POUR OBTENIR Université De Bordeaux

Academie De L'

Des Polonaise

Sciences

Spécialité Biochimie

Par Anna

Magdalena Kabala

Sous La

Jean-Paul Di Rago

Róża Kucharczyk

M Wysocki

Mme Szczepanowska

M Becker

M Choquet

M Di Rago

THÈSE EN COTUTELLE PRÉSENTÉE

Keywords: ATP synthase, syndrome NARP, cancer, biogenese de l'ATP synthase ATP synthase, NARP syndrome, cancer, biogenesis of ATP synthase

Biogenese de l'ATP synthase mitochondriale et des dysfonctions générant des maladies

La F 1 F O -ATP synthase mitochondriale produit la majorité de l'énergie cellulaire chez les eucaryotes aérobes sous forme d'ATP par le processus des oxydations phosphorylantes.

Chez la plupart des espèces, cette enzyme possède une origine génétique double, nucléaire et mitochondriale. Dans la première partie de ce travail, je décris la construction de modèles de levure de mutations du gène mitochondrial ATP6 de l'ATP synthase découvertes chez des patients atteints de maladies neurologiques (9185T>C and 9191T>C) ou dans des tumeurs (8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C). Le gène ATP6 code une sousunité essentielle (a/6) du domaine F O de l'ATP synthase. J'ai trouvé que la mutation 9185T>C n'affecte pas l'assemblage de l'ATP synthase, mais conduit à une diminution de la vitesse de synthèse d'ATP d'environ 30%. La mutation 9191T>C empêche presque entièrement l'incorporation de la sous-unité a/6 dans l'ATP synthase. Les cinq mutations identifiées dans les tumeurs ont un effet modeste à nul, indiquant que ces mutations ne favorisent pas la tumorigenèse en affectant le processus énergétique mitochondrial, comme évoqué précédemment. J'ai ensuite étudié la régulation de la synthèse des sous-unités a/6 et 9 dans les mitochondries de levures. La sous-unité 9 est présente sous la forme d'un anneau de 10 copies qui interagit avec la sous-unité 6. Durant la catalyse, la rotation de cet anneau provoque des changements conformationnels favorisant la synthèse d'ATP dans le secteur F 1 de l'ATP synthase. Je montre que la synthèse de ces protéines est couplée à leur assemblage, de manière à ce qu'elles soient produites dans une stoechiométrie adéquate et pour éviter l'accumulation d'intermédiaires d'ATP synthase potentiellement délétères

 ............................................................................. III.3.1 Growth phenotypes of ATP9 relocation strains lacking AEP1, AEP2 and III.3.2.2 AEP1,AEP2................................................................................. III.3.3 In vivo analysis of mitochondrially encoded proteins translation in ATP25, AEP1 and AEP2 deprived strains in WT and atp9::ARG8m Mitochondria (gr. mitosthread, gr. chondrosgrain) were described as ubiquitous cytoplasmic structures in 1890 by Altmann. He compared them to bacteria living inside the cell and designated "bioblasts". Investigations of the next century showed that mitochondria are semi-autonomous organelles surrounded with two membranes. They contain their own genome (mitochondrial DNA, mtDNA) which is expressed in the organelle. Mitochondria were shown to contain respiratory chain complexes, the enzymes of the citric acid cycle, fatty acid oxidation and oxidative phosphorylation [START_REF] Ernster | Mitochondria: a historical review[END_REF]. In 1967 Peter Mitchell published his chemiosmotic hypothesis of oxidative phosphorylation [START_REF] Mitchell | Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism[END_REF]Mitchell 1967a;Mitchell and Moyle 1967b) The first comparison of mitochondria to bacteria by Altmann was not completely wrong. In 1970 this hypothesis was revived by Lynn Margulis and now we know that mitochondria have originated from once free-living α-proteobacteria from the Rickettsiales order [START_REF] Gray | Mitochondrial evolution[END_REF]. Nowadays there are two hypothesis (Fig. 1) describing eukaryogenesis, i.e. evolution of eukaryotic cell containing mitochondria [START_REF] Koonin | The origin and early evolution of eukaryotes in the light of phylogenomics[END_REF]). The first one, "the archezoan scenario", assumed that an α-proteobacterium was engulfed by a primitive amitochondrial eukaryote called archezoan. In the second one, "the symbiogenesis scenario", an archeal cell took up an α-proteobacterium which resulted in generation of the mitochondria followed by appearance of the nucleus and other components of the cell [START_REF] Koonin | The origin and early evolution of eukaryotes in the light of phylogenomics[END_REF][START_REF] Gray | Mitochondrial evolution[END_REF]. For the time being, both hypotheses seem possible.

I.1.1 Mitochondrial structure, morphology and inheritance

Mitochondria are bounded with two membranes. OM (for outer membrane) and IM (for inner membrane). The region between them is referred to as intermembrane space (IMS) (Fig. 2) [START_REF] Fritz | Connection of the mitochondrial outer and inner membranes by Fzo1 is critical for organellar fusion[END_REF][START_REF] Logan | The mitochondrial compartment[END_REF]). The OM is permeable for molecules of the size smaller than 5kDa that can pass easily through a pore channel called the porine and containing a protein importing complex (TOM -Translocase of the Outer Membrane) [START_REF] Becker | Mitochondrial protein import: from transport pathways to an integrated network[END_REF][START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]) The IM separates mitochondrial matrix from intermembrane space and is largely impermeable. It contains various transporters for metabolites referred to as MCF (Mitochondrial Carrier Family) and protein translocases (TIMtranslocase of the inner membrane) [START_REF] Becker | Mitochondrial protein import: from transport pathways to an integrated network[END_REF][START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]. It is built from about 75% of proteins and 25% of lipids and is enriched with cardiolipin. Its surface is much larger than that of outer membrane because it forms cavities into matrix called cristae. The IM contains the complexes of respiratory chain and ATP synthase.

The mitochondrial matrix is an aqueous compartment with high density of proteins (about 270 to 560g/l [START_REF] Partikian | Rapid diffusion of green fluorescent protein in the mitochondrial matrix[END_REF]). It contains particles of mtDNA, proteins needed for its expression and many enzymes participating in metabolic reactions.

Mitochondrial morphology varies between cell types and is dependent on the physiological state of the cell. Mitochondria can be elongated and form a network as the endoplasmic reticulum like in HeLa cells or be more granular like in hepatocytes (Fig. 3A) [START_REF] Collins | Mitochondria are morphologically and functionally heterogeneous within cells[END_REF]. They are also very dynamic structures. They move constantly throughout the cytoplasm along actin filaments, change their number, size and shape due to fusion and fission processes [START_REF] Okamoto | Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes[END_REF][START_REF] Scheffler | Mitochondria[END_REF]). In yeast Saccharomyces cerevisiae mitochondria form a branched tubular network (Fig. 3B andC) [START_REF] Okamoto | Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes[END_REF] which is constantly changing in response to cellular needs. On a fermentable carbon source yeast cells contain a relatively small number of mitochondria but the network becomes much larger when moved to a non-fermentable carbon source [START_REF] Westermann | Mitochondrial dynamics in model organisms: what yeasts, worms and flies have taught us about fusion and fission of mitochondria[END_REF]. In fission yeast mutants mitochondria form a big tubular network, while in the fusion yeast mutants they are fragmented (Fig. 3C). The processes of fusion and fission play a role in inheritance of mitochondria as they cannot be synthesized de novo. Fission occurs before transmission of the mitochondria from the mother to the daughter cell during cell division. In Saccharomyces cerevisiae the mitochondria are transported along actin cables to the bud and immobilized there until the end of the bud formation [START_REF] Fagarasanu | Orchestrating organelle inheritance in Saccharomyces cerevisiae[END_REF][START_REF] Westermann | Mitochondrial dynamics in model organisms: what yeasts, worms and flies have taught us about fusion and fission of mitochondria[END_REF]. In animals and plants they are transmitted maternally [START_REF] Logan | The mitochondrial compartment[END_REF]. The imbalance between fusion and fission may disrupt proper inheritance of mitochondria and mtDNA, and result in development of mitochondrial diseases [START_REF] Okamoto | Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes[END_REF][START_REF] Westermann | Mitochondrial dynamics in model organisms: what yeasts, worms and flies have taught us about fusion and fission of mitochondria[END_REF][START_REF] Zhao | Regulation of mitochondrial dynamics: convergences and divergences between yeast and vertebrates[END_REF]. Scale bars, 5µm. Based on [START_REF] Collins | Mitochondria are morphologically and functionally heterogeneous within cells[END_REF]. B: Mitochondrial network visualized by matrix-targeted GFP in MR6 strain grown on galactose (this work). C: Wild-type and mutant cells defective in fusion (fzo1Δ) and fission (dnm1Δ). Mitochondria are visualized by matrix-targeted GFP.

Bar, 5µm. Based on [START_REF] Okamoto | Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes[END_REF].

I.1.2 Functions of mitochondria

Many metabolic pathways indispensable for cell life occur in mitochondria (Fig. 4).

One of the most important metabolic pathway is the production of cellular energy in the form of ATP by oxidative phosphorylation (the process is described in details later). Mitochondria are the place of β-oxidation of fatty acids where acetyl-CoA is generated. It furnishes the tricarboxylic acid cycle (TCA, known also as the citric acid cycle or the Krebs cycle) where the precursors of other metabolic pathways, such as amino acids or heme synthesis, are produced. Heme synthesis, a part of the urea cycle, biosynthesis of lipids (for example cardiolipin) and steroidogenesis also take place in mitochondria. Another very important metabolic pathway that occurs in mitochondria is the synthesis of iron sulfur centers that serve in such process as electron transfer, redox reactions and iron homeostasis [START_REF] Scheffler | Mitochondria[END_REF][START_REF] Galluzzi | Mitochondrial control of cellular life, stress, and death[END_REF]. Mitochondria, together with the endoplasmic reticulum, participate in calcium storage and homeostasis [START_REF] Pozzan | The comeback of mitochondria to calcium signalling[END_REF][START_REF] Nicholls | The integration of mitochondrial calcium transport and storage[END_REF]. They were also shown to trigger the intrinsic pathway of programmed cell death [START_REF] Galluzzi | Mitochondrial control of cellular life, stress, and death[END_REF].

I.1.3 The mitochondrial genome

The size of mitochondrial DNA (mtDNA) varies between metazoans, plants and fungi but genetic information is similar for most of them. Human mtDNA is about 16.5 kb. It encodes 2 ribosomal RNAs, 22 tRNAs, 7 subunits of complex I (ND1-6, ND4L), 1 subunit of complex III (cytochrome b), 3 subunits of complex IV (COI, COII, COIII) and 2 subunits of ATP synthase (ATP6, ATP8). It contains also a non-coding "control region" where the origin of replication and promoter sites of transcription are localized (Fig. 5A) [START_REF] Anderson | Sequence and organization of the human mitochondrial genome[END_REF][START_REF] Scheffler | Mitochondria[END_REF].

The mtDNA of S. cerevisiae is about 78 kb. It encodes 2 ribosomal RNAs, Var1p that is a protein of small unit of the ribosome, 24 tRNAs, 9S RNA component of RNase P implicated in tRNA maturation and intron cleavage, 1 subunit of complex III (cytochrome b), 3 subunits of complex IV (COXI, COXII, COXIII), 3 subunits of ATP synthase (ATP6, ATP8, ATP9) (Fig. 5B). In some yeast strains mtDNA encodes also a subunit of mitochondrial endonuclease (ENS2) [START_REF] De Zamaroczy | The primary structure of the mitochondrial genome of Saccharomyces cerevisiae--a review[END_REF][START_REF] Foury | The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae[END_REF].

In most organisms sequences of individual mtDNA particles can differ between each other, a state known as heteroplasmy [START_REF] Chinnery | The inheritance of mitochondrial DNA heteroplasmy: random drift, selection or both?[END_REF]. In S. cerevisiae heteroplasmy is instable and after about 20 generations all particles of mtDNA are the same, i.e. in the state of homoplasmy [START_REF] Berger | Mitochondrial DNA inheritance in Saccharomyces cerevisiae[END_REF][START_REF] Scheffler | Mitochondria[END_REF]). Moreover yeast cells, as facultative aerobes, can survive mutations in mtDNA that lead to dysfunction in respiration if they are grown on fermentable carbon sources. Sometimes the mutations cause also mtDNA instability. Yeast cells with point mutations causing a respiration defect are called mit -strains, the ρ -strains have large deletions (>50%) in mtDNA sequence and ρ 0 strains are totally devoid of mtDNA.

I.1.4 Nuclear DNA-encoded mitochondrial proteins

About 99% of mitochondrial proteins are encoded in the nucleus, synthesized on cytosolic ribosomes and imported into mitochondria [START_REF] Becker | Mitochondrial protein import: from transport pathways to an integrated network[END_REF][START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]. Most of the mitochondrial proteins are synthesized with a 10 to 60 amino acid pre-sequence that is recognized by the outer membrane transport machinery (the TOM complex) and directed to the inner membrane transport machinery (the TIM23 complex).

Active transport into the matrix is driven by the pre-sequence translocase-associated motor (PAM). Once the pre-sequence reaches the matrix, it is cleaved by the mitochondrial processing peptidase (MPP). The protein either is moved into the inner membrane directly, or enters the matrix where it stays, or is directed to the inner membrane by the OXA export machinery (Fig. 6A, from [START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]). The inner membrane proteins synthesized without the pre-sequence bind carrier precursors that are directed to the TOM complex. In the intermembrane space they use small TIM chaperones and TIM22 complex to be inserted into the inner membrane (Fig. 6B, from [START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]). The proteins of the intermembrane space are characterized by cysteine-rich motives and are transported by the TOM complex in a reduced form. In the intermembrane space they are oxidized by the mitochondrial intermembrane space import and assembly (MIA) machinery which traps them inside (Fig. 6 C, from [START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]). The outer membrane proteins are β-barrel or contain an α-helical transmembrane segments. The former are transported into the intermembrane space by the TOM complex, and with help of small TIM chaperons are sorted out by the sorting and assembly machinery (the SAM complex) (Fig. 6D, from [START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]). The latter are inserted in the outer membrane directly by the mitochondrial import complex (MIM) (Fig. 6E, from [START_REF] Harbauer | The protein import machinery of mitochondria-a regulatory hub in metabolism, stress, and disease[END_REF]).

I.1.5 Respiratory chain and oxidative phosphorylation

The production of cellular energy in the form of ATP by ATP synthase is driven by proton gradient on the IM. The proton gradient is generated by complexes of respiratory chain during electron transfer to oxygen (Fig. 7). Complex I (NADH-ubiquinone oxidoreductase) transfers electrons from NADH to ubiquinone. Complex II (succinate:ubiquinone reductase) oxidizes succinate to fumarate and also uses ubiquinone as an electron acceptor. Electrons from the reduced ubiquinone are transferred to cytochrome c by complex III (the bc 1 complex, cytochrome c reductase). Complex IV (cytochrome c oxidase) catalyzes the final transport of electrons from cytochrome c to oxygen. During the electron transport complexes I, III and IV pump protons from the matrix to the intermembrane space, creating a proton gradient (Fig. 7) used by the ATP synthase to synthesize ATP from ADP and Pi [START_REF] Mitchell | Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism[END_REF]Mitchell and Moyle 1967b). Synthesized ATP is transported out of the mitochondria in exchange for ADP by adenine nucleotide translocator (ANT).

Yeast S. cerevisiae does not have complex I but two NADH dehydrogenases, located on the matrix (Ndi1p) and on the intermembrane space (Nde1p) sides of the IM (Fig. 7B). The electron activity of these proteins is not coupled to proton translocation across IM. The yeast's respiratory chain contains also present glycerol-3-phosphate dehydrogenase (Gut2p) which reduces ubiquinone and L-lactate dehydrogenase (LDH), which in turn reduces cytochrome c (Fig. 7B) [START_REF] Rigoulet | Electron competition process in respiratory chain: regulatory mechanisms and physiological functions[END_REF]. The schema made by Małgorzata Rak.

I.2 THE STRUCTURE OF F 1 F O -ATP SYNTHASE

The ATP synthase (Fig. 8) is an energy producing enzyme present in almost all organisms from prokaryotes to eukaryotes and is composed of two functional domains, F 1 and F O . Its structure is well conserved from the bacteria to humans, however the eukaryotic enzyme has some additional subunits as compared to the prokaryotic one [START_REF] Ackerman | Function, structure, and biogenesis of mitochondrial ATP synthase[END_REF]Kucharczyk, Zick et al. 2009c;[START_REF] Jonckheere | Mitochondrial ATP synthase: architecture, function and pathology[END_REF]. The table below (Table 1) presents components of ATP-synthase from bacterium Escherichia coli, yeast Saccharomyces cerevisiae and humans (Homo sapiens).

Domain Function E. coli H. sapiens S. cerevisiae

Protein Gene

F 1 Catalytic head α α α ATP1 β β β ATP2 Central stalk/ rotor γ γ γ ATP3 ε δ δ ATP16 - ε ε ATP15 F O Proton channel a a Atp6p ATP6 c c Atp9p ATP9 Peripheral stalk δ OSCP OSCP ATP5 b b Atp4p ATP4 - d d ATP7 - F 6 h ATP14 - - i/j ATP18 Stator - A6L Atp8p ATP8 - f f ATP17 F O associated proteins Dimerisation - e e ATP21/TIM11 - g g ATP20 - - k ATP19 Regulatory proteins Inhibitors of hydrolytic activity - IF1 IF 1 /Inh1p INH1 - - Stf1p STF1 - - Stf2p STF2 - - Stf3p STF3

I.2.1 The F 1 domain

F 1 domain of ATP synthase is located at matrix side of the inner mitochondrial membrane and contains two functional parts: the α/β hexamer (the catalytic head) and the central stalk (the rotor). The α/β hexamer is built of three α subunits and three β subunits in alternant arrangement. It is the catalytic center of the enzyme. The central stalk is built of subunits γ, δ and ε (Fig. 8). The N-and C-terminal parts of γ subunit protrude into the central cavity of the α/β hexamer while the rest of the protein is in contact with subunits δ and ε, and the Atp9p-ring [START_REF] Abrahams | Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria[END_REF]).

The α/β hexamer as a catalytic center contains three nucleotide binding sites in each subunit β, where each subunit is at a different conformational stage in a given moment. Each subunit β changes the conformational states in one cycle of ATP synthesis. The conformational states are: β E (empty), β HC and β DP (that bind ADP and Pi) ,and β TP (that binds ATP). These conformational changes results in ATP synthesis or ATP hydrolysis (Fig. 9) [START_REF] Boyer | The binding change mechanism for ATP synthase--some probabilities and possibilities[END_REF][START_REF] Gao | A structure-based model for the synthesis and hydrolysis of ATP by F1-ATPase[END_REF]. The changes are driven by subunit γ rotation. The rotation of γ is a result of the rotation of the Atp9p-ring which in turn is provoked by proton movement through the proton channel [START_REF] Stock | The rotary mechanism of ATP synthase[END_REF].

