize to Golgi membranes. Furthermore, GABARAPL2 binds the AAA-ATPase NSF, which recycles SNARE proteins after they promoted membrane fusion through formation of SNARE-pin complexes [START_REF] Sagiv | GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28[END_REF]. The autophagic function of GABARAPL2 was discovered through its high sequence homology to GABARAP, LC3s, and yeast Atg8 as well as through its ATG7-dependent activation [START_REF] Tanida | The human homolog of Saccharomyces cerevisiae Apg7p is a Protein-activating enzyme for multiple substrates including human Apg12p, GATE-16, GABARAP, and MAP-LC3[END_REF]. Together with the other members of the GABARAP subfamily, GABARAPL2 was found to act late in autophagy by promoting either sealing of autophagosomes or their fusion with lysosomes [START_REF] Weidberg | LC3 and GATE-16/GABARAP subfamilies are both essential yet act differently in autophagosome biogenesis[END_REF]. Whether its function to promote autophagosome-lysosome fusion and its interaction with NSF are interdependent remains, however, to be elucidated. Interestingly, GABARAPL2 was the only member of the GABARAP subfamily that promoted membrane tethering in vitro, further supporting the notion that GABARAPL2 participates in sealing of autophagosomes [START_REF] Weidberg | LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis[END_REF]. Furthermore, GABARAPL2 binds p62-oligomers to promote their delivery to autophagosomal membranes, implying that GABARAPL2 is involved in aggrephagy [START_REF] Shvets | Dissecting the involvement of LC3B and GATE-16 in p62 recruitment into autophagosomes[END_REF].

Another function of GABARAPL2 in selective autophagy involves its interaction with the autophagy receptor NDP52 during xenophagy. However, in contrast with the canonical function of cargo receptors in tethering substrates to autophagosomes, the interaction of GABARAPL2 with NDP52 was found to promote myosin VI dependent maturation of autophagosomes [START_REF] Verlhac | Autophagy receptor NDP52 regulates pathogen-containing autophagosome maturation[END_REF].

Physical approaches to decipher hATG8-functions

Many, if not all steps in autophagy involve at least one hATG8 protein. Moreover, siRNA and knockout studies revealed high degree of functional redundancy. GABARAP proteins can, for example, compensate for one another and for LC3s, but LC3s cannot replace GABARAPs [START_REF] Nguyen | Atg8 family LC3/GABARAP proteins are crucial for autophagosome-lysosome fusion but not autophagosome formation during PINK1/Parkin mitophagy and starvation[END_REF].

But how can these hATG8s promote on the one hand distinct functions in autophagy while being on the other hand functionally redundant? One possible answer to this question is that they are not acting alone, but recruit downstream factors with selective functions during autophagosome nucleation, expansion, maturation, sealing and fusion with lysosomes. This requires, on the other hand, that hATG8s recruit their interaction partners in a spatiotemporally coordinated and precise fashion. Many of these binding partners possess peptide motifs (LIR, CLIR, GIR), that bind into binding grooves of hATG8s. Due to the fact that the LIR-binding sites in hATG8s are structurally and electrostatically similar, many LIRs bind all hATG8s with similar affinity [START_REF] Noda | Atg8-family interacting motif crucial for selective autophagy[END_REF]. However, recently, two different types of more specific motifs, one selective for LC3C (CLIR) and the other one for GABARAPs (GIR) have been identified [START_REF] Rogov | Structural and functional analysis of the GABARAP interaction motif (GIM)[END_REF][START_REF] Muhlinen | LC3C, Bound Selectively by a Noncanonical LIR Motif in NDP52, Is Required for Antibacterial Autophagy[END_REF]. Based on the extensive characterization of theses motifs, peptide-sensors have been developed which allow subcellular localizations of distinct hATG8 in cells to be studied [START_REF] Stolz | Fluorescence-based ATG8 sensors monitor localization and function of LC3/GABARAP proteins[END_REF].

Many proteins that possess LIR or related motifs belong to the family of cargo receptors. These proteins as well as their interactions with hATG8s have been well characterized, both in vivo and in vitro. The formation of protein aggregates and their interactions with the cargo receptor p62 have, for example, been reconstituted from purified components in vitro. One of these studies revealed that protein aggregates induce the formation of p62 oligomers both of which then coalesce into "aggregosomes" that are degraded by autophagy [START_REF] Zaffagnini | p62 filaments capture and present ubiquitinated cargos for autophagy[END_REF]. Earlier studies revealed that p62 also spontaneously oligomerize to form helical, filamentous structures [START_REF] Ciuffa | The Selective Autophagy Receptor p62 Forms a Flexible Filamentous Helical Scaffold[END_REF]. Moreover, these p62 filaments are themselves degraded through autophagy, which is essential to maintain proper p62levels in cells [START_REF] Geetha | Structure and functional properties of the ubiquitin binding protein p62[END_REF]. Both, p62 decorated protein aggregates as well as p62-oligomers must tightly interact with autophagic membranes to be efficiently engulfed by autophagosomes. This process depends on the interaction of p62 with hATG8s [START_REF] Pankiv | p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy[END_REF] which is, however, rather weak.