The catalytic head can either synthesize or hydrolyze ATP. The synthesis of ATP requires energy that is produced by the respiratory chain as the proton gradient, whereas hydrolysis does not. Hence, when the respiratory chain is not capable of maintaining the proton gradient, the inhibitory proteins (IF 1 /Inh1p and Stf1p in S. cerevisiae) fix in the catalytic sites of α/β hexamer to block the hydrolysis of ATP, in order to avoid futile consumption of ATP [START_REF] Schwerzmann | Regulation of the mitochondrial ATP synthase/ATPase complex[END_REF][START_REF] Robinson | The structure of F(1)-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF(1)[END_REF].

I.2.2 The F O domain and the peripheral stalk

F O domain is composed of subunit 9-ring (10 monomers in S. cerevisiae, 8 in mammals), subunits 6, 8, i/j and f (Fig. 8) (Rak, Zeng et al. 2009b;Kucharczyk, Zick et al. 2009c). Subunit 9 has two transmembrane α-helices with a connecting loop directed in the matrix. The loops in the ring interact with the central stalk, whereas the membrane part of Atp9 proteins contacts Atp6p. The Atp9p-ring and subunit 6 form the proton channel of ATP synthase. Subunit 6 has five transmembrane α-helices with N-terminal directed to the inner membrane space and C-terminal to the matrix. Atp6p is connected with the peripheral stalk with the membrane part of subunit 4 (Rak, Zeng et al. 2009b).

The peripheral stalk (Fig. 8) extends from the inner membrane to the top of the α/β hexamer and its function is to stabilize the connection between F 1 and the proton channel during rotation of the motor (the Atp9p-ring and the central stalk). It is built of four subunits: 4, d, h and OSCP (the oligomycin-sensitivity conferring protein). Subunit 4 has two transmembrane domains that anchor it in the inner mitochondrial membrane and make a connection with Atp6p. In the matrix, Atp4p is connected with subunits d, h and OSCP. The last one is located on the top of the catalytic head [START_REF] Walker | The peripheral stalk of the mitochondrial ATP synthase[END_REF].

As mentioned before, ATP synthesis/hydrolysis is driven by the rotation of the subunits 9 ring. This rotation is provoked by protonation and deprotonation of Glu 59 in Atp9p

and Arg 186 in Atp6p (residues in S. cerevisiae proteins), which results in the proton translocation through the inner mitochondrial membrane [START_REF] Stock | Molecular architecture of the rotary motor in ATP synthase[END_REF][START_REF] Stock | The rotary mechanism of ATP synthase[END_REF][START_REF] Walker | The ATP synthase: the understood, the uncertain and the unknown[END_REF]). The exact mechanism of this process is still unknown.

I.3 BIOGENESIS OF THE F 1 F O -ATP SYNTHASE

The F 1 F O -ATP synthase from yeast S. cerevisiae is composed of subunits encoded both in nuclear and mitochondrial genomes. Atp6p, Atp9p and Atp8p are encoded in mtDNA while the rest is encoded in nuclear DNA (nDNA). Biogenesis of the complex requires coordination of expression of genes from two genomes and preserving the non-equal stoichiometry of subunits (10 subunits 9, 3 α and β, and one of each other subunit) (Fig. 11).

I.3.1 Assembly of the F 1 domain

Five subunits of the F 1 domain in stoichiometry α 3 β 3 γδε are encoded in nDNA, synthesized on cytoplasmic ribosomes and transported to the mitochondrial matrix where they are assembled. We know three proteins needed for the assembly of the catalytic head: Atp11p, Atp12p and Fmc1p (Ackerman and Tzagoloff 1990a;Wang and Ackerman 2000a;Wang, Sheluho et al. 2000b;[START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF]. Atp12p is a chaperon for subunit α (Wang, Sheluho et al. 2000b) while Atp11p is a chaperon for subunit β (Wang and Ackerman 2000a). Atp12p and Atp11p bind to the nucleotide binding sites of α and β, respectively, to prevent them from aggregation (Wang, Sheluho et al. 2000b). It was shown that in the strains lacking ATP1 subunits β form large insoluble aggregates, and the same happens for subunit α when ATP2 is absent. A similar situation was observed in strains deleted from either ATP11 or ATP12 genes (Ackerman and Tzagoloff 1990a). During assembly of the α/β hexamer, Atp11p bound to subunit β is released in exchange for subunit α whereas Atp12p bound to subunit α is released in exchange for β (Fig. 12) (Wang, Sheluho et al. 2000b;[START_REF] Ackerman | Atp11p and Atp12p are chaperones for F(1)-ATPase biogenesis in mitochondria[END_REF]. Assembly of catalytic head requires also the presence of subunit γ as Atp12p-α and Atp11p-β accumulate in the absence of γ [START_REF] Paul | Cloning of the yeast ATP3 gene coding for the gamma-subunit of F1 and characterization of atp3 mutants[END_REF][START_REF] Ludlam | Chaperones of F1-ATPase[END_REF]. Fmc1p is needed for the F 1 assembly at elevated temperature (35 -37°C). At the nonpermissive temperature, α and β subunits aggregate in a fmc1Δ strain but not when it is grown at 30°C. The thermo-sensitive phenotype of fmc1Δ is reversed, if Atp12p is over-expressed. It was proposed that Fmc1p stabilizes Atp12p at elevated temperature [START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF].

I.3.2 Assembly of the F O domain

The subunits of F O domain in yeast S. cerevisiae are encoded in mtDNA and synthesized on mitochondrial ribosomes (see Fig. 5 and 11). Expression and assembly of these proteins require a number of factors, shown in Fig. 13.

Figure 13 -Proteins needed for F O domain biogenesis. 9subunit 9, 6subunit 6, 10aapressequence of subunit 6. Based on (Rak, Zeng et al. 2009b).

I.3.2.1 Expression of ATP9 and the Atp9p-ring assembly

ATP9 (OLI1) gene is transcribed with VAR1 and tRNA Ser (Fig. 14). This polycistron is transcribed from two initiation sites localized approximately 550bp and 630bp upstream of ATP9 ORF. The first transcript is cleaved by endonucleases to obtain mature forms of ATP9 (0.9kb) and VAR1 (1.9kb) mRNA, and tRNA Ser [START_REF] Christianson | Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase[END_REF][START_REF] Zassenhaus | Origins of transcripts of the yeast mitochondrial var 1 gene[END_REF][START_REF] Finnegan | Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase[END_REF]. So far there are three known proteins involved in regulation of expression of ATP9:

Aep1p/Nca1p, Aep2/Atp13p and Atp25 [START_REF] Ackerman | ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase[END_REF][START_REF] Payne | Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae[END_REF][START_REF] Ziaja | Nuclear control of the messenger RNA expression for mitochondrial ATPase subunit 9 in a new yeast mutant[END_REF][START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF]. All of them are encoded in nuclear DNA.

Aep1p and Aep2p (ATP Expression protein 1 and 2, respectively) were first identified in 1991 [START_REF] Payne | Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae[END_REF]) by the study of two mutants that could not grow on glycerol at 36°C (ts379 and ts1860). At the non-permissive temperature, these strains showed no synthesis of Atp9p and Atp8p, and a diminished synthesis of Atp6p. Further experiments revealed that a ts379 mutant did not produce mature ATP9 mRNA about 1h after the temperature shift while in ts1860 it was present until 5h after the temperature shift. As no changes in ATP8 and ATP6 mRNA were detected, the authors proposed that lack of Atp8p and decrease in Atp6p synthesis are secondary effects of the absence of Atp9p. The authors postulated that the mutated protein in the ts379 strain, named Aep2p, is involved in stability or maturation of ATP9 mRNA, whereas mutated protein in ts1860, named Aep1p, acts on the level of translation. Further analysis [START_REF] Payne | Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae[END_REF] showed that Aep1p has the size of ca. 59kDa. It is a hydrophilic protein with no membrane spanning domains. There are more basic than acidic residues in Aep1p indicating that it might interact with nucleic acids.

Mature Aep2p is a protein of about 58kDa arising from the 64kDa precursor [START_REF] Finnegan | The mature AEP2 gene product of Saccharomyces cerevisiae, required for the expression of subunit 9 of ATP synthase, is a 58 kDa mitochondrial protein[END_REF]. It is globally hydrophilic but contains two non-polar regions that can be membrane-spanning domains [START_REF] Ackerman | ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase[END_REF]. Another study suggested that Aep2p interacts with 5'UTR of ATP9 mRNA and plays a direct role in translation as a RNA-binding protein [START_REF] Ellis | Suppression of a nuclear aep2 mutation in Saccharomyces cerevisiae by a base substitution in the 5'-untranslated region of the mitochondrial oli1 gene encoding subunit 9 of ATP synthase[END_REF]. Both Aep1p and Aep2p have N-terminal mitochondrial targeting sequences, are expressed at very low levels and have no homology to known proteins [START_REF] Ackerman | ATP13, a nuclear gene of Saccharomyces cerevisiae essential for the expression of subunit 9 of the mitochondrial ATPase[END_REF][START_REF] Finnegan | Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase[END_REF][START_REF] Payne | Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae[END_REF][START_REF] Finnegan | The mature AEP2 gene product of Saccharomyces cerevisiae, required for the expression of subunit 9 of ATP synthase, is a 58 kDa mitochondrial protein[END_REF].

Atp25p was also studied with the use of a thermo-sensitive mutant that failed to grow on glycerol at elevated temperature. There was no Atp9p synthesis in this mutant due to lack of mature ATP9 mRNA about 4h after the temperature shift [START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF]. The predicted size of the protein was about 70kDa but detailed analysis showed that Atp25p is cleaved into fragmentsabout 30kDa N-terminal and about 35kDa C-terminal. The cleavage site was determined as serine 293. The authors claim that C-terminus of Atp25 is an inner membrane protein facing the matrix side, while the N-terminus is associated to the inner membrane facing the inner membrane space. Both parts are necessary to restore the respiratory growth of the atp25Δ strain, however they have different functions. The Cterminus is necessary to stabilize ATP9 mRNA whereas the N-terminus is a chaperon protein that promotes oligomerization of Atp9-ring [START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF].

I.3.2.2 Expression of ATP6 and ATP8

ATP6 and ATP8 genes are transcribed together with COX1 (de [START_REF] De Zamaroczy | The primary structure of the mitochondrial genome of Saccharomyces cerevisiae--a review[END_REF][START_REF] Foury | The complete sequence of the mitochondrial genome of Saccharomyces cerevisiae[END_REF]. The polycistron may also contain ENS2 gene, downstream of ATP6 and ATP8, encoding DNA endonuclease, but its presence depends on the strain (Fig. 15) [START_REF] Nakagawa | A maturase-like subunit of the sequence-specific endonuclease endo.SceI from yeast mitochondria[END_REF]. The primary transcript is cleaved twice and gives COX1 mRNA and two ATP8/ATP6 mRNAs. The first 5.2kb ATP8/ATP6 mRNA originates after cleavage at 3'end of COX1 mRNA, the second one originates from the cleavage site located 600bp downstream the 3'end of COX1 mRNA and has 4.6kb [START_REF] Beilharz | Physiological alteration of the pattern of transcription of the oli2 region of yeast mitochondrial DNA[END_REF][START_REF] Christianson | Identification of multiple transcriptional initiation sites on the yeast mitochondrial genome by in vitro capping with guanylyltransferase[END_REF][START_REF] Simon | Organization and processing of the mitochondrial oxi3/oli2 multigenic transcript in yeast[END_REF]. Both ATP8/ATP6 transcripts are produced in equal quantity and are used for translation. So far there are four known specific proteins implicated in expression of ATP6 and ATP8 genes: Nca2p, Nca3p, Aep3p and Atp22p [START_REF] Pelissier | Regulation by nuclear genes of the mitochondrial synthesis of subunits 6 and 8 of the ATP synthase of Saccharomyces cerevisiae[END_REF][START_REF] Camougrand | NCA2, a second nuclear gene required for the control of mitochondrial synthesis of subunits 6 and 8 of ATP synthase in Saccharomyces cerevisiae[END_REF][START_REF] Pelissier | NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae[END_REF][START_REF] Helfenbein | ATP22, a nuclear gene required for expression of the F0 sector of mitochondrial ATPase in Saccharomyces cerevisiae[END_REF][START_REF] Ellis | Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae[END_REF]). Nca2p and Nca3p were identified during the study of mutants that did not grow on glycerol at 14°C [START_REF] Pelissier | Regulation by nuclear genes of the mitochondrial synthesis of subunits 6 and 8 of the ATP synthase of Saccharomyces cerevisiae[END_REF]). These mutant strains had increased level of the primary transcript containing COX1/ATP8/ATP6, diminished levels of 5.2kb ATP8/ATP6 transcript in comparison with its short version and lower Atp6p and Atp8p synthesis. The mature COX1 transcript was not affected. Though the Nca2p and Nca3p proteins probably influence the maturation/stability of ATP8/ATP6 mRNA [START_REF] Pelissier | Regulation by nuclear genes of the mitochondrial synthesis of subunits 6 and 8 of the ATP synthase of Saccharomyces cerevisiae[END_REF][START_REF] Camougrand | NCA2, a second nuclear gene required for the control of mitochondrial synthesis of subunits 6 and 8 of ATP synthase in Saccharomyces cerevisiae[END_REF][START_REF] Pelissier | NCA3, a nuclear gene involved in the mitochondrial expression of subunits 6 and 8 of the Fo-F1 ATP synthase of S. cerevisiae[END_REF].

Aep3p was shown to stabilize ATP8/ATP6 transcripts. In the aep3Δ strain the level of longer transcript was severely diminished while the shorter one was not detected, although the levels of the primary transcript and mature COXI mRNA were not changed. The authors postulated also the role of Aep3p in Atp6p and Atp8p synthesis because neither of the two proteins were synthesized in the aep3Δ strain [START_REF] Ellis | Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae[END_REF]). Aep3p was also described as a component of the mitochondrial translation initiation complex, although its second function is stimulation of the yeast mitochondrial initiation factor 2 (ymIF2), a crucial factor for translation initiation, to bind unformylated Met-tRNA f Met as the first amino acid [START_REF] Lee | Yeast AEP3p is an accessory factor in initiation of mitochondrial translation[END_REF]. Moreover the Aep3 protein was shown to be a PPR protein [START_REF] Ellis | Aep3p stabilizes the mitochondrial bicistronic mRNA encoding subunits 6 and 8 of the H+-translocating ATP synthase of Saccharomyces cerevisiae[END_REF][START_REF] Lee | Yeast AEP3p is an accessory factor in initiation of mitochondrial translation[END_REF]. PPR proteins are characterized by one or more degenerated pentatricopetptide repeat motifs that bind RNA. They are believed to be involved in stabilization and processing of mitochondrial mRNA.

Atp22 was first described as a factor necessary for assembly of F O domain located in the inner membrane facing the matrix side [START_REF] Helfenbein | ATP22, a nuclear gene required for expression of the F0 sector of mitochondrial ATPase in Saccharomyces cerevisiae[END_REF]. In atp22Δ strains both ATP8/ATP6 transcripts are present, but only Atp8p is synthesized. Lack of Atp22p could be circumvented by the presence of heteroplasmy with ATP6 gene under control of COX1 5'UTR, whereas other mitochondrial genes where expressed from WT mtDNA. Hence Atp22p is concerned as a translational activator specific for Atp6p (Zeng, Hourset et al. 2007a).

There is one more factor that influence stabilization/processing of ATP8/ATP6 transcript, Nam1p. This protein is located in the mitochondrial matrix and is weakly associated to the inner membrane [START_REF] Wallis | The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix[END_REF]. It was shown to interact with mitochondrial RNA polymerase (Rpo41p) [START_REF] Wallis | The NAM1 protein (NAM1p), which is selectively required for cox1, cytb and atp6 transcript processing/stabilisation, is located in the yeast mitochondrial matrix[END_REF][START_REF] Rodeheffer | Nam1p, a protein involved in RNA processing and translation, is coupled to transcription through an interaction with yeast mitochondrial RNA polymerase[END_REF]). In the nam1 mutant, the levels of COB, COX1 and ATP8/ATP6 transcripts were diminished. In the intron-less mtDNA however, only ATP8/ATP6 are affected [START_REF] Groudinsky | The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae[END_REF]. Therefore Nam1p plays a role not only in maturation/stabilization of the intron containing transcripts but also in maturation/stabilization of ATP8/ATP6 transcript [START_REF] Groudinsky | The NAM1/MTF2 nuclear gene product is selectively required for the stability and/or processing of mitochondrial transcripts of the atp6 and of the mosaic, cox1 and cytb genes in Saccharomyces cerevisiae[END_REF].

The synthesis of Atp6 and Atp8 proteins depends also on the presence of the assembled F 1 catalytic head (Rak and Tzagoloff 2009a). The authors suggest that this control is Atp22p-dependent as over-expression of the ATP22 gene in a strain lacking F 1 restored synthesis of Atp6p and Atp8p (Rak and Tzagoloff 2009a).

I.3.3 Assembly of the F 1 F O -ATP synthase

The ATP synthase assembly is a gradual process (Fig. 16 and17). As described in section I.3.1, the F 1 domain assembles independently from the F O domain, as evidenced by the presence of F 1 in ρ -/ρ 0 cells. The Atp9p-ring is believed to assemble independently from other ATP synthase subunits [START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]Rak, Zeng et al. 2009b;[START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF]. Newly synthesized Atp9p interacts with Oxa1p (mitochondrial inner membrane insertase), which helps Atp9p insertion into the membrane. This interaction is not essential, because in the oxa1Δ mutant the ATP synthase can still assemble, however with a lower efficiency [START_REF] Jia | Oxa1 directly interacts with Atp9 and mediates its assembly into the mitochondrial F1Fo-ATP synthase complex[END_REF]). The oligomerization of the Atp9p-ring is dependent on the presence of the N-terminal part of Atp25p [START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF]). After assembly, the Atp9p-ring associates with the F 1 domain (Kucharczyk, Zick et al. 2009c;[START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF].

The assembly of the peripheral stalk was long believed to occur spontaneously (selfassembly). However, recently a complex of inner membrane proteins (INAC), composed of Ina22 and Ina17 proteins, was shown to facilitate assembly of the peripheral stalk and its assembly with F 1 [START_REF] Lytovchenko | The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo-ATP synthase[END_REF]. It is however not clear at what moment of the ATP synthase assembly the peripheral stalk interacts with the F 1 and F O domains (Fig. 16 and17).

How subunit 8 assembles in not well understood. There is a study showing that assembly of Atp8p with the Atp9p ring occurs before assembly of Atp6p [START_REF] Hadikusumo | The definition of mitochondrial H+ ATPase assembly defects in mit-mutants of Saccharomyces cerevisiae with a monoclonal antibody to the enzyme complex as an assembly probe[END_REF]. A more recent analysis demonstrates however, that Atp8p and Atp6p associate before being incorporated into the ATP synthase (see Fig. 17) [START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF]. In other models, Atp6p is believed to be the last subunit to be incorporated into ATP synthase, with the help of two proteins: Atp10 (Ackerman and Tzagoloff 1990b; Tzagoloff, [START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]) and Atp23p (Fig. 18) [START_REF] Osman | Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase[END_REF]Zeng, Neupert et al. 2007c). Atp10p is a mitochondrial matrix protein that interacts co-or post-translationally with newly synthesized Atp6p (Ackerman and Tzagoloff 1990b;[START_REF] Paul | A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10[END_REF].

Atp10p may protect Atp6p against proteases until its incorporation and/or directly intervene in the assembly with Atp9p. In the atp10Δ mutant strain, the Atp6p is rapidly degraded, but the formation of the Atp9p ring is not affected [START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]). Analysis of atp10Δ suppressors revealed that interaction between the two proteins probably involves the C-terminal part of Atp6p [START_REF] Paul | A single amino acid change in subunit 6 of the yeast mitochondrial ATPase suppresses a null mutation in ATP10[END_REF].

Subunit 6 in yeast S. cerevisiae is synthesized with a 10aa pre-sequence on the Nterminal that is cleaved during the assembly [START_REF] Michon | NH2-terminal sequence of the isolated yeast ATP synthase subunit 6 reveals post-translational cleavage[END_REF]). Atp23p is an intermembrane metalloprotease that is responsible for Atp6p maturation [START_REF] Osman | Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase[END_REF]Zeng, Neupert et al. 2007c). Furthermore, presence of Atp23p is necessary for Atp6p assembly with the Atp9p-ring [START_REF] Osman | Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase[END_REF]Zeng, Neupert et al. 2007c).

Interestingly, the cleavage of 10aa pre-sequence is not essential for Atp6p assembly. The presence of the atp23-E168Q mutation that blocks the proteolytic activity of Atp23p did not influence the assembly of the ATP synthase complex [START_REF] Osman | Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase[END_REF]Zeng, Neupert et al. 2007c). The pre-sequence is supposed to favor the interaction between Atp6p and the Atp9p-ring. In a strain encoding Atp6p lacking the pre-sequence, ATP synthase complexes were assembled but two times less efficiently than in WT (Zeng, Kucharczyk et al. 2007b).

The ATP synthase was also shown to form dimers and oligomers in the IM (Fig. 16).

The process is dependent on subunits e, g and k but is not essential for proper functioning of the enzyme (Kucharczyk, Zick et al. 2009c).

I.4. ATP SYNTHASE AND DISEASES

A number of human diseases have been associated with defects in the ATP synthase.

Some of these diseases were shown to result from mutations affecting the assembly of ATP synthase. One was found in the nuclear gene encoding Atp12, a protein involved in the assembly of the F 1 sector. A patient carrying this mutation presented with dysmorphic features and atrophies in the nervous system, enlarged liver, hypotonia and increased urinary lactate, fumarate, methylglutaconic acid and amino acids. A biochemical analysis revealed decreased activity and content of the ATP synthase in the liver and skeletal muscles, and fibroblasts. The patient died at the age of 14 months (De Meirleir, [START_REF] De Meirleir | Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12[END_REF]). In 2008, a mutation in the TMEM70 gene (c.317-2A>G) was identified in a patient with neonatal mitochondrial encephalocardiomyopathy and ATP synthase assembly deficiency [START_REF] Cizkova | TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy[END_REF]. Since then a lot of other mutations were described in this gene [START_REF] Shchelochkov | Milder clinical course of Type IV 3methylglutaconic aciduria due to a novel mutation in TMEM70[END_REF][START_REF] Cameron | Complex V TMEM70 deficiency results in mitochondrial nucleoid disorganization[END_REF][START_REF] Jonckheere | Restoration of complex V deficiency caused by a novel deletion in the human TMEM70 gene normalizes mitochondrial morphology[END_REF][START_REF] Spiegel | TMEM70 mutations are a common cause of nuclear encoded ATP synthase assembly defect: further delineation of a new syndrome[END_REF][START_REF] Tort | Screening for nuclear genetic defects in the ATP synthase-associated genes TMEM70, ATP12 and ATP5E in patients with 3methylglutaconic aciduria[END_REF][START_REF] Torraco | TMEM70: a mutational hot spot in nuclear ATP synthase deficiency with a pivotal role in complex V biogenesis[END_REF][START_REF] Atay | A novel homozygous TMEM70 mutation results in congenital cataract and neonatal mitochondrial encephalo-cardiomyopathy[END_REF]. The most common symptoms in TMEM70 patients are lactic acidosis, cardiomyopathy, dysfunctions of central nervous system and 3-methylglutaconic aciduria [START_REF] Houstek | TMEM70 protein -a novel ancillary factor of mammalian ATP synthase[END_REF]). The TMEM70 protein has no homologous in yeast S.

cerevisiae. The exact role of this protein in the assembly of the ATP synthase remains unknown [START_REF] Kratochvilova | Mitochondrial membrane assembly of TMEM70 protein[END_REF].

Diseases were also associated to mutations in genes encoding subunits of the ATP synthase. Two of these mutations were located in the nucleus. One was in the ATP5E gene encoding subunit ε (a tyrosine to cysteine change at position 12). This patient showed respiratory problems, hyperlactacidemia, 3-methylglutaconic aciduria, ataxia, exercise intolerance and muscle weakness, and severe peripheral neuropathy. The biochemical analysis revealed severely reduced amount of the ATP synthase in fibroblasts [START_REF] Mayr | Mitochondrial ATP synthase deficiency due to a mutation in the ATP5E gene for the F1 epsilon subunit[END_REF]. Another mutation was found in the ATP5A1 gene encoding subunit α (arginine to cysteine change at position 329). The patient showed severe neonatal encephalopathy and died after one week. Considerable reduction of the ATP synthase amount and activity in the patient's fibroblasts, probably arose from the impaired assembly of α/β hexamers [START_REF] Jonckheere | A complex V ATP5A1 defect causes fatal neonatal mitochondrial encephalopathy[END_REF].

Diseases were also associated to the mitochondrial ATP6 gene encoding subunit a (MTATP6) [START_REF] Jonckheere | Mitochondrial ATP synthase: architecture, function and pathology[END_REF]. Mutations 8893T>G, 8893T>C, 9176T>G/C, 9035T>C, 9185T>C and 9191T>C in the MTATP6 gene resulted in the NARP (Neurogenic Ataxia and Retinitis Pigmentosa) syndrome or maternally inherited Leigh syntdrome (MILS), the 8851T>C mutation resulted in FBSN (Familial Bilateral Stratial Sclerosis) (Kucharczyk, Zick et al. 2009c;[START_REF] Jonckheere | Mitochondrial ATP synthase: architecture, function and pathology[END_REF]. The main clinical phenotypes include progressive loss of vision, ataxia, dementia, muscle weakness, mental retardation, stroke-like episodes, neurological problems and myopathies [START_REF] Zeviani | Mitochondrial disorders[END_REF]Kucharczyk, Zick et al. 2009c). Pathogenic mutations were found also in the A6L gene (MTATP8)

encoding subunit 8, 8529G>A and 8928T>C. These mutations affect tryptophan at position 55 and result in defective assembly and decreased activity of the ATP synthase [START_REF] Jonckheere | A novel mitochondrial ATP8 gene mutation in a patient with apical hypertrophic cardiomyopathy and neuropathy[END_REF][START_REF] Ware | Infantile cardiomyopathy caused by a mutation in the overlapping region of mitochondrial ATPase 6 and 8 genes[END_REF]. In one patient the 8411A>G mutation was described, but the effect on the ATP synthase remains unknown (Mkaouar-Rebai, [START_REF] Mkaouar-Rebai | A de novo mutation in the adenosine triphosphatase (ATPase) 8 gene in a patient with mitochondrial disorder[END_REF]. A very rare mutation, the deletion ΔTA9205, was identified in two patients and resulted in improper maturation of ATP8-ATP6-COX3 transcript due to removal of the termination codon between the ATP8 and ATP6 genes [START_REF] Seneca | A mitochondrial DNA microdeletion in a newborn girl with transient lactic acidosis[END_REF][START_REF] Jesina | Diminished synthesis of subunit a (ATP6) and altered function of ATP synthase and cytochrome c oxidase due to the mtDNA 2 bp microdeletion of TA at positions 9205 and 9206[END_REF]).

I.5. YEAST MODELS OF PATHOGENIC atp6 MUTATIONS

Point mutations of the human mtDNA are often heteroplasmic, i.e. co-existing with various amounts of wild type mtDNA within cells and tissues. The severity of diseases depends on the proportion of mutated vs. wild type mtDNA. For example, a milder NARP syndrome develops, if the level of heteroplasmy of 8993T>G mutation is between 70 and 90%. Higher mutation loads result in the more severe MILS disease. Mutations of mtDNA are usually considered to be highly recessive, which may render them difficult to evaluate the degree at which they affect the mitochondrial function. Furthermore, given the high mutational rate of the mitochondrial genome and the presence of numerous family-or population-specific polymorphisms, it may be difficult to distinguish between a neutral mtDNA variant and a disease-causing mutation. Also, it has been frequently recognized that the effects of deleterious mtDNA mutations may be exacerbated by mtDNA nucleotide changes that are not pathogenic per se or by some unknown nuclear genetic background specificities [START_REF] Cai | Mitochondrial variants may influence the phenotypic manifestation of Leber's hereditary optic neuropathy-associated ND4 G11778A mutation[END_REF][START_REF] Swalwell | A homoplasmic mtDNA variant can influence the phenotype of the pathogenic m.7472Cins MTTS1 mutation: are two mutations better than one?[END_REF]).

In the absence of methods for manipulating mammalian mitochondrial genomes, yeast S. cerevisiae has been considered as an alternative model system to study specific mtDNA mutations found in patients. It is currently the only organism in which a mitochondrial genetic transformation is possible by the ballistic delivery into mitochondria of in vitro constructed mtDNA bearing mutations, followed by their integration into the WT mtDNA by homologous DNA recombination (Bonnefoy and Fox 2001b). Yeast is unable to maintain heteroplasmic DNA stably [START_REF] Okamoto | The sorting of mitochondrial DNA and mitochondrial proteins in zygotes: preferential transmission of mitochondrial DNA to the medial bud[END_REF], so it is very easy to obtain homoplasmic mtDNA and investigate the exact biochemical consequences of the mutation of interest.

R. Kucharczyk and J.-P. di Rago have used this approach to define the impact of five mutations in the mitochondrial ATP6 gene found in patients with NARP and Leigh syndromes on ATP synthase (Rak, Tetaud et al. 2007a;Kucharczyk, Rak et al. 2009a;Kucharczyk, Salin et al. 2009b;[START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF][START_REF] Kucharczyk | Defining the pathogenesis of human mtDNA mutations using a yeast model: the case of T8851C[END_REF]. Three of these mutations (9176T>G, 8851T>C and 8993T>G) severely compromised the ability of yeast to grow on the respiratory carbon source, whereas it was much less, if at all, affected by the remaining two (9176T>C and 8993T>C). In vitro, the five mutations decreased the rate of mitochondrial ATP synthesis, by 30 to >95%. The three atp6 mutants that grew well on the respiratory carbon source were much more sensitive in vivo to oligomycin, a specific inhibitor of ATP synthase, because less drug is required to reach the threshold of the ATP synthesis activity (20%) below which the yeast respiratory growth is compromised [START_REF] Mukhopadhyay | Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media[END_REF]. The possibility to prepare large amounts of yeast mitochondria allowed to investigate the impact of the 8851T>C mutation with purified ATP synthase. It revealed a block in the rotation of the enzyme motor. It is to be noted that the pathogenic character of this mutation was uncertain due to the existence of only a very limited number of known cases. Its study in yeast provided strong evidence that it was truly responsible for the severe neurological disorders and premature death, a few weeks only after birth of the patients in whom the 8851T>C mutation was found. The 9176T>G mutation was shown to affect very severely the assembly of yeast ATP synthase by preventing incorporation of Atp6p, and we presented evidence that a similar effect occurs in human 9176T>G cells (Kucharczyk, Salin et al. 2009b). Interestingly, these studies in yeast were in line with what was known about the respective severities of these five mutations in humans they affect [START_REF] De Meirleir | Bilateral striatal necrosis with a novel point mutation in the mitochondrial ATPase 6 gene[END_REF][START_REF] Dionisi-Vici | Fulminant Leigh syndrome and sudden unexpected death in a family with the T9176C mutation of the mitochondrial ATPase 6 gene[END_REF][START_REF] Baracca | Catalytic activities of mitochondrial ATP synthase in patients with mitochondrial DNA T8993G mutation in the ATPase 6 gene encoding subunit a[END_REF][START_REF] Carrozzo | The T9176G mutation of human mtDNA gives a fully assembled but inactive ATP synthase when modeled in Escherichia coli[END_REF][START_REF] Carrozzo | A mitochondrial ATPase 6 mutation is associated with Leigh syndrome in a family and affects proton flow and adenosine triphosphate output when modeled in Escherichia coli[END_REF][START_REF] Mattiazzi | The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants[END_REF][START_REF] Houstek | Mitochondrial diseases and genetic defects of ATP synthase[END_REF][START_REF] Morava | Clinical and biochemical characteristics in patients with a high mutant load of the mitochondrial T8993G/C mutations[END_REF][START_REF] Baracca | Biochemical phenotypes associated with the mitochondrial ATP6 gene mutations at nt8993[END_REF][START_REF] Cortes-Hernandez | ATP6 homoplasmic mutations inhibit and destabilize the human F1F0-ATP synthase without preventing enzyme assembly and oligomerization[END_REF].

I.6 MITOCHONDRIAL DNA MUTATIONS AND CANCER

One of the features of cancer cells is an enhanced level of lactic acid even in the presence of oxygen. The phenomenon is known as "aerobic glycolysis" or "Warburg effect" as it was first observed by Otto Warburg, who studied cancer cells. He proposed that this is a consequence of dysfunction of mitochondria (Warburg 1956a;[START_REF] Warburg | On the origin of cancer cells[END_REF]). The intense studies of cancer cells showed that mitochondria in those cells have abnormal structures and impaired metabolism [START_REF] John | Dysfunctional mitochondria, not oxygen insufficiency, cause cancer cells to produce inordinate amounts of lactic acid: the impact of this on the treatment of cancer[END_REF][START_REF] Ramanathan | Perturbational profiling of a cell-line model of tumorigenesis by using metabolic measurements[END_REF][START_REF] Chen | Oxygen consumption can regulate the growth of tumors, a new perspective on the Warburg effect[END_REF][START_REF] Galluzzi | Mitochondrial gateways to cancer[END_REF]. They produce more ROS, have altered calcium homeostasis and redox state of the cell, which change the activities of transcription factors, such as hypoxia-inducible factor 1α (HIF1α), and impair the intrinsic way of apoptosis [START_REF] Galluzzi | Mitochondrial gateways to cancer[END_REF][START_REF] Wallace | Mitochondria and cancer[END_REF]. Mutations in the genes encoding isocytrate dehydrogenase, succinate dehydrogenase and fumarate hydratase were found in various cancers. They lead to accumulation of metabolic by-products (R-enantiomer of 2hydroxyglutarate, succinate and fumarate, respectively) that were shown to have oncogenic activity [START_REF] Wallace | Mitochondria and cancer[END_REF][START_REF] Gaude | Defects in mitochondrial metabolism and cancer[END_REF].

Mitochondrial DNA mutations were found in various tumors (1736 according to [START_REF] Lu | Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis[END_REF], but it is not confirmed whether they are involved in tumorigenesis [START_REF] Copeland | Mitochondrial DNA alterations in cancer[END_REF][START_REF] Brandon | Mitochondrial mutations in cancer[END_REF][START_REF] Lu | Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis[END_REF]). Their direct impact was studied in cybrid cells, where mtDNA carrying mutations had been introduced to ρ 0 cells [START_REF] King | Human cells lacking mtDNA: repopulation with exogenous mitochondria by complementation[END_REF]. Introduction of cybrid cells that carried mutations in the mtDNA at positions 8993T>G or 9176T>G of the MTATP6 gene into nude mice, gave an advantage at the early stage of tumor growth probably by preventing apoptosis [START_REF] Shidara | Positive contribution of pathogenic mutations in the mitochondrial genome to the promotion of cancer by prevention from apoptosis[END_REF]. Moreover the PC3 prostate cancer cell line bearing the 8993T>G mutation in mtDNA generated tumors seven times larger than the same line without this mutation [START_REF] Petros | mtDNA mutations increase tumorigenicity in prostate cancer[END_REF]. The 13997G>A and 13885insC mutations in the ND6 gene caused higher metastatic potential due to enhanced ROS production [START_REF] Ishikawa | ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis[END_REF]. Human colorectal cancer cell line with the homoplasmic mtDNA mutation in ND5 gene was shown to have decreased oxygen consumption and ATP synthesis, increased lactate and ROS production, enhanced apoptotic potency and they were all dependent on glucose. Interestingly, the same cell line with heteroplasmic mtDNA with the same mutation produced less ROS, probably due to activation of antioxidant enzymes, but showed enhanced growth of the tumor while the tumor growth in homoplasmic cell line was inhibited [START_REF] Park | A heteroplasmic, not homoplasmic, mitochondrial DNA mutation promotes tumorigenesis via alteration in reactive oxygen species generation and apoptosis[END_REF]. The mitochondrial functions are, however, indispensable for cancer cells viability, as complete elimination of mtDNA reduces the growth of cancer cells on soft agar and tumor formation in nude mice [START_REF] Morais | Tumor-forming ability in athymic nude mice of human cell lines devoid of mitochondrial DNA[END_REF][START_REF] Cavalli | Diminished tumorigenic phenotype after depletion of mitochondrial DNA[END_REF]. Still, the role of mtDNA mutations in tumor development is not well known.

GOALS OF THE WORK

The first part of my work aimed at defining the consequences on yeast ATP synthase of two mutations of the mitochondrial MTATP6 gene found in patients suffering from the NARP syndrome. Although the pathogenicity of these mutations has been established, nothing was known about their impact on the ATP synthase.

I then describe the construction of yeast models of mutations in ATP6 that have been found in tumors. It has been hypothesized that these mutations may be at the origin or favor development of tumors by altering mitochondrial oxidative phosphorylation but the consequences of these mutations have never been characterized.

Yeast models of human ATP synthase can be used for the screening of drugs active against these disorders. I have investigated the suppressor mechanism of one of the hits that have been selected, chlorhexidine. My experiments aimed to determine how this drug influence the assembly of the ATP synthase in a mutant lacking a protein (Fmc1p) involved in this process.