Reconstitutions using p62 oligomers and model membranes which were decorated with LC3B showed that oligomerization of p62 is important to overcome the obstacle of weak binding.

Oligomerization of p62 leads to a spatiotemporally concentration of LIR motifs, which in turn strongly facilitates membrane binding by interacting with many LC3B-molcules [START_REF] Wurzer | Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy[END_REF].

Another approach to reveal biophysical properties of ATG8 proteins in vitro involved the reconstitution of the Ub-like conjugation system in order to conjugate hATG8s to model membranes in vitro. First reconstitutions focused on the yeast system and revealed that conjugating yeast Atg8 to small unilamellar vesicles (LUVs) induces clustering and hemifusion of LUVs [START_REF] Nakatogawa | Atg8, a ubiquitin-like protein required for autophagosome formation, mediates membrane tethering and hemifusion[END_REF].

Similar studies in which human ATG8s have been chemically crosslinked to LUVs revealed identical activities of LC3 and GABARAPL2 [START_REF] Weidberg | LC3 and GATE-16 N Termini Mediate Membrane Fusion Processes Required for Autophagosome Biogenesis[END_REF]. Moreover, the yeast E3-ligase complex comprising Atg12, Atg5 and Atg16 has also been found to tether membranes [START_REF] Romanov | Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation[END_REF]. Which steps in autophagy relay on these membrane tethering activities is currently unknown. Similar reconstitutions of the yeast conjugation system also revealed a cargo receptor unrelated function Atg8. In these studies, Atg8 was found to assemble together with the E3-ligase complex into membrane scaffolds with structural characteristic that resembled those of canonical membrane coats including clathrin or COP [START_REF] Kaufmann | Molecular mechanism of autophagic membrane-scaffold assembly and disassembly[END_REF]. These studies also showed that Atg8-scaffolds are important for the expansion of autophagosomes by a yet to be investigated molecular mechanism [START_REF] Kaufmann | Scaffolding the expansion of autophagosomes[END_REF].

Taken together, in vitro reconstitutions and physical approaches contributed tremendously to our current knowledge how autophagosomes are generated and how they capture different types of cargo. However, more complex systems involving even more components of the autophagic machinery will be required to reveal further insight into the molecular mechanisms of autophagy.

Authors Contributions

J.M. and T.W. wrote the manuscript. is their C-terminal ubiquitin (Ub)-like domain shown in blue (LC3 family) and green (GABARAP family). In contrast to Ub, ATG8s process an N-terminal helix that is part of an interaction region for LIR motifs. For LC3A, LC3B, GABARAP and GABARAPL1 interactions with LIR motifs are shown as surface (ATG8s) and ball-and-stick representations (LIR motif). All images were prepared using PyMOL software (with PDB codes 3WAL: LC3A, 5D94: LC3B, 3WAM : LC3C, 1GNU: GABARAP, 2L8J: GABARAP L1 and 4CO71: GABARAP L2 for secondary structure and surface representations as well as 5cx3: LC3A FYCO1 LIR, 5yis: LC3B AnkB LIR, 5yir: GABARAP-AnkB LIR and 2l8j: GABARAPL1 NBR1-LIR for hATG8-LIR motif interactions).

Figure 1 :

 1 Figure 1: Schematic drawing of the ATG8-conjugation cascade. After priming by ATG4 (not shown) ATG8 proteins are conjugated to a cysteine in the catalytic cleft of ATG7, transferred to ATG3, and finally conjugated to PE within autophagic membranes. Two pools of ATG8, conjugated to the inner or the outer membranes of phagophores, can be distinguished.

Figure 2 :

 2 Figure 2 : Overall structural comparison of human ATG8 proteins. Common to all hATG8s

Acknowledgements 16

We thank Lisa Dempfle for comments on the manuscript.

Funding

Research in the laboratory Membrane Biochemistry and Transport was supported by the ERC grant Autophagy in vitro (638603) and the Deutsche Forschungsgemeinschaft (WO1770/2-1).