Unexpectedly, some of the atp6 mutations causing the NARP syndrome were found to up-regulate the Atp6p synthesis. My study of these mutations reveals a mechanism by which the synthesis of Atp6p is coupled to its assembly. Finally, I present experiments aiming to better understand how the Atp9p-ring is regulated in relation to its assembly. I present evidence for a mechanism in which Atp9p regulates its own synthesis.

MATERIALS AND METHODS

II. 1 STRAINS AND CULTURE CONDITIONS II.1.1 Bacterial strains

Bacterial strains DH5α or XL1-Blue were used to amplify plasmids. Bacterial strain XL1-Gold was used to restore plasmids after ligation.

II.1.2 Yeast strains

All yeast strains used in this work are listed in Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 aep1 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp1 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp25::KanMX Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp12 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1

::HIS3 Ø Ø Ø WT (Rak, Tetaud et al. 2007b) MR6 ρ 0 Ø Mata
::HIS3 Ø Ø Ø ρ 0 (Rak, Tetaud et al. 2007b) RKY20 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p L183P (Kucharczyk, Rak et al. 2009a) RKY26 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp9::ARG8 m (Bietenhader, Martos et al. 2012) AMY10 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 pAM19 ATP9-5 2μ ρ + atp9::ARG8 m (Bietenhader, Martos et al. 2012) MR10 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6::ARG8 m (Rak,
AKY1 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p I170V this study AKY2 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p L232P this study AKY5 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p S250P (Kabala,
::KanMX Ø Ø Ø WT this study AKY7 MR6 aep2
::HIS3 aep2::KanMX Ø Ø Ø WT this study AKY8 Ø Matα kar1-1 ade2-101 ura3-52 leu2 arg8::URA3 Ø Ø Ø ρ -atp6p I170V this study AKY9 Ø Matα kar1-1 ade2-101 ura3-52 leu2 arg8::URA3 Ø Ø Ø ρ -atp6p L232P this study AKY13 Ø Matα kar1-1 ade2-101 ura3-52 leu2 arg8::URA3 Ø Ø Ø ρ -atp6p L250P (Kabala, Lasserre et al. 2014) AKY14 Ø Matα kar1-1 ade2-101 ura3-52 leu2 arg8::URA3 Ø Ø Ø ρ -atp6p L252P (Kabala, Lasserre et al. 2014) AKY27 RKY20 atp23 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp23::KanMX Ø Ø Ø ρ + atp6p L183P this study AKY28 MR6 atp23 Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-
::KanMX Ø Ø Ø WT this study AKY37 RKY20 atp1
pAM19 ATP9-5 2μ WT this study pAK19 C-terminal ATP25-HA 2μ pG29ST32 N-terminal ATP25-HA 2μ AKY140 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp25::KanMX pAM19 ATP9-5 2μ ρ + atp9::ARG8 m this study pAK19 C-terminal ATP25-HA 2μ AKY141 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp25::KanMX pAM19 ATP9-5 2μ ρ + atp9::ARG8 m this study pAK19 C-terminal ATP25-HA 2μ pG29ST32 N-terminal ATP25-HA 2μ RKY39 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p-W136R this study RKY39 1G Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p-R136I this study RKY39 5G Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p-R136G this study RKY39 6G Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p- W136R,R179I this study RKY39 16G Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p-R136K this study RKY60 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p
::HIS3 Ø Ø Ø ρ + atp6p-P163S this study RKY62 Ø Mata
::HIS3 Ø Ø Ø ρ + atp6p-K90E this study RKY66 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 Ø Ø Ø ρ + atp6p-L252P (Kabala, Lasserre et al. 2014) RKY89 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 atp25::KanMX4 pAM19 ATP9-5 2μ ρ + atp9::ARG8 m this study pRK56 ATP25-HA 2μ RKY92 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-1 CAN1 arg8::HIS3 aep1::KanMX4 pAM19 ATP9-5 2μ ρ + atp9::ARG8 m this study pRK54 AEP1 2μ RKY93 Ø Mata ade2-1 his3-11,15 trp1-1 leu2-3,112 ura3-
::KanMX4 Ø Ø Ø ρ + atp6::ARG8 m this study FG152 FG151/pAM19

II.1.3 Bacterial media

Lysogeny Broth (LB): bacto-tryptone 10 g/l, bacto-yeast extract 5 g/l, NaCl 10 g/l.

Solid medium was prepared with 15 g/l of bactoagar. Optionally antibiotic ampiciline was added to obtain final concentration of 100 g/l (LBA).

SOC: bacto-peptone 20 g/l, bacto-yeast extract 5 g/l, 10 mM NaCl, 2.5 mM KCl, 1/100 th volume of 2 M glucose. Before use 1/100 th volume of sterile filtered 1 M MgCl 2 /1 M MgSO 4 was added.

II.1.4 Yeast media

YPDA: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glucose 20 g/l, adenine 50 mg/l, for solid medium agar 20 g/l.

YPDA for -80°C: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glucose 20 g/l, adenine 50 mg/l, glycerol 250 ml/l.

YPGalA: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, galactose 20 g/l, adenine 50 mg/l, for solid medium agar 20 g/l.

YPGA: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glycerol 20 ml/l, adenine 50 mg/l, for solid medium agar 20 g/l.

DOX: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glycerol 20 ml/l, adenine 50 mg/l, for solid medium agar 20 g/l, doxycycline 40 µg/ml.

YPGEA: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glycerol 20 ml/l, ethanol 30 ml/l, adenine 50 mg/l, for solid medium agar 20 g/l.

BET: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glucose 20 g/l, adenine 50 mg/l, for solid medium agar 20 g/l, ethidium bromide 40 μg/l.

G418: bacto-yeast extract 10 g/l, bacto-peptone 10 g/l, glucose 20 g/l, adenine 50 mg/l, agar 20 g/l, G418 sulphate 200 or 250 mg/l.

Complete synthetic medium (CSM):

glucose or galactose 20 g/l or glycerol 20 ml/l, yeast nitrogen base 1.7 g/l, ammonium sulfate 5 g/l, CSM-AA (a mix of amino acids and nucleotides without selected amino acids) as recommended, supplementary amino acids of need: adenine 50 mg/l, tryptophan 50 mg/l, leucine 100 mg/l, arginine 50 mg/l, uracil 20 mg/l.

W0 without amino acids and ammonium sulfate: galactose 20 g/l, yeast nitrogen base 1.7 g/l, supplementary amino acids of need: adenine 50 mg/l, tryptophan 50 mg/l, leucine 100 mg/l, arginine 50 mg/l, uracil 20 mg/l.

BIOL3: glucose 50 g/l, yeast nitrogen base 1.67 g/l, ammonium sulfate 5 g/l, CSMleucine 0.8 g/l, sorbitol 182.7 g, adenine 40 mg/l, agar 30 g/l.

II.1.5 Culture conditions for bacteria

Bacteria cells were incubated at 37°C on plates or in liquid with shaking at 180 rpm.

Optic density (OD) at 600 nm was measured to estimate the number of bacteria cells in the cultures.

II.1.6 Culture conditions for yeast

Yeast cells were incubated at 28°C, 35°C -37°C on plates or in liquid with shaking at 150-180 rpm. Optic density (OD) at 650 nm was measured to estimate quantity of yeast cells in the cultures. 1 OD corresponds to 1.2 10 7 of cells per milliliter.

II.1.7 Preserving bacteria and yeast

Bacteria as well as yeast strains were preserved in -80°C. Bacteria in LB medium containing 50% v/v glycerol, yeast cells in YPDA medium contains 25 -50% v/v glycerol.

II.2. GENETIC TECHNIQUES

II.2.1 Preparation of competent bacterial cells for transformation

Pre-culture of bacteria was prepared in LB liquid medium at 18°C for 2 days. Fresh 250 ml of SOC medium was inoculated and incubated with shaking (225-250 rpm) at 18°C

till OD 600 reached 0.6. Cultures were kept on ice for 10 minutes and centrifuged for 5 minutes at 4°C, 2,500 g. The pellet was re-suspended in TB buffer (10 mM PIPES, 15 mM CaCl 2 , 250 mM KCl, 55 mM MnCl 2 4H 2 O, pH 6.7), put in ice for 10 minutes and re-centrifuged in the same conditions as before. The pellet was re-suspended in 20 ml freshly prepared TB-DMSO (7% DMSO in ice-cold TB), incubated on ice for 10 minutes. After that time small portions of bacterial suspensions were frozen in liquid nitrogen. Competent cells were preserved at -80°C.

II.2.2 Transformation of bacteria by heat-shock

Plasmid DNA was introduced into cells by heat shock transformation. Previously prepared competent bacterial cells were thawed in ice. About 1 µg of plasmid or ligation mixture was added to 50 µl of bacteria and incubated for 30 minutes on ice. Heat shock was obtained by moving bacteria to 42°C for 2 minutes, and again into ice for 10 minutes. After the heat shock cells were regenerated at 37°C in liquid LB medium for 1 hour and spread on LBA plate to select cells with the plasmid of interest. Colonies were visible after one night incubation at 37°C.

II.2.3 Crossbreeding of two yeasts strains

Crossbreeding of yeast occurred between strains with opposite sexual mating types (MATa and MATα). It was realized by two methods. The first one, the so-called "drop on drop", took place on a YPDA plate where 10 µl drops of each strain pre-cultured in liquid medium, were laid one on another. In the second one, about 250 µl of one strain from fresh liquid culture were spread on a YPDA plate. Another strain was transferred on the plate by replica from the plate that contained single colonies. The crossed cells were replicated on selective medias to select the clones of interest.

II.2.4 Estimation of percentage of ρ -/ρ 0 cells in a culture

Some mutations in both nuclear and mitochondrial DNA cause instability of mtDNA.

In order to verify the stability of the mitochondrial genome, percentage of ρ -/ρ 0 in cell cultures was estimated. About 100 colonies from liquid cultures were spread on YPDA plates. A fermentable source of carbon permits growth of ρ -/ρ 0 as well as ρ + cells. After appearance of single colonies they were replicated on medium permitting the growth of ρ + cells only. Strains containing WT mtDNA were replicated on YPGA plates, whereas for strains containing ARG8m auxotrophic marker introduced into mtDNA, selection medium was CSM-R. Only cells containing intact mtDNA could grow.

Identification of ρ + cells for strains with deletion of nuclear genes resulting in lack of growth on non-fermentable carbon source despite the presence of mtDNA, was achieved by crossing single colonies grown on YPDA plates with a D273.10B ρ 0 strain and by replication of diploids on the YPGA medium. Crossbreeding with the D273.10B ρ 0 strain complemented deletion in nuclear DNA but did not deliver any additional mtDNA. As a result, only cells that contained mtDNA from the analyzed strain could grow on the YPGA medium.

In each case the percentage of ρ -/ρ 0 cells was calculated between the number of colonies that grew on selective medium and the number of colonies that grew on YPDA plate.

II.2.5 Transformation of plasmid or linear DNA into yeast cells

10 7 yeast cells from the fresh liquid culture were centrifuged and washed once with sterile water. The cells were re-suspended in 360 µl of the sterile solution containing: 34%

(m/v) of polyethylene glycol 3350, lithium acetate 0.1 mol/L, denaturated DNA of salmon sperm 0.28 mg/ml, 1 -2 µg of plasmid or linear DNA. Cells were incubated at 28°C with shacking for 30 minutes in order to loosen the cell wall. Heat shock for strains presenting WT respiratory growth was done at 42°C for 20 minutes (respiratory efficient cells) or at 37°C for 10 minutes (respiratory deficient cells). After the heat shock cells were centrifuged and re-suspended in 1ml of liquid YPDA and incubated at 28°C with shaking for maximal 1 hour to regenerate them after transformation. Then the cells were centrifuged again and spread on a solid selection medium (Gietz and Schiestl 2007)

II.2.6 Deletion of yeast nuclear genes

Deletions of yeast nuclear genes were made by homologous recombination of the KanMX4 cassette into a locus of the gene of interest. Yeast bearing this gene acquired resistance to geneticin (G418). Deletion cassettes were amplified by PCR using as a matrix a total DNA isolated from strains from the Euroscarf collection of gene deletions. The KanMX4 gene was amplified with 200 -300bp flanks containing up-stream and down-stream sequences of the gene of interest by "upper" and "lower" primers (Table 4, Fig. 19).

Transformation with the cassette was performed as described above. Cells in which the KanMX4 gene was integrated into nuclear DNA were selected on glucose medium supplemented with G418 (200 µg/ml). The correct integration was verified by PCR. Total DNA from the selected clones was isolated and PCR was performed using primers: complement to internal sequence of the KanMX4 gene and to the up-stream sequence of the gene of interest before the sequence of "upper" primer used to amplify the cassette ("verif") (Fig. 19). Sequences of the primers used in the study are presented in Table 4.

II.2.7 Transformation of plasmid DNA into yeast mitochondria by the biolistic method

Plasmid encoding the mutated versions of the ATP6 gene and wild type sequence of the COX2 gene was co-transformed with the LEU2-bearing plasmid Yep351 into DFS160 ρ 0 strain by microprojectile bombardement with the use of the biolistic PDS-100/He particle delivery system (Bio-Rad) as described previously (Bonnefoy and Fox 2001b). Mitochondrial transformants were identified among leucine prototrophes by their ability to produce respiring clones when crossed to the non-respiring strain NB40-3c that contains a deletion of the mitochondrial COX2 gene.

II.2.8 Cytoduction of mitochondrial genome between two yeasts strains

Cytoduction of mitochondrial DNA into ρ 0 cells is possible thanks to the mutation of the kar1-1 in KAR1 gene. Cells with this mutation can form zygotes but cannot form a diploid nucleus. Efficiency of diploid cells in crossing with wild type KAR1 strains is 5% [START_REF] Conde | A mutant of Saccharomyces cerevisiae defective for nuclear fusion[END_REF]. Cytoplasm of two parental cells mix in the zygote, enabling the transfer of mtDNA into ρ 0 strain. After crossing of the donor strain of mtDNA bearing the kar1-1 (DFS160/60) mutation and the acceptor ρ 0 strain of desired nuclear background by the "drop on drop" method, the cells were cultured in 50 ml of YPDA liquid medium containing 5 -10% glucose for a night. Cells were transferred into fresh YPDA (5% glucose) medium twice, to separate the nucleus and mitochondria of ρ + and ρ 0 types. 1,000 cells were spread on YPDA plates at the density of 100 cells per plate and their genotypes were analyzed by replication on selection media, to identify the type of nucleus and the presence/type of mtDNA.

II.2.9 Construction of atp6-mutant strains

The QuikChange XL Site-directed Mutagenesis Kit of Stratagene, was used for the mutagenesis reaction performed on the BamHI-EcoRI fragment of the ATP6 locus cloned into pUC19 (plasmid pSDC8, see (Zeng, Kucharczyk et al. 2007b)). The mutated ATP6 fragments were liberated and ligated with pJM2 [START_REF] Steele | Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation[END_REF] cut with BamHI and EcoRI, yielding plasmids that contained the yeast mitochondrial COX2 gene as a marker for mitochondrial transformation and BamHI-EcoRI fragments of mutated ATP6 genes. The 3' part of the wild type ATP6 locus was excised with SapI and EcoRI from pSDC9 (Rak, Tetaud et al. 2007a) and ligated between the same sites of previously obtained plasmids (pRK28, pRK29, pRK27, pRK30, pRK36, pSDC14-atp6-S250P, pSDC14-atp6-L252P, Table 3), which resulted in final plasmids used for yeast transformation (pRK32, pRK33, pRK31, pAK4-6, pAK5-1, pRK37, pRK38 Table 3). The plasmids were introduced by co-transformation with the nuclear selectable LEU2 gene containing plasmid Yep351 into the  0 strain DFS160 by microprojectile bombardment with the use of the biolistic PDS-1000/He particle delivery system (Bio-Rad) as described previously (Bonnefoy and Fox 2001b).

Mitochondrial transformants ( -synthetic) were identified among the Leu+ nuclear transformants by their ability to produce respiring clones when mated to the non-respiring NB40-3c strain bearing a deletion in the mitochondrial COX2 gene. The  -synthetic clones were crossed to the atp6::ARG8m deletion strain MR10 (Rak, Tetaud et al. 2007b) for the production of clones harboring the MR10 nucleus and where the ARG8m ORF [START_REF] Steele | Expression of a recoded nuclear gene inserted into yeast mitochondrial DNA is limited by mRNA-specific translational activation[END_REF] had been replaced by recombination with the mutated atp6 genes. The + clones were identified by its arginine auxotrophy and the ability to grow on respiratory medium. The presence of the desired mutation in atp6 locus was verified by sequencing.

II.3. TECHNIQUES IN MOLECULAR BIOLOGY

II.3.1 List of plasmids used in the study

II.3.2 List of primers used in the study

Gene Sequence of primers for construction of the strains

ATP1 (YBL099W)

Upper: 5' GTAGAGATATTATACTGTCATAGCTC 3' Lower: 5' GAATAAACTAGAGGCTATTGTGG 3' Verif: 5' GGTCATATCAAATTCACATCAGAC 3'

ATP7 (YKL016C)

Upper: 5' CACTTGCTGCTTAGCGTAGGTG 3' Lower: 5' TTCACGTTCATGCTTGATGCTTG 3' Verif: 5' GATTGGATATTTGTCTTGATGCC 3'

ATP10 (YLR393W)

Upper: 5' CTCGCCACATCACTATGCAGC 3' Lower: 5' GCTGCGAACAGAGGATATGAGC 3' Verif: 5' CCACCTGACTAATTGCATTACAG 3'

ATP11 (YNL315C)

Upper: 5' GTGCCGTATCAGTAGTCGTAGAAGC 3' Lower: 5'GCTGCTGGGAGGTACTCAACATCAG 3' Verif: 5'GTACTCATCGAGCACCCTTTGC 3'

ATP12 (YJL180C)

Upper: 5' GTGTCCTGGCGTTTCTTAAGCTCAC 3' Lower: 5' CACACGGAAGCTGTATCGCACTC 3' Verif: 5' GCTAGCTGCTGATTGACCATATCC 3'

ATP15 (YPL271W)

Upper: 5' CACACTAAGTCTGGCAACGCGC 3' Lower: 5' GCGATTGGCGATGATGGGAC 3' Verif: 5' GCCATGAACTTGAGGAATTGG 3'

ATP23 (YNR020C)

Upper: 5' GTTCGTCATATGATAATGCTTGG 3' Lower: 5'GACCAAGTGTCATGTGTACGTTG 3' Verif: 5' GAACAACACACCTAGAGCACAC 3'

FMC1 (YIL098C)

Upper: 5' GCTTGATACGTTTGGACAGTAGTTC 3' Lower: 5' TACTTCATTCTGGGATGCCTATC 3' Verif: 5'GCTAGTGCCAACTCGTCGTG 3'

AEP1 (YMR064W)

Upper: 5' CGTAGCACTTTGTTGTTCCATGC 3' Lower: 5' CATTTGTCGCAACGGAATTATCTG 3' Verif: 5' GGTTCACCCGATTTCACTGG 3'

AEP2 (YMR282C)

Upper: 5' CCTTTGTACCAAATATACTGAAG 3' Lower: 5' CATCGTTTTAAAGTACAACTCC 3' Verif: 5' GCTTTACGATCCACATTCCC3'

ATP25 (YMR098C)

Upper: 5' CTAACCTCTCTTCTAATATACTTGC 3' Lower: 5' GCATTCAGGTCTGAGTAATGAGC 3' Verif: 5' GAAATGGGGTCACAATCATCC 3' KanMX 5' GGATGTATGGGCTAAATGTACG 3'

atp6-K90E

forward: 5`GCTTAAAGGACAAATTGGAGGTGAAAATTGAGGTTTATATTTCCCTATG 3' reverse: 5'CATAGGGAAATATAAACCTCAATTTTCACCTCCAATTTGTCCTTTAAGC 3'

atp6-P157T

forward: 5`GGTTGAGTATTCTTCTCATTATTCGTAACTGCTGGTACACCATTACC 3' reverse: 5' GGTAATGGTGTACCAGCAGTTACGAATAATGAGAAGAATACTCAACC 3'

atp6-P163S

forward: 5`TCATTATTCGTACCTGCTGGTACACCATTATCATTAGTACCTTTATTAGTTATTA TTGAAACTTTATCTTATTTCGCTAGA 3' reverse: 5' TCTAGCGAAATAAGATAAAGTTTCAATAATAACTAATAAAGGTACTAATGATAA TGGTGTACCAGCAGGTACGAATAATGA 3'

atp6-I170V

forward: 5`CCATTAGTACCTTTATTAGTTGTTATTGAAACTTTATCTTATTTCGCTAGAGC 3' reverse: 5' GCTCTAGCGAAATAAGATAAAGTTTCAATAACAACTAATAAAGGTACTAATGG 3'

Gene

Sequence of primers for construction of the strains atp6-L232P forward: 5`GCTATGATCTTAGCCATTATGATGCCAGAATTCACTGGCCGTCGTTTTACAACG TCG 3' reverse: 5' CGACGTTGTAAAACGACGGCCAGTGAATTCTGGCATCATAATGGCTAAGATCAT AGC 3'

atp6-S250P

forward: 5' GTCTGGGCTATTTTAACAGCACCATATTTAAAAGATGCAGTATACTTACAT 3' reverse: 5'ATGTAAGTATACTGCATCTTTTAAATATGGTGCTGTTAAAATAGCCCAGAC

atp6-L252P

forward: 5' GTCTGGGCTATTTTAACAGCATCATATCCAAAAGATGCAGTATACTTACAT 3' reverse: 5' ATGTAAGTATACTGCATCTTTTGGATATGATGCTGTTAAAATAGCCCAGAC 3'

II.3.3 Isolation of plasmids from bacterial cells

Plasmids were extracted from bacteria with the alcalic lysis method. Bacteria cells cultured in liquid LBA medium were centrifuged and treated with GTE solution (50 mM glucose, 25 mM Tris-HCl pH 8, 10 mM EDTA pH 8) containing RNAse (0.5 µg/µl final concentration). After 5 minutes lysis buffer (0.2 M NaOH, 1% SDS) was added and cells were kept for 5 minutes on ice. To stop the lysis and precipitate proteins 7.5 M NH 3 Ac were added and suspension was shaken strongly 3 -4 times and incubated for 5 minutes more on ice and then centrifuged for 6 minutes at 14,000 rpm. The supernatant containing plasmid DNA was recuperated and followed by precipitation of plasmid DNA with 96% ethanol. minutes and centrifuged at 13,000 g for 5 minutes. The supernatant was collected, mixed with 5 minutes, washed with 1 ml of 70% ethanol, re-centrifuged for 3 minutes and dried at 37°C

II.3.4 Extraction of yeast

for 10 minutes. The obtained DNA was re-suspended in 30 µl of sterile water.

II.3.5 Precipitation of DNA with ethanol

DNA was precipitated by ethanol in the presence of sodium cations. A tenth part of 3 M sodium acetate (v/v) and two and a half of 100% ethanol (v/v) were added to the solution containing DNA, mixed and kept at -20°C for at least 30 minutes. The pellet obtained after centrifugation at 16,000 g, 4°C for 30 minutes, washed with 1 ml of 70% ethanol and re-centrifuged at 16,000 g for 3 minutes. Finally the precipitated DNA was dried at 37°C for 10 minutes and re-suspended in sterile water.

II.3.6 Electrophoresis of DNA in agarose gel

Agarose gels, 0.8 -1% (m/v), were used to separate DNA. Agarose was dissolved in a 1X TAE buffer (40 mM Tris, 20 mM acetic acid, 1 mM EDTA, pH 8). The same buffer was used during migration. Ethidium bromide or GelRed from Biotium were used to visualize DNA in UV. Force (50 -100 V) and time (20 -40 minutes) of the migration were dependent on size of the gel.

II.3.7 Purification of DNA from agarose gel

DNA after enzymatic digestion was separated in agarose gel as described before.

Fragments of interest were cut out from the gel and purified with the use of the freeze-squeeze method or by the QIAEX II Gel Extraction Kit from Qiagen. The extraction with the kit was proceeded as recommended with a manufacture protocol. The freeze-squeeze purification method is based on the Tautz and Renz article [START_REF] Tautz | An optimized freeze-squeeze method for the recovery of DNA fragments from agarose gels[END_REF]. A DNA fragment cut from the gel was immediately frozen in liquid nitrogen and placed in a 0.5 ml eppendorf tube filled with polypropylene wool. The tube had been previously pounced and placed in 1.5 ml eppendorf tube. Then the frozen agarose slice was thawed at 37°C for at least 10 minutes and centrifuged for 5 minutes at 16,000 g. The DNA was precipitated with ethanol.

II.3.8 Nucleic acids measurement

The quantity of nucleic acid was measured in spectrophotometer at λ 260 nm.

II.3.9 Enzymatic digestion

DNA was incubated with 1 -5 units of the enzyme per µg of DNA and with an appropriate digestion buffer at 37°C for at least 2 hours. The enzyme was inactivated by the DNA precipitation procedure. The Klenow enzyme or T4-DNA polymerase were used as recommended with manufactures protocols to produce the blunt ends in linear DNA after digestion.

II.3.10 Ligation of DNA fragments

T4 DNA-ligase was used to clone the linear DNA into the vector. Before ligation, the vector and the insert were digested with appropriate restriction enzymes, separated in the agarose gel and purified from the gel. The insert and the vector were mixed at the 3:1 ratio, respectively. The mass added to the reaction mix was calculated in accordance with the following formula:

. The reaction was proceeded at RT for 1h or at 16°C o/n.

II.3.11 Polymerase chain reaction (PCR) of DNA fragments

To amplify DNA the Taq recombinant polymerase (Life Technology) or Pfu-Ultra polymerase (Stratagene) were used. The PCRs were prepared as recommended with manufactures protocols.

Freshly grown colonies were also used as templates for verification of the deletion strains. A little portion of a colony was suspended in 20 µl of 0.2 M NaOH and warmed for 30 minutes at 95°C. 0.5 µl of the solution containing DNA released from cells was added to the reaction mixture as a template.

II.4. TECHNIQUES IN BIOCHEMISTRY

II.4.1 Isolation of mitochondria from yeast cells

Isolation of mitochondria from yeast cells [START_REF] Guerin | Preparation of yeast mitochondria (Saccharomyces cerevisiae) with good P/O and respiratory control ratios[END_REF]) includes enzymatic digestion of the cell wall, mechanic disruption of the cells and differential centrifugations to obtain a fraction rich in mitochondria. Mitochondria were extracted with the use of iso-osmotic buffers which makes it possible to use them for functional studies.

Yeast cells were cultured in 2 liters of liquid YPGalA (for respiratory deficient strains)

or YPEGA (for respiratory efficient strains) to exponential phase of growth (OD 650 between 2 and 4). The whole procedure was carried out at 4°C. Cells were centrifuged for 5 minutes at 5,740 g and washed with sterile water (4°C). The pellet was re-suspended in solution containing 0.1 M Tris, 0.5 M β-mercaptoethanol, pH 9.3. 20 ml of solution was used per 1 g of dry mass of yeast. Dry mass of yeast was calculated in accordance with the following formula:

. Yeasts were incubated for 10 minutes at 32°C in order to reduce the disulfide bonds in proteins of the cell wall with β-mercaptoethanol. The solution was completed with a buffer containing: 0.5 M KCl, 10 mM Tris pH 7, and centrifuged for 5 minutes at 3,840 g. This step was repeated twice. The obtained pellet was re-suspended in 10 ml par 1g of dry mass of the digestion buffer (1.35 M sorbitol, 1 mM EGTA, 10 mM citric acid, 30 mM disodium phosphate, pH 5.8) mixed with zymolyase (15 mg/g of dry mass). Digestion was done at 32°C

for 20 minutes. To verify if the process had been completed, 4 µl of digested yeast cells were mixed with 15 µl of sterile water. In the absence of the cell wall protoplasts break due to osmotic shock, which can be seen with the use of an optic microscope. If digestion was not completed, the time was prolonged. When the process was finished, the cell suspension was completed with a protoplasts washing buffer (0.75 M sorbitol, 0.4 M mannitol, 10 mM Trismaleate, 0.1% (m/v) BSA, pH 6.8) and centrifuged for 5 minutes at 12,000 g. Protoplasts were washed twice by delicate re-suspending in the same protoplasts washing buffer with use of 15 cm 3 potter and centrifuged for 5 minutes at 12,000g. In the same manner protoplasts were re-suspended in a homogenization buffer (0.6 M mannitol, 2 mM EGTA, 10 mM Trismaleate, 0,2% (m/v) BSA, pH 6.8). Lysis of the protoplasts was achieved by mixing 3 times for 5 seconds in a Mixer blender (Waring). The next steps of the procedure make it possible to separate mitochondria from other cellular parts by differential centrifugation. After lysis, the extract was centrifuged for 8 minutes at 750 g. The collected supernatant was centrifuged for 10 minutes at 12,000 g. The pellet containing the isolated mitochondria was washed once in a recuperation buffer (0.6 M mannitol, 2 mM EGTA, 10 mM Trismaleate, pH 6.8) with the use of a potter. Washed, pure mitochondria were transferred into glass tube and kept on ice during respiration, ATP synthesis and mitochondrial membrane potential measurements.

Remaining mitochondria were frozen in liquid nitrogen in form of drops and conserved at -80°C.

II.4.2 Measurement of respiration in isolated mitochondria

The Oxygraph system with the Clark's electrode from Heito (France) was used to measure the respiration in freshly isolated mitochondria. This electrode was separated from the reaction chamber with a membrane that is permeable only to oxygen, which is reduced when it reaches the cathode. The reduction creates a potential difference that represents the oxygen present in the reaction chamber and, therefore, may be visualized. The electrode, surrounded by the membrane, was placed in a reaction glass chamber of 1 ml and maintained at 28°C. 150 µg/ml of the mitochondrial proteins were deposed into the chamber filled with a respiration buffer (0.65 M mannitol, 0.36 mM EGTA, 5 mM Trisphosphate, 10 mM Trismaleate, pH 6.8) and constantly agitated.

Activities of the respiratory chain complexes and ATP synthase were determined as the rate of oxygen consumption in the presence of different substrates and inhibitors. The following substrates and inhibitors were used: 4 mM NADH, 150 µM ADP, 4 µM CCCP, oligomycin 3 µg/ml, 12.5 mM ascorbate, 1.4 mM TMPD. The oxygen consumption rate was presented in nmol of oxygen consumed per minute per milligram of proteins.

NADH was added to the chamber as first. It is the substrate of complex I that permits generation of the proton gradient and stimulates the oxygen consumption by the respiratory chain. At this stage ATP synthase can use the substrates that have been already present in mitochondria, hence we measured the basal level of the respiratory chain activity known as state 4.

In the presence of NADH and ADP, ATP synthase uses the proton gradient to synthesize ATP from ADP and Pi. As a consequence, the respiratory chain increases its activity and consumes oxygen faster. At this stage we measured the respiratory chain activity in phosphorylating conditions (state 3).

The maximal activity of respiratory chain was measured in the presence of NADH and CCCP, a compound that dissipates the proton gradient on the inner mitochondrial membrane.

The maximal activity of complex IV was determined by adding ascorbate and TMPD which deliver electrons directly to cytochrome c, which transfers them to cytochrome c oxidase. Therefore complex IV functions independently from other respiratory chain complexes. Introduction of CCCP dissipates the proton gradient, so the oxygen consumption dependents only on the maximal activity of complex IV.

Use of NADH and oligomycin was the internal control for preparation of mitochondria. The experiment shows whether the basal respiratory chain activity is not a consequence of proton leak through F O domain of ATP synthase. Oligomycin blocks the proton transfer through the channel of ATP synthase. If the rate of oxygen consumption in the presence of NADH is higher than the consumption in the presence of NADH and oligomycin, it means that there is a proton leak through F O .

II.4.3 Measurement of mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨ) was measured in isolated mitochondria using fluorescent dye rhodamine 123 [START_REF] Emaus | Rhodamine 123 as a probe of transmembrane potential in isolated rat-liver mitochondria: spectral and metabolic properties[END_REF]). Fluorescence emitted by rhodamine 123 was quenched because of its entering into mitochondria in response to ΔΨthe higher the membrane potential was, the more of rhodamine entered the mitochondria.

Therefore changes in the membrane potential could be visualized by the changing fluorescence of the dye in the reaction buffer. The capacity of the respiratory chain and ATP

synthase to maintain the membrane potential was measured in the presence of different substrates and inhibitors, as it was done for the purpose of measurement of oxygen consumption. The measurements were done in a spectrofluorometer (Flax de Safas). Wave

II.4.4 Quantification of ATP produced by isolated mitochondria

A volume of freshly isolated mitochondria corresponding to 150 µg/ml of mitochondrial proteins was added to the oxygraph chamber containing 1 ml of the respiratory buffer (0.65 M mannitol, 0.36 mM EGTA, 5 mM Trisphosphate, 10 mM Trismaleate, pH 6.8) at 28°C. The substrate of respiratory chain was NADH (4 µM). The ATP synthesis was done in an excess of ADP (1 mM). After addition of ADP, 100 µl of the reaction mixture was taken from the chamber every 15 seconds within 1 minute (for well respiring strains) or every 30 seconds within 2 minutes (for respiratory deficient strains) and rapidly precipitated with 25 µl of: 7% (v/v) perchloric acid and 25 mM EDTA. To measure the ATP synthesis independent of the ATP synthase, the enzyme was blocked with oligomycin (3 µg/ml) for 3 minutes before addition of ADP and samples were collected as described. All samples were centrifuged for 5 min at 15,000 g at 4°C, 110 µl of supernatants were preserved and pH was adjusted to 7 with addition of 2 M KOH and 0.

II.4.5 Estimating ATPase activity of ATP synthase

ATPase activity of ATP synthase was determined in thawed mitochondria.

Mitochondria were thawed on ice and the protein concentration was measured de novo with the use of Lowry's method. A volume of mitochondria corresponding to 150 µg of mitochondrial proteins was mixed with 900 µl of the ATPase buffer (0.2 M KCl, 3 mM MgCl 2 , 10m M Tris-HCl, pH 8.4) and incubated at 30°C. A series of five tubes was provided for each sample: two without oligomycin, two with 10 µg/ml oligomycin and one with 1% TCA. All tubes were incubated for 3 minutes at 30°C (time necessary to inhibit F O domain with oligomycin). Then ATP that corresponds to 5 µM final concentration was added to the reaction mix and the tubes were incubated for 2 more minutes at 30°C. The reaction was stopped by addition of 5% TCA. The quantity of phosphate produced during the reaction of ATP hydrolysis to ADP and Pi was measured as described by Somlo [START_REF] Somlo | Induction and repression of mitochondrial ATPase in yeast[END_REF]). Tubes were centrifuged for 10 minutes at 4°C, 9,300 g. Half of the supernatant of each tube was added to 5 volumes of the MARIKA reaction solution (5.4 mM Ammonium HeptaMolybdate

II.4.6 Extraction of total proteins from yeasts cells

Total protein extract was prepared from 10 OD of yeast cells in exponential phase of growth (OD 650 2 -4). The cells were centrifuged for 3 minutes, 2,000 g. The pellet was resuspended in 500 µl of lysis buffer (1.85 M NaOH, 7.4% (v/v) β-mercaptoethanol and incubated at 4°C for 10 minutes. The same volume of 5 % TCA was added and the samples were incubated at 4°C for 10 minutes. After centrifugation for 5 minutes at 4°C, 16,000 g, the pellet was washed first with 3 M Tris at pH 8.9 and then with sterile water. The final pellet was re-suspended in 50 µl of 5% SDS and neutralized with 3 M Tris at pH 8.9, if needed (final pH of the samples should be about 7 -8). In the next step the sample was warmed for 15 minutes at 42°C and centrifuged for 5 minutes at 1,000 g at RT. The supernatant was collected and stocked at -20°C.

To verify if Atp9p synthesized in excess has formed the inclusion bodies in mitochondria, the pellet from the final centrifugation was re-suspended in highly denaturing buffer (7 M urea, 2 M Triton, 4 % Chaps, 65 mM DTT) and centrifuged for 5 minutes at 16,000 g. The supernatant was collected and stocked at -20°C. The pellet was treated in the same manner one more time and the final supernatant was kept and stocked at -20°C. Protein concentration in each sample was determined with Lowry's method.

II.4.7 Measuring of protein content with the use of colorimetric method of Lowry

Protein concentration in total extracts from yeast cells or mitochondria was based on the method described by Lowry [START_REF] Lowry | Protein measurement with the Folin phenol reagent[END_REF]). The samples were diluted 1/10 (for measurements of proteins from the yeast cells) or 1/20 (for measurements of mitochondrial proteins) in 5% SDS. 5 µl of dilution was mixed with 70 µl of 5% SDS and 750 µl of the reaction solution (0.01% (m/v) CuSO 4 , 0.02% (m/v) NaK, 3.92% (m/v) Na 2 CO 3 dissolved in 0.1 M NaOH). After a three-minute incubation at 37°C, 75 µl of Folin solution (diluted 1/3) was added to each sample and incubated for 6 more minutes at 37°C. At the same time the calibration curve was made with known concentration of the bovine albumin (from 0 to 10 µg of proteins) treated in the same way as the samples. The absorbance at wave length of 750 nm was measured and concentration of the proteins in samples was determined in milligrams per milliliter.

II.4.8 Separation of mitochondrial membrane complexes by BN-PAGE

The already isolated mitochondria were quickly thawed and the protein concentration was measured de novo by Lowry's method. A volume corresponding to 250 -500 µg of mitochondrial proteins were centrifuged for 5 minutes at 4°C, 16,000 g. The pellets of mitochondria were re-suspended in 50 µl of extraction buffer (30 mM HEPES, 150 mM potassium acetate, 12% (m/v) glycerol, 2 mM aminocaproic acid, 1 mM EDTA, cocktail of inhibitors of proteases from Roche) containing 2 g of digitonin per 1 g of proteins. Digitonin is a mild detergent that extracts mitochondrial membrane complexes in their native forms [START_REF] Kun | Stabilization of mitochondrial functions with digitonin[END_REF]). The mix was incubated for 30 minutes on ice and centrifuged for collected and mixed with a charging buffer (final concentrations: 0.1% (m/v) of Coomassie blue G250 and 32 mM 6-aminocaproic acid).

Mitochondrial membrane complexes were separated by migration in BN-PAGE gel.

This technique makes it possible to separate different supramolecular forms of ATP synthase preserving at the same time its enzymatic activities. After extraction from the inner mitochondrial membrane, complexes were separated in non-denaturing gradient polyacrylamide gel in a migration buffer containing Coomassie Blue. The protocol was based on the publication of Schägger and von Jagow [START_REF] Schagger | Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form[END_REF]. Commassie blue G250 interacts with proteins and gives them negative charge. Electrophoretic mobility of proteins depends on the size of complexes and the amount of Coomassie blue interacting with them. Two solutions of different concentrations of acrylamide (3 and 13%) were used to create a polyacrylamide gradient gel. They were placed in two compartments connected in an interior. One of the compartments was connected to a peristaltic pomp. The pomp creates an effect of aspiration and a 3% solution overfilled gradually to a 13% solution. Homogenization of the two solutions was guaranteed by agitation in the compartment of the 13% solution.

Thanks to the pomp system, the lower part of the gel contained only the 13% solution, then there was a mix of the solutions gradually changing the percentage of acrylamide to 3% solution at the top of the gel. The 3% solution contained: 2.96% of acrylamide/bis-acrylamide mix (29.1:0.9), 1X gel buffer (25 mM imidazol, 0.5 M 6-aminocaproic acid, pH 7), 0.04% APS, TEMED. The 13% solution contained: 200 g/l of glycerol, 13% of acrylamide/bisacrylamide mix (29.1:0.9), 1X gel buffer (25 mM imidazol, 0.5 M 6-aminocaproic acid, pH 7), 0,03% APS, TEMED. The gradient gel mixer was filled with 12 ml of 3% solution and 11.5 ml of 13% solution. The stacking gel was composed of: 2.96% of acrylamide/bisacrylamide mix (29.1:0.9), 1X gel buffer (25 mM imidazol, 0.5 M 6-aminocaproic acid, pH 7), 0.08% APS, TEMED. Cathode buffer was composed of: 7.5 mM imidazol, 50 mM tricine, 40 mg/l of Coomassie blue G250, adjusted to pH 7 at 4°C. Anode buffer was composed of: 25 mM imidazol adjusted to pH 7 at 4°C. Migration was proceeded at 10 mA constant for about 8h.

II.4.9 Visualization of ATPase activity on gel

ATP synthase can be detected after separation of inner mitochondrial membrane complexes in non-denaturing conditions (BN-PAGE) by its ATPase activity. In mitochondria the main enzyme with ATPase activity is the F 1 domain of ATP synthase. The method of revealing this activity on gel was described by Grandier-Vazeille and Guérin (Grandier-Vazeille and Guerin 1996). The gel was incubated for 3 hours with shaking in a revelation buffer (270 nM glycine, 35 mM Tris at pH 8.4). After this time the gel was incubated with the same buffer completed with 14 mM MgSO 4 , 0.2% Pb(NO 3 ) and 8 mM ATP until the moment of apparition of white bands. These bands corresponded to the precipitated plumb and phosphate due to hydrolysis of ATP by ATP synthase.

After revealing of ATPase activity, the revelation buffer was removed and the gel was de-colored with a solution containing 30% ethanol and concentrated HCl. Next the gel was colored with Coomassie blue to visualize all proteins presented in the gel.

II.4.10 Separation of the proteins in denaturing conditions (SDS-PAGE)

The method was based on the publication of Schägger and von Jagow [START_REF] Schagger | Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa[END_REF]. Proteins were deposed in an acrylamide/bis-acrylamide gel containing SDS.

SDS is an anionic detergent keeping the proteins in denatured, non-native and negatively charged forms. Electrophoretic mobility of proteins in the gel depends on their size.

Separation of proteins in denaturing conditions was performed in commercially available pre-casted gels and migration buffers from kit NuPAGE Novex 4-12% Bis-Tris Gel from Invitrogen. Migration was done at constant 200 V for 35 -55 minutes. Immediately thereafter the migration proteins were transferred onto a nitrocellulose membrane.

II.4.11 Transfer of the proteins onto a nitrocellulose membrane

Proteins separated on the gel were transferred onto a nitrocellulose membrane with the use of the iBlot Transfer Stack kit and iBlot Dry Blotting System from Invitrogen. The proteins were fixed on the membrane due to its non-specific affinity to amino acids.

II.4.12 Immunodetection of proteins

The nitrocellulose membrane with fixed proteins was incubated with shaking for at least 30 minutes in a 1X PBS-T buffer (2.7 mM KCl, 10 mM Na 2 HPO 4 2H 2 O, 1.76 mM KH 2 PO 4 , 0.14 M NaCl, 0.05% (v/v) Tween-20, adjusted to pH 7.4) mixed with 5% (m/v) dehydrated skim milk. A primary antibody in the same buffer was added immediately thereafter and incubation was continued for at least two hours in RT. The overnight incubation was done at 4°C. Concentration of the antibody dependents on its type and is presented in Table 5. After hybridization with a primary antibody the membrane was washed in 1X PBS-T buffer for 5 minutes. This step was repeated five times. Next, the membrane was incubated with a secondary antibody for at least 1.5 h. The secondary antibody, conjugated with horseradish peroxidase, was either anti-rabbit or anti-mouse following the type of the primary antibody. It was diluted in the same 1X PBS-T buffer with 5% (m/v) dehydrated skim milk. Next the membrane was washed as after the incubation with the primary antibody. The presence of bound antibodies was detected thanks to the reaction catalyzed by horseradish peroxidase in the presence of its substrate ECL from GE Healthcare. Exposition time was from 30 seconds to 30 minutes.

Antibody

II.4.13 In vivo labelling of the products of mitochondrial translation

In vivo labeling of the products of mitochondrial translation was based on the publication of Barrientos [START_REF] Barrientos | Shy1p is necessary for full expression of mitochondrial COX1 in the yeast model of Leigh's syndrome[END_REF]. Yeast cells were cultured in a liquid medium for 5 -7 generations. The media used were YPGalA for strains without plasmids and CSM galactose supplemented with appropriate amino acids for strains bearing plasmids.

10 OD of the cells at the exponential phase of growth (OD 650 2 -4) were centrifuged for 3 minutes at 2,000 g and washed twice with minimal galactose medium without ammonium sulfate and amino acids. Cells were finally re-suspended in the same minimal galactose medium without ammonium sulfate supplemented with auxotrophic amino acids (without methionine and cysteine) and incubated for 2 hours at 28°C with shacking. After starvation, 6 OD of the cells were taken for further analysis and centrifuged for 3 minutes at 2,000 g. The cell pellets were re-suspended in 500 µl of the same medium. Freshly prepared 1 mM cycloheximide was added and the mix was incubated for 5 minutes at 28°C. 50 microcuries of the methionine/cysteine, where methionine is labeled with 35 S (1000 Ci/mmol, Promix Amersham Bioscience), was added. The samples were then incubated for 20 minutes at 28°C.

Next, the cells were centrifuged for 1 minute 10,000 g and 75 µl of the lysis buffer (1.85 M NaOH, 14% (v/v) β-mercaptoethanol, 10 mM phenylmethylsulfonyl fluoride) was added. The lysis was stopped with 1 ml of 25% TCA and centrifuged for 5 minutes at 14,000 g. The pellet was washed once with 3 M Tris pH 8.9 and once with sterile water. The final pellet was re-suspended in 50 µl of Laemmli buffer (2% SDS, 10% glycerol, 2.5% β-mercaptoethanol, 0.06 M Tris-HCl, pH 6.8, 0.002% (m/v) of bromophenol blue). Emitted radioactivity was measured in the presence of scintillation reagent with a scinitillator.

The following two gel systems were used to visualize the products of mitochondrial translation: 17.5% SDS-PAGE [START_REF] Laemmli | Cleavage of structural proteins during the assembly of the head of bacteriophage T4[END_REF] to separate Atp8p and Atp9p, 12% urea/glycerol gel to separate Atp6p and Cox3p and Cox2p and cytochrome b. The same amount of radioactivity for each sample was deposed in the gel. 17.5% SDS-PAGE resolving gel was composed of: 17.5% acrylamide/bis-acrylamide (37.5:1), 0.375 M Tris-HCl pH 8.9, 0.1% SDS, 0.05% APS and TEMED. The stacking gel was composed of: 5% acrylamide/bisacrylamide (37.5:1), 0.06 M Tris-HCl pH 6.8, 0.1% SDS, 0.05% APS and TEMED. The migration was made in the buffer composed of: 0.4 M Tris, 0.2 M glycine and 0.1% SDS at 35 mA constant for 4 hours. 12% urea/glycerol resolving gel was composed of:

12% acrylamide/bis-acrylamide (37.5:1), 4 M urea, 25% (m/v) glycerol, 0.75 M Tris-HCl pH 8.9, 0,26% APS and TEMED. The stacking gel was composed of: 6% acrylamide/bis-acrylamide (37.5:1), 0.125 M Tris-HCl pH 6.8, 0.1% SDS, 0.025% APS and TEMED. The migration was made in the buffer composed of: 0.4 M Tris, 0.2 M glycine and 0.1% SDS at 150 V constant for 16 hours. This method of protein separation has been adapted from the publication of Kunkele et al. [START_REF] Kunkele | The preprotein translocation channel of the outer membrane of mitochondria[END_REF]). After migration both gels were dried and radioactively marked proteins were visualized by autoradiography in PhosphorImager after a one-week exposition.

RESULTS

III.1. MUTATIONS IN ATP6 GENE AND HUMAN DISEASES

In the first part of my work I was interested in analyzing the effects of mutations in mitochondrial ATP6 gene found in cells from patients. Two of them are newly identified mutations causing the NARP (neurogenic myopathy, ataxia, retinitis pigmentosa) syndrome.

Five were found in various human tumors in conserved positions between yeast and human subunit 6. Mitochondrial ATP6 gene encodes subunit 6 of F 1 F O -ATP synthase that forms a proton channel together with the ring of subunit 9. Mutations in subunit 6 may impair the proton transport through inner mitochondrial membrane leading to malfunctioning of F 1 F O -ATP synthase and decreased ATP production.

III.1.1 Analysis of the two newly identified NARP mutations

NARP syndrome is one of mitochondrial diseases caused by mutations in mtDNA in MTATP6 gene. Previously in our laboratory, we modeled in yeast five other NARP ATP6 mutations: 8993T>G (Rak, Tetaud et al. 2007a), 8993T>C (Kucharczyk, Rak et al. 2009a), 9176T>G (Kucharczyk, Salin et al. 2009b), 9176T>C [START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF]) and 8851T>C [START_REF] Kucharczyk | Defining the pathogenesis of human mtDNA mutations using a yeast model: the case of T8851C[END_REF]. As two novel mutations (9185T>C and 9191T>C) were described [START_REF] Moslemi | Two new mutations in the MTATP6 gene associated with Leigh syndrome[END_REF] we decided to introduce them into yeast mtDNA and investigated their effects on F 1 F O -ATP synthase. The obtained results are described in [START_REF] Kabala | Defining the impact on yeast ATP synthase of two pathogenic human mitochondrial DNA mutations, T9185C and T9191C[END_REF]) which I have attached and summarized below.

9185T>C and 9191T>C mutations in human MTATP6 change leucine 220 into proline and leucine 222 into proline, respectively, in human subunit 6. These amino acids correspond to serine 250 and leucine 252 residues in yeast protein, respectively. Yeast clones with atp6-S250P mutation grow well on non-fermentable carbon source (glycerol) but are sensitive on oligomycin which indicates diminished production of ATP by ATP synthase.

atp6-L252P mutation disabled yeast growth on respiratory carbon source (Fig. 1 in the article below).

In the next step I measured: oxygen consumption, ATP synthesis and hydrolysis, and inner membrane potential in isolated mitochondria. For strain bearing atp6-S250P mutation, about a 10-15% drop in oxygen consumption at state 3 was observed, while for atp6-L252P

this drop was about 67-85%. The ATP synthesis rates were also diminished: about 30% and 90% in atp6-S250P and atp6-L252P, respectively. The hydrolysis rates were similar in both mutants as compared to WT (Table 2 in the article below). Interestingly, in the atp6-L252P strain the hydrolysis rate did not change after addition of oligomycin, which suggests the presence of free F 1 domain. Analysis of ATP synthase assembly/stability in native gels confirmed it, both by coomasie-blue staining and hydrolysis activity in gel. No change in ATP synthase stability could be seen in the atp6-S250P strain (Fig. 2A in the article below). Like in WT we could observe fully assembled monomeric and dimeric ATP synthase complexes.

Inner mitochondrial membrane potential was evaluated by measuring of the proton pumping activity coupled to F 1 -mediated ATP hydrolysis. In the atp6-S250P strain the level of proton pumping was comparable to that of WT in contrast to the atp6-L252P strain, from which this activity was almost absent (Fig. 3 in the article below).

In the two examined mutant strains I analyzed also synthesis of mitochondrially encoded proteins by in vivo labeling. No change in this activity could be observed (Fig. 4 in the article below). Shift of Atp6p migration may be caused by mutations that change protein mobility in the gel. Although the level of synthesis is normal in both strains, in the atp6-L252P strain steady state level of subunit 6 is strongly diminished (Fig. 2B in the article below) while no change is observed in the atp6-S250P strain as compared to WT. This result also suggests a dysfunction of F 1 F O -ATP synthase assembly in the atp6-L252P strain as

Atp6p is rapidly degraded when not assembled into the complex.

Both mutations are located in the last helix of the Atp6 protein in the vicinity of amino acids that are well-conserved between different species and make contact sites with subunits 9 ring [START_REF] Fillingame | Coupling proton movements to c-ring rotation in F(1)F(o) ATP synthase: aqueous access channels and helix rotations at the a-c interface[END_REF]Kucharczyk, Zick et al. 2009c). Results obtained for the atp6-S250P mutation indicate relatively mild effects on F 1 F O -ATP synthase. There is about 30% less ATP produced in this strain but neither defects in assembly of the enzyme nor proton leakage could have been observed. We suggested that this mutation could create some local structural modifications that influence proton transport through the channel as it was proposed before for the 9176T>C mutant [START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF]). The 30% drop in ATP production may be sufficient to mildly compromise human health. In contrast, the atp6-L252P mutation affects the ATP synthase very severely. Probably due to the mutation Atp6p cannot be efficiently assembled into the enzyme or interact with the ring of subunits 9.

This can explain severe clinical phenotype in the patient similar to those presented in the patient bearing the 9176T>G mutation (Kucharczyk, Salin et al. 2009b).

III.1.2 Use of yeast Saccharomyces cerevisiae as a model organism for drug screening

In the following article [START_REF] Couplan | A yeast-based assay identifies drugs active against human mitochondrial disorders[END_REF], which is summarized below, we use the fmc1 deletion mutant as a model of diseases caused by mutations in human ATP12

(De Meirleir, [START_REF] De Meirleir | Respiratory chain complex V deficiency due to a mutation in the assembly gene ATP12[END_REF]) and TMEM70 [START_REF] Cizkova | TMEM70 mutations cause isolated ATP synthase deficiency and neonatal mitochondrial encephalocardiomyopathy[END_REF]) genes that are characterized by a low content of ATP synthase. The fmc1 mutant grows poorly on glycerol at 35°C. We used this mutant for screening of drugs that can improve its ability to grow on glycerol at this temperature. Two active drugs were selected: chlorhexidine (CH) and oleate (OA), which were then tested on five yeast atp6-NARP models and in a human cybridbased model of NARP.

We demonstrated that DHLA, a compound already used as a treatment in patients with mitochondrial encephalopathy [START_REF] Mattiazzi | The mtDNA T8993G (NARP) mutation results in an impairment of oxidative phosphorylation that can be improved by antioxidants[END_REF][START_REF] Dimauro | Approaches to the treatment of mitochondrial diseases[END_REF], recovered respiratory growth of the fmc1 mutant at 35°C. It was also true for OA, a compound that induces ODC1 gene in yeast. ODC1 gene was previously shown as a suppressor of respiratory growth defect in the fmc1Δ strain [START_REF] Tibbetts | Yeast mitochondrial oxodicarboxylate transporters are important for growth on oleic acid[END_REF]. The next step was to test about 12,000 compounds from chemical libraries and identify those that rescue the fmc1 mutant. Chlorhexidine from Prestwick Chemical Library, a collection of drugs already tested in humans, was identified. It was also active in the atp6-NARP 8993T>G and 8851T>C strains.

Deletion of the FMC1 gene from yeast strain results in multiple defects when cells are grown at 35°C. We observed lower oxygen consumption by respiratory chain complexes due to low amounts of these complexes in mitochondria, weak energization of inner mitochondrial membrane, decreased level of ATP synthesis, strongly reduced content in ATP synthase, presence of inclusion bodies in mitochondrial matrix and almost no cristae in the mitochondria. Figure 2 in the article below shows that CH attenuates all these phenotypes whereas had no influence on WT strain.

To answer the question whether CH has a global effect in fmc1Δ mutant we analyzed the transcriptional response in the WT, fmc1Δ mutant treated and not treated with the drug.

The obtained results revealed that the most affected genes with a reduced expression due to the fmc1 deletion are connected with the mitochondrial respiration and the retrograde signaling pathway. Presence of CH in the medium partially reversed this effect. With respect to WT, we found that CH dramatically increase the expression of QRC9 (Fig. 3B in the article below), a gene that encodes a subunit of cytochrome bc 1 complex. Interestingly, over-expression of the QRC9 gene from a plasmid was alone able to improve the respiratory growth of fmc1Δ, indicating that this gene plays a key role in the CH rescue (Fig. 3C in the article below).

Further analysis pertained to the effect of DHLA, OA and CH in human NARP cybrid cells. We could observe a significant increase in growth rates of these cells in glucosedeprived medium (Fig. 4A in the article below). No such effect could be observed for WT cells As mitochondrial disorders often result in lactic acidosis due to enhanced glycolysis, we measured the levels of glycolytic byproducts (pyruvate and lactate) secreted by the human NARP cells upon treatment with CH. The levels of these metabolic intermediates were diminished in comparison to non-treated cybrid cells (Fig. 4B and C in the article below).

Results presented in the publication below show that yeast can be successfully used as a model for screening of drugs active against ATP synthase disorders.

III.1.3 Analysis of the five mutations in ATP6 gene identified in human tumors

In humans two subunits, MTATP6 and A6L, are encoded in the mitochondrial genome.

Numerous mutations in these genes -55 in MTATP6 and 9 in A6Lhave been found in cancer cells [START_REF] Lu | Implications of mitochondrial DNA mutations and mitochondrial dysfunction in tumorigenesis[END_REF]). We have analyzed conservation of residues in Atp6p, between yeast and humans. We have investigated in yeast S. cerevisiae five mutations that affect highly conserved residues: 8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C [START_REF] Maximo | Mitochondrial DNA somatic mutations (point mutations and large deletions) and mitochondrial DNA variants in human thyroid pathology: a study with emphasis on Hurthle cell tumors[END_REF][START_REF] Tan | Comprehensive scanning of somatic mitochondrial DNA mutations in breast cancer[END_REF][START_REF] Petros | mtDNA mutations increase tumorigenicity in prostate cancer[END_REF][START_REF] Abu-Amero | Association of mitochondrial DNA transversion mutations with familial medullary thyroid carcinoma/multiple endocrine neoplasia type 2 syndrome[END_REF][START_REF] Costa-Guda | Mitochondrial DNA mutations in oxyphilic and chief cell parathyroid adenomas[END_REF]. All mutations and the corresponding amino acid changes in human and yeast proteins have been presented in Table 6. To characterize their impact on ATP synthase, we have created and biochemically characterized yeast strains bearing equivalents of these mutations.

III.1.3.1 Respiratory growth and genetic stability of the yeast atp6 "cancer" mutants

Growth analysis of the atp6 mutants revealed that they all grew well on rich glucose and glycerol media, at both 28 and 36°C (Fig. 20). Growth was also analyzed on glycerol medium supplemented with 0.25 µg/ml and 0.5 µg/ml oligomycin. Oligomycin is an inhibitor of the F O domain. In these conditions the atp6-P157T mutant grew slower whereas atp6-L232P stopped growing with respect to wild type yeast. This higher in vivo sensitivity is possibly due to the fact that a smaller dose of the drug is required to reach the 20% oxidative phosphorylation threshold under which the production of ATP becomes limiting for respiratory growth [START_REF] Mukhopadhyay | Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media[END_REF][START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF]. In contrast, the atp6-P163S, atp6-K90E and atp6-I170V mutants were more resistant to oligomycin than the wild type. This result is in accordance with the previous studies showing that mutations at the level of residues I171 and S175 within the loop connecting helix 3 and helix 4 of subunit 6 can compromise the binding of oligomycin to ATP synthase [START_REF] Macino | Assembly of the mitochondrial membrane system: sequence analysis of a yeast mitochondrial ATPase gene containing the oli-2 and oli-4 loci[END_REF][START_REF] John | Amino acid substitutions in mitochondrial ATPase subunit 6 of Saccharomyces cerevisiae leading to oligomycin resistance[END_REF].

The logarithmic growth of all atp6 mutant strains on liquid glucose and glycerol media was comparable to the wild type strain growth in both temperatures. However, they needed more time than WT (2 days vs. a couple of hours) to adapt in glycerol at 36°C before starting to grow.

In yeast, ATP synthase mutations often increase the production of  -/ 0 petites cells with large deletions in the mtDNA or a total loss of mtDNA [START_REF] Contamine | Maintenance and integrity of the mitochondrial genome: a plethora of nuclear genes in the budding yeast[END_REF][START_REF] Bietenhader | Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution[END_REF]. The parental strain MR6 (WT) never produced more than 5% of  -/ 0 cells when cultured in complete glucose or galactose media at 28 and 35°C. All but one mutant produced the same percentage of petites as the WT strain. The atp6-P163S mutant had two times higher propensity to produce  -/ 0 cells at the elevated temperature.

III.1.3.2 Mitochondrial oxygen consumption

First, I measured oxygen consumption in isolated mitochondria using NADH as an electron donor for respiratory chain, at state 4 (i.e. without addition of ADPbasal respiration), at state 3 (i.e. in the presence of ADPphosphorylating conditions), and in the presence of the membrane potential uncoupler CCCP (i.e. maximal respiration). I also used ascorbate/TMPD to deliver electrons directly to complex IV, the last complex of the electron transport chain. Mitochondria were isolated from yeast cultured on rich liquid glycerol/ethanol at 28°C or, to avoid the selection of spontaneous revertants during the two days of adaptation to the glycerol medium, in rich liquid galactose at 36°C. Galactose is a fermentable carbon source, which unlike glucose does not elicit repression of mitochondrial function.

The respiratory activities have been presented in Table 7A and B. The oxygen consumption by mitochondria isolated from strains cultured at 28°C were similar between the WT and atp6 mutant strains at state 3 and 4, before and after dissipation of mitochondrial membrane potential by CCCP, and at the level of complex IV (Table 7A). In contrast, these activities at state 3 and 4 differed from the wild type strain for the atp6-L232P, atp6-P163S and atp6-K90E mutants, when mitochondria were isolated from the cultures at 36°C, by 30%, 60% and 20%, respectively. (Table 7B). A decrease in oxygen consumption at the level of complex IV from 19 to 44% was observed in the atp6-P163S mutant. Accordingly complex IV was significantly less abundant in mutant atp6-P163S, whereas it was unaffected in the other atp6 mutants. These observations are in line with the previous studies showing that less complex IV accumulates in yeast ATP defective synthase mutants (Rak, Tetaud et al. 2007b;[START_REF] Soto | Synthesis of cytochrome c oxidase subunit 1 is translationally downregulated in the absence of functional F1F0-ATP synthase[END_REF].

III.1.3.3 Mitochondrial ATP synthesis

I determined the influence of the atp6 mutations on the rate of ATP synthesis in isolated mitochondria in the presence of a large excess of external ADP to avoid accumulation of intra-mitochondrial ATP, which would otherwise prevent ATP synthase from functioning.

Mitochondria isolated from the atp6-P157T, atp6-P163S, atp6-I170V and atp6-L232P mutant strains cultivated at 28°C produced about 15 to 28% less ATP than those isolated from the WT strain (Table 7A). Mitochondria isolated from the atp6-P157T, atp6-I170V and atp6-L232P

mutant strains cultivated at 36°C also produced about 15 to 28% less ATP than the WT mitochondria (Table 7B). Mitochondria isolated from the atp6-P163S mutant at 36°C produced about 42% less ATP than the WT mitochondria. Mitochondria isolated from the atp6-K90E mutant strain produced about 15 to 50% more ATP in comparison to the WT mitochondria, at both temperatures (Table 7A andB).

III.1.3.4 Mitochondrial ATP hydrolysis

I measured the hydrolytic activity of ATP synthase in isolated mitochondria in non-osmotic conditions buffered at pH 8.4 and in the presence of saturating amounts of ATP to obtain the maximal activity of the enzyme. With the exception of atp6-K90E all atp6 mutants had similar ATPase activity as compared to WT. The atp6-K90E mutant presented elevated activityabout 50% at both temperatures (Table 7A andB). In mitochondria isolated from cells grown at 28°C ATPase activity was inhibited by oligomycin at the level of ca. 66% in WT and 73 -89% in the atp6 mutants. When mitochondria were isolated from cells grown at 36°C, inhibition by oligomycin was at the level of ca. 60% for WT and 48 -65% for four atp6 mutants. The atp6-L232P mutant was inhibited only at the level of ca. 24% (Table 7A andB).

A. from wild type and mutants strains grown for 5 -6 generations in YPGEA at 28°C (A) or YPGalA at 36°C (B). Reaction mix for assays contained 0.15 mg/ml protein, 4 mM NADH, 150 µM ADP (for respiration assays) or 750 µM ADP (for ATP synthesis), 12.5 mM ascorbate (Asc), 1.4 mM TMPD, 4 µM CCCP, 3 µg/ml oligomycin (oligo). All strains contained 2 -5% of ρ -/ρ 0 cells at 28°C, the percent of ρ -/ρ 0 cells at 36°C is indicated in the table. The values reported are averages of triplicate assays ± standard deviation. Respiratory and ATP synthesis activities were measured using freshly isolated, osmotically protected mitochondria buffered at pH 6.8. For the ATPase assays, mitochondria kept at -80°C were thawed and the reaction performed in absence of osmotic protection and at pH 8.4.

Strain

III.1.3.5 Measurements of inner mitochondrial membrane potential

Mitochondrial membrane potential (ΔΨ) was measured in isolated mitochondria using fluorescent dye rhodamine 123 (see Materials and methods). Mitochondria were energized similarly in the WT and atp6 mutants at 28°C and 36°C. Addition of ADP caused a rapid increase in fluorescence due to ATP synthesis by F 1 F O -ATP synthase in all analyzed strains at both temperatures, but the time lag of restoration of the potential to the level established before the addition of ADP was longer for atp6-P163S at both temperatures and for atp6-L232P at 36°C (Fig. 21). The proton pumping activity coupled to F 1 -mediated ATP hydrolysis was comparable between the WT and all atp6 mutants at both temperatures. Energization of the mitochondrial inner membrane by ADP (panels A and C) or ATP (panels B and D) in intact mitochondria isolated from strains grown in YPGEA at 28°C or YPGalA at 36°C, was monitored by Rhodamine 123 fluorescence quenching. The additions were 0.5 µg/ml Rhodamine 123, 0.15 mg/ml mitochondrial proteins (Mito), 10 mM ethanol (EtOH), 75 µM ADP, 1 mM ATP, 0.2 mM potassium cyanide (KCN), 6 µg/ml oligomycin (OLIGO) and 3 µM CCCP.

III.1.3.6 Assembly/stability of the ATP synthase

Finally, I investigated the influence of the atp6 mutations on ATP synthase assembly/stability. To visualize the complex, I extracted mitochondrial membrane proteins with 2% digitonin from thawed mitochondria isolated from 28°C and 36°C cultures. I separated them in native polyacrylamide gel which I stained with coomasie brilliant blue (BN-PAGE) or analyzed the hydrolyzing activity of the complex in the gel (Fig. 22A andB). I also transferred proteins on nitrocellulose membrane and revealed them with antibodies (Fig. 22C and D).

Complexes extracted from mitochondria derived from cultures at 28°C demonstrate similar amounts of fully assembled ATP synthase dimers and monomers in the WT and all atp6 mutants (Fig. 22A). They are also well visualized in gel via their ATPase activity, which revealed also free F 1 (Fig. 22A). This reflects partial damaging of the complex during extraction as the same amount of free F 1 is present in the WT and atp6 mutants. No difference could be observed in the enzyme amount after transferring native complexes to membrane and staining them with anti-Atp1p antibody (Fig. 22C). Analysis of steady state levels of three ATP synthase subunits, Atp1p, Atp4p and Atp6p, in denaturing conditions (separation in SDS polyacrylamide gels) revealed that they were also in a normal amount in all mutant strains as compared to WT (Fig. 22E).

Complexes extracted from mitochondria derived from cultures at 36°C demonstrate a diminished amount of ATP synthase monomers in the atp6-P163S mutant, when the complexes were transferred to a nitrocellulose membrane and stained with antibodies against Atp9p and Atp1p (Fig. 22D). The ATPase signal corresponding to the monomeric form of the enzyme in this mutant had a much lower intensity than WT (Fig. 22B). We can also observe an increased signal of free F 1 in atp6-L163S mutant in the ATPase activity (Fig. 22B) and on the membrane stained with antibody against Atp1p (Fig. 22D). This may indicate a smaller stability of ATP synthase in this mutant. All other mutants showed no changes in ATP synthase stability in comparison to WT.

RESULTS

123

I analyzed also synthesis of mitochondrially encoded proteins by in vivo labeling with a mix of [ 35 S] methionine/cysteine, in the presence of cycloheximide to inhibit cytosolic protein synthesis. This revealed that proteins encoded by the mitochondrial genome were synthesized efficiently in all atp6 mutant strains (Fig. 23).

The analysis of five atp6-"cancer" yeast mutant strains showed no significant effect on ATP synthase and respiratory chain at 28°C and only a mild effect in the atp6-P163S, atp6-L232P and atp6-K90E mutant strains at 36°C, although the mutations were identified in the conserved positions between yeasts and humans.

III.2. REGULATION OF EXPRESSION OF ATP6 IN RELATION TO ITS ASSEMBLY INTO THE F 1 F O -ATP SYNTHASE COMPLEX

As I have demonstrated in section III.1, yeast S. cerevisiae can be successfully used to analyze the effects of mutations in the ATP6 gene responsible for human diseases.

Interestingly, in one mutant atp6-L183P (Kucharczyk, Rak et al. 2009a), Atp6p was synthesized much (3 -4 times) more rapidly than in the WT due to increased translation, and not because of a higher accumulation of ATP6 transcripts (Fig. 24A, B andC). Despite its up-regulation, the mutated protein was less abundant in the steady state than the wild type Atp6p, whereas other subunits of ATP synthase were not affected in the mutant (Fig. 24D).

This indicates that incorporation of Atp6p into ATP synthase is partially compromised by the L183P change and that unassembled Atp6p is proteolytically degraded, as usually observed in mutants where Atp6p cannot assemble. As a result, partial ATP synthase assemblies lacking Atp6p form in the atp6-L183P mutant. These assemblies are quite fragile and partially dissociate in BN-PAGE gel into free F 1 and Atp9p-rings (Fig. 24E). Such an up-regulation of Atp6p was not observed in other yeast NARP mutants (W136R [START_REF] Kucharczyk | Defining the pathogenesis of human mtDNA mutations using a yeast model: the case of T8851C[END_REF]), L183R (Rak, Tetaud et al. 2007a) and L247P [START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF]); see Fig. 26A). In these mutants the assembly of ATP synthase was not affected. Thus, it was possible that the up-regulation of Atp6p in atp6-L183P mutant was provoked by the defective incorporation of Atp6p into ATP synthase. To test this hypothesis I have generated other atp6 mutants, as revertants from the atp6-W136R mutant, and analyzed their properties, as described hereafter.

III.2.1 Isolation of revertants from the atp6-W136R mutant

To generate novel atp6 mutants, I have isolated revertants from atp6-W136R on glycerol plates. To this end ten independent cultures of the atp6-W136R mutant in liquid rich glucose medium were prepared and 10 8 cells from each culture were spread on the glycerol medium. After a few days of incubation at 28°C, clones with improved respiratory growth (Fig. 26) were taken from each plate and their ATP6 gene was sequenced, which led to identification of 4 different intragenic reversions: atp6-R136I, atp6-R136G, atp6-R136K, atp6-W136R, R179I.

III.2.2 Influence of the novel atp6 mutations on ATP synthase assembly/stability and Atp6p synthesis

Among the four revertants, we have found only one (atp6-W136R, R179I) where a free Atp9p-ring was present in BN-PAGE gels (Fig. 26B). In this revertant, the steady state accumulation of Atp6p was decreased in comparison to the WT and to the three other revertants (Fig. 26E). I next analyzed the in vivo labeling of mitochondrial translation products. Interestingly, in the atp6-W136R, R179I strain Atp6p synthesis was up-regulated as in the atp6-L183P strain, whereas the synthesis of this protein was normal in the three other revertants (Fig. 26C andD). were extracted and loaded on the SDS-PAGE gel (100 000 cpm per lane). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager. E: SDS-PAGE analysis of total mitochondrial proteins. Mitochondrial proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp6p and porine.

III.2.3 Hypothesis

As described in the literature, Atp6p is the last subunit to be incorporated into the ATP synthase complex and unassembled Atp6p is known to be highly sensitive to proteolytic degradation, which indicates that this protein is post-translationally controlled to avoid accumulation of unassembled Atp6p. However, Atp6p seems to be also subject to translational controls: in yeast strains failing to express or assemble the -F 1 and -F 1 proteins, translation of Atp8p and Atp6p is strongly down regulated, presumably as a means to prevent the accumulation of free F O particles that have the potential to uncouple the mitochondrial membrane (Rak and Tzagoloff 2009a;[START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF]. The data presented above suggest that atp6 mutations that compromise the assembly of Atp6p results in a higher rate of Atp6p synthesis. This led us to hypothesize that the Atp6p-less ATP synthase complex that forms before incorporation of Atp6p controls the synthesis of Atp6p as a means to couple the synthesis of Atp6p to its assembly (Fig. 27). To test this hypothesis I have combined the atp6-L183P mutation with mutations that prevent synthesis or assembly of other components of the ATP synthase, and we have then determined whether the synthesis of the mutated Atp6p is still up-regulated. The goal was to know which part of ATP synthase is necessary for this up-regulation.

III.2.3.1 Construction of deletion mutant strains

Deletions of genes encoding subunits of ATP synthase or genes encoding proteins that take part in ATP synthase biogenesis were done in the WT and in the atp6-L183P mutant. As A common feature of ATP synthase mutants is an enhanced production of ρ -/ρ 0 cells. I therefore systematically scored the number of ρ -/ρ 0 cells in the cultures of the different strains I constructed (see Table 8).

III.2.3.2 The catalytic head of F 1 domain is necessary for up-regulation of Atp6p synthesis

As expected, strains atp1Δ, atp11Δ, atp12Δ, and fmc1 I have constructed grew poorly on glucose supplemented with ethidium bromide (EtBr), a drug that prevents propagation of mtDNA (Fig. 29). This is because F 1 is required to energize the mitochondrial inner membrane when yeast is unable to respire [START_REF] Giraud | The absence of the mitochondrial ATP synthase delta subunit promotes a slow growth phenotype of rho-yeast cells by a lack of assembly of the catalytic sector F1[END_REF][START_REF] Lefebvre-Legendre | F1-catalysed ATP hydrolysis is required for mitochondrial biogenesis in Saccharomyces cerevisiae growing under conditions where it cannot respire[END_REF]. It is to be noted that phenotype of the fmc1Δ strain manifests Analysis of translation of mitochondrially encoded proteins clearly showed that there is neither Atp6p nor Atp8p synthesis in the examined deletion strains without distinction of the mtDNA background (Fig. 30A). It was demonstrated before that synthesis of Atp6 and Atp8 proteins is F 1 -dependent (Rak and Tzagoloff 2009a). My result confirmed those data (Fig. 30A). In fmc1Δ strains grown at 35 -37°C, where there is also a substantial but not complete lack of F 1 , the synthesis of Atp6p and Atp8p was only moderately affected, if at all (Lefebvre-Legendre, [START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF]. Interestingly, up-regulation of Atp6p synthesis was not longer observed in atp6-L183P cells lacking Fmc1p (Fig. 30B). Hence we can say that sufficient amount of F 1 -catalytic head is required for this up-regulation. for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12% urea/glycerol polyacrylamide gel and 17.5% polyacrylamide gel (the same amount of radioactivity per line). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager.

III.2.3.3 The Atp9p-ring is also necessary for up-regulation of Atp6p synthesis

Combining the atp6-L183P mutation to ATP9 deletion is not easy to achieve.

Therefore, to test the influence of Atp9p on the synthesis of Atp6p in the atp6-L183P mutant, I deleted AEP1 and AEP2 genes, which are both required to express Atp9p. However, lack of any of their protein products very strongly affects the stability of mtDNA. In the laboratory, it was shown that the nuclear ATP9-5 gene from Podospora anserina can partially complement the yeast atp9Δ strain and stabilizes mtDNA. I introduced a plasmid bearing ATP9-5 gene into the WT and atp6-L183P strains and then transformed them with aep1::KanMX4 and aep2::KanMX4 deletion cassettes. The ATP9-5 gene is under control of Tet-O promoter and its expression can be shut down in the presence of doxycycline. The phenotype analysis showed that growth on glycerol of the aep1Δ and aep2Δ strains is dependent on the presence of Atp9-5 protein, because they stopped growing on glycerol in the presence of doxycycline (Fig. 31A). As it will be shown below, these strains as expected failed to express the mitochondrial ATP9 gene. The up-regulation of Atp6p conferred by the atp6-L183P mutation was largely lost upon deletion of AEP1 and AEP2 (Fig. 31B). Even though, they produce functional Atp9-rings from the nuclear ATP9-5 gene, they do so very poorly. Thus, it can be inferred that a normal expression of Atp9p is needed for the up-regulation of Atp6p in the atp6-L183P mutant, similarly to what was observed with a null mutation in FMC1 where expression of F 1 is dramatically but not totally compromised (see above). urea/glycerol gel (the same amount of radioactivity per each line). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager.

III.2.3.4 Assembly factors Atp10p and Atp23p also play a role in the regulation of

Atp6p synthesis

I next examined, whether proteins known to take part in the assembly/maturation of Atp6p (Atp10p and Atp23p) are required for the up-regulation of Atp6p synthesis in the atp6-L183P mutant. Atp10p protects newly synthesized Atp6 against mitochondrial proteases until its assembly and/or helps its incorporation into the ATP synthase (Ackerman and Tzagoloff 1990b;[START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]). Atp23p has a double function (Zeng, Neupert et al. 2007c). As a metalloprotease, it cleaves off the first 10 aa of Atp6p, a peptide that facilitates the interaction of Atp6p with Atp9p. Its second function is required to fold Atp6p [START_REF] Osman | Prohibitins interact genetically with Atp23, a novel processing peptidase and chaperone for the F1Fo-ATP synthase[END_REF]Zeng, Neupert et al. 2007c). In the laboratory we have a plasmid encoding catalytically inactive Atp23p (atp23-E168Q). The presence of this plasmid in atp23Δ strains permits yeast cells to fold Atp6p despite their inability to mature Atp6p. It allowed me to determine which function, perhaps both or none, is required for Atp6p up-regulation in the atp6-L183P mutant.

As expected, the atp10Δ and atp23Δ strains I constructed were unable to grow on glycerol (Fig. 32A andB). In accordance with literature [START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]), Atp6p synthesis was almost normal in atp10Δ strains with a wild type mtDNA (Fig. 33A). The level of Atp6 synthesis in atp10Δ with the atp6-L183P mtDNA was normal too, indicating that the up-regulation of Atp6p, seen with mutations in Atp6p that compromise its assembly, depend upon Atp10p. The steady state levels of Atp6p in the atp10Δ strains were strongly diminished due to rapid degradation (Fig. 33B), as was shown before [START_REF] Tzagoloff | Atp10p assists assembly of Atp6p into the F0 unit of the yeast mitochondrial ATPase[END_REF]).

Fig. 33A shows that presence of Atp23p is not necessary for WT Atp6p synthesis. As expected Atp6p cannot be maturated and the protein migrates a bit slower than its maturated form, as shown in (Zeng, Neupert et al. 2007c). However atp23Δ in atp6-L183 mtDNA background results in the loss of Atp6p synthesis up-regulation. In atp23Δ strain where I had introduced a plasmid encoding catalytically inactive form of Atp23p (atp23-E168Q) Atp6p

up-regulation was still present. I can conclude that the assembly function of Atp23p but not its proteolytic activity is required for the up-regulation of Atp6p synthesis in the atp6-L183P mutant. After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager. B:

SDS-PAGE analysis of total mitochondrial proteins. Mitochondrial proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp6p, Ade13p and porine.

III.2.3.5 Influence of the central and the peripheral stalks on up-regulation of Atp6p synthesis

The central stalk is composed of three subunits: γ, δ and ε encoded by ATP3, ATP16

and ATP15 genes, respectively. Deletion of the first two results in 100%  -/ 0 cells, due to massive proton leaks through the F O , whereas cultures of atp15Δ cells retain a significant proportion of   cells. Thus, to test the influence of the central stalk on the synthesis of Atp6p in the atp6-L183P mutant I deleted the ATP15 gene. Peripheral stalk stabilizes the connection between catalytic the head and the proton channel in ATP synthase. To determine how it influences Atp6p synthesis in the atp6-L183P mutant, I deleted the ATP7 gene, encoding subunit d. The loss of ATP7 also increases the production of  -/ 0 cells, but usually not to 100%.

As expected, neither atp15Δ nor atp7Δ could grow on non-fermentable carbon source (Fig. 34A andB). In the absence of subunit ε Atp6p is still synthesized both in the WT as well as in the atp6-L183P backgrounds (Fig. 34C). Deletion of ATP15 did not specifically influence Atp6p synthesis in the WT background. However in the atp6-L183P cells, the effect of Atp6p synthesis up-regulation disappeared and the synthesis was comparable to that of WT.

Unfortunately the atp7Δ strain produced too many ρ -/ρ 0 cells rendering the results less clear even though they suggested that synthesis of Atp6p was no longer up-regulated in the atp6-L183P mutant (Fig. 34C). I conclude that the presence of central and peripheral stalks in ATP synthase sub-complex is necessary for appearance of Atp6p synthesis up-regulation in the atp6-L183P mutant.

Considered together the results strongly support our hypothesis that a partial ATP synthase assembly lacking only Atp6p (see Fig 27) drives the synthesis of Atp6p, which provides a means to couple the synthesis of this protein to its assembly, and that this control depends on the two proteins (Atp10p and Atp23p) known to be involved in the assembly of Atp6p.

III.3. REGULATION OF EXPRESSION OF ATP9

The results shown in the previous section indicate that Atp6p synthesis is coupled to its assembly. In this section, I present results indicating that the synthesis of Atp9p, the protein partner of Atp6p within the ATP synthase, is also regulated in relation with its assembly.

In yeast S. cerevisiae expression of mitochondrially encoded ATP9 gene is controlled by at least three proteins: Aep1, Aep2 and Atp25. Aep1p and Aep2p interact with 5'UTR of ATP9 mRNA and are essential for translation. Aep2p also takes part in mRNA maturation, its stability and/or initiation of translation [START_REF] Finnegan | Characterization of a yeast nuclear gene, AEP2, required for accumulation of mitochondrial mRNA encoding subunit 9 of the ATP synthase[END_REF][START_REF] Payne | Properties of two nuclear pet mutants affecting expression of the mitochondrial oli1 gene of Saccharomyces cerevisiae[END_REF][START_REF] Payne | Characterization of a second nuclear gene, AEP1, required for expression of the mitochondrial OLI1 gene in Saccharomyces cerevisiae[END_REF]). Atp25p is cleaved in two fragments of similar length: the C-terminal part is responsible for ATP9 mRNA stability, N-terminal part would be a chaperon needed for formation of the Atp9-ring [START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF]. It is not known, whether this cleavage occurs before or after the import of Atp25p into mitochondria. It is believed that Atp9-ring assembles separately and then interacts with the F 1 . The F 1 -Atp9-ring sub-complex finally associates with a second sub-complex containing the peripheral stalk, Atp8p and Atp6p, to form fully assembled ATP synthase [START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF].

It is to be mentioned that so far detailed analysis of the roles of these proteins in the ATP9 gene expression was very difficult because of strong instability of mtDNA in strains from which AEP1, AEP2 or ATP25 genes had been deleted. What we know about their functions was largely obtained using thermo-sensitive mutants in these genes. In the laboratory we showed that the nuclear ATP9-5 gene from Podospora anserina can partially complement a yeast strain in which the ATP9 gene has been replaced in-frame by ARG8m [START_REF] Bietenhader | Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution[END_REF]. It was expected that Atp9-5 protein expression does not require the presence of Aep1p, Aep2p and C-terminal part of Atp25p, as these proteins are only required to express the mitochondrial ATP9 gene, whereas the N-terminal part of Atp25p could still be needed to assemble the Atp9-5 rings. To test this hypothesis, we have deleted AEP1, AEP2 and ATP25 genes in a wild type yeast strain (MR6) transformed with a plasmid expressing ATP9-5 under the control of the Tet-O doxycycline repressible promoter (pAM19). We have also deleted these genes in the atp9 strain transformed with the same plasmid (pAM19), to see whether expression of the ARG8m marker requires Aep1p, Aep2p, and Atp25p or not.

III.3.1 Growth phenotypes of ATP9 relocation strains lacking AEP1, AEP2 and ATP25,

and containing a wild type mtDNA

III.3.1.1 ATP25

On non-fermentable carbon source (Fig. 35), the atp25Δ + ATP9-5 strain grew, although at a slower rate than the WT. This growth was inhibited by doxycycline, which indicated that it was totally dependent on the expression of ATP9-5. As expected, the atp25Δ + ATP9-5 strain recovered the normal respiratory growth upon transformation with a plasmid containing a wild type ATP25 gene, even in the presence of doxycycline, due to the restoration of expression of the mitochondrial ATP9 gene. When transformed with a plasmid expressing only the C-terminal part of the ATP25 gene, growth was not improved and remained entirely dependent on ATP9-5. Interestingly, growth of atp25Δ + ATP9-5 was significantly improved by the N-terminal part of ATP25, but only in the absence of doxycycline, presumably because the assembly of Atp9-5p becomes more efficient. Finally, after transformation with both the N-and C-terminal fragments, atp25Δ + ATP9-5 could grow in the presence of doxycycline but at a much slower rate than wild type yeast. This result indicates that expression of ATP9 is much less efficient when the two regions of Atp25p are expressed separately (see below).

III.3.1.2 AEP1, AEP2

Strains lacking the AEP1 or AEP2 gene could grow on glycerol if transformed with the ATP9-5 gene, and this growth was fully inhibited by doxycycline, which demonstrated that it was entirely dependent on ATP9-5. Upon transformation with AEP1 or AEP2 plasmid, growth was fully restored and was no longer inhibited by doxycycline, because of ATP9 expression restoration (Fig. 36).

III.3.2 Growth phenotypes of ATP9 relocation strains lacking AEP1, AEP2 and ATP25, and in which ATP9 is replaced by ARG8m

III.3.2.1 ATP25

WT strain (MR6) is respiratory competent but cannot grow on medium without arginine, because it lacks the ARG8 gene in nuclear DNA and does not have its mitochondrial version (ARG8m) in mtDNA. The strain (RKY26) where ATP9 has been replaced with ARG8m is respiratory deficient but can grow on glucose plates lacking arginine (-Arg). There is no difference in growth on -Arg medium between atp9Δ and atp9Δ + ATP9-5. Upon deletion of the ATP25 gene, the strain atp9Δ + ATP9-5 became arginine auxotroph, and this phenotype was suppressed by a transformation with a plasmid containing wild type ATP25 (Fig. 37A). Presence of the N-terminal part of ATP25 on plasmid did not restore growth on medium without arginine. This is not surprising because as a chaperon protein which facilitates oligomerization of the Atp9-ring the N-terminal fragment of Atp25p should not be needed for ARG8m expression. On the opposite the C-terminal part of Atp25p, which is known to stabilize ATP9 mRNA was able to restore arginine prototrophy (Fig. 37A). It is to be noted that only a part of the coding sequences of ATP9 have been replaced by ARG8m. It is possible that the 5' and 3' untranslated regions of the ATP9 transcripts that are preserved in the atp9 deletion strain are the sequences on which the C-terminal part of Atp25p is acting.

However the growth on arginine of the modified yeast strain was slower as compared to atp9Δ and atp9Δ + ATP9-5, and was not significantly improved upon addition of the Nterminal fragment of Atp25p (Fig. 37A). Perhaps the interaction of the C-terminal fragment of Atp25p is less efficient with the atp9::ARG8m transcripts than with intact ATP9 transcripts.

III.3.2.2 AEP1 and AEP2

Lack of Aep1p or Aep2p in the atp9Δ strain prevents growth on medium without arginine. Introduction of plasmids with the AEP1 or AEP2 genes restores arginine prototrophy. Thus, as the C-terminal fragment of Atp25p, the Aep1p and Aep2p proteins are required for expression of the ARG8m gene integrated at the mitochondrial ATP9 locus (Fig. 37B andC).

III.3.3 In vivo analysis of mitochondrially encoded proteins translation in ATP25, AEP1 and AEP2 deprived strains in WT and atp9::ARG8m mtDNA backgrounds

For these analyses, strains were cultivated in synthetic galactose media deprived of nutrients (uracil and leucine), making the presence of different plasmids indispensable for growth. Consistent with the growth tests described above, Atp9p (WT mtDNA) or Arg8m (atp9::ARG8m mtDNA) was not synthesized in the strains lacking AEP1, AEP2 or ATP25, whereas all other proteins encoded by the mitochondrial genome were normally synthesized (Fig. 38A -D). In atp25 + ATP9-5 strains with WT mtDNA and transformed with the C-terminal fragment of ATP25, the synthesis of Atp9p was almost undetectable, and remained poor when the N-terminal fragment was added. However trace amounts of Atp9p could be detected by Western blot in these strains (Fig. 39A). Similarly, the synthesis and steady state accumulation of Arg8m were very poor in strains containing only the C-terminal fragment of ATP25, and only a modest improvement was observed when both fragments of Atp25p were present (Fig. 39B), despite the ability of these strains to grow in media lacking arginine.

Western blots were performed to determine the amount of the C-terminal and N-terminal fragments of Atp25p in the cells, when these were expressed separately instead of an intact ATP25 gene. This could be done due to the inclusion of an HA epitope in these fragments. Somewhat surprisingly, both fragments were in a very low amounts compared to cells that express a wild type ATP25 gene (Fig. 39C). We do not know the reason of this. One possibility is that these fragments are more susceptible to degradation (outside and/or inside mitochondria) when they are not produced by the cleavage of a whole Atp25p protein.

Anyway, these data provide an explanation for the poor expression of Atp9p or Arg8p in the strains expressing these two fragments separately, alone or in combination.

III.3.4 Atp9-5 protein stimulates expression of mitochondrial ATP9 locus

One of the control strains used in the experiments described above (but not presented yet) was the WT strain where I introduced the plasmid encoding ATP9-5 gene. During the analysis of synthesis of mitochondrially encoded proteins I observed an unexpected phenomena. In the presence of the Atp9-5 protein, synthesis of mitochondrial Atp9p augmented about twice (Fig. 40A). This effect disappeared, if the strain was grown in a medium supplemented with doxycycline (Fig. 40B), indicating that the up-regulation of Atp9p is in some way provoked by the nuclearly encoded Atp9-5 protein. I next prepared total cellular protein extracts and separated them in denaturing gels, transferred them to nitrocellulose membranes and stained them with an antibody against yeast Atp9p. It is worth to mention that this antibody does not recognize Atp9-5p. The obtained result shows that the steady state level of Atp9p was exactly the same in the two analyzed strains, WT and WT + ATP9-5 (Fig. 40C). I checked, whether an excess of synthesized Atp9p aggregates by treating the protein samples with denaturing agents (as described in Materials and Methods) before loading on gel. No increase in the state accumulation was observed in WT + ATP9-5 (Fig. 40C), showing that the Atp9p proteins synthesized in excess compared to WT do not accumulate in cells but are eliminated instead, presumably by proteolytic degradation.

I then aimed to know, whether the mitochondrial expression of the ARG8m marker under control of the ATP9 regulatory sequences would also be stimulated by ATP9-5. As shown in Fig. 40D, the synthesis of Arg8p was indeed significantly stimulated, at least 2-fold.

In contrast to Atp9p, steady-state accumulation of Arg8p was increased in similar proportion (Fig. 40E), indicating that the excess of newly synthesized Arg8 proteins is not degraded.

Previous work has shown that in a atp9Δ strain, Atp6p is apparently poorly synthesized ( [START_REF] Bietenhader | Experimental relocation of the mitochondrial ATP9 gene to the nucleus reveals forces underlying mitochondrial genome evolution[END_REF], see also Fig. 42). Interestingly, the synthesis of Atp6p was fully restored upon transformation of atp9 with ATP9-5 (Fig. 42). This observation is consistent with the results presented in section III.2.3.3, showing that Atp9p positively influences the synthesis of Atp6p.

III.3.5 Expression of ATP9-5 in wild type yeast dramatically affects the stability of Atp6p.

As described above, expression of ATP9-5 in wild type yeast stimulates Atp9p synthesis in mitochondria. Although Atp6p synthesis remained normal, the steady-state accumulation of this protein was strongly diminished (see Fig. 40A andC). A possible explanation is the following. The Atp9-5 protein differs from yeast Atp9p by a number of amino acids, notably in the second transmembrane helix. This domain interacts with Atp6p, which allows protons to be transferred from Atp6p to the Atp9p-ring. Although the mitochondrial ATP9 gene is efficiently expressed in WT + ATP9-5, even better than in untransformed WT, it is possible that the co-expression of ATP9 and ATP9-5 is detrimental.

Indeed, although Atp9-5p and Atp9p are not structurally identical, they may associate with each other and form mixed oligomers that interact less efficiently with Atp6p than pure yeast Atp9p-rings. Because of this, the Atp6p protein may become more susceptible to degradation.

As explained below, the decreased stability of Atp6p in WT + ATP9-5 might be responsible for the up-regulation of ATP9 by ATP9-5.

III.3.6 Is Atp9p stability F 1 -dependent?

It has been proposed that ATP synthase F 1 domain activates Atp6p and Atp8p translation (Rak and Tzagoloff 2009a). Although expression of Atp9p is usually considered as F 1 -independent, we observed that the steady-state accumulation of this protein is dramatically reduced in -F 1 and/or -F 1 deficient strains (Fig. 41). Since Atp9p synthesis is not obviously impacted, it can be inferred that this effect is caused by post-translational degradation. The steady-state accumulation of Atp9p was not affected in the atp6 strain, indicating that it is the interaction between the Atp9 and/or Atp9p ring with F 1 that is critical for protection of Atp9p from degradation.

III.3.7 The up-regulation of Atp9p by Atp9-5 is enhanced by lack of Atp6p and is F 1dependent

I demonstrated above that in the presence of nuclear Atp9-5 protein synthesis of mitochondrial Atp9p is enhanced. Interestingly, this effect was dramatically exacerbated when the mitochondrial ATP6 gene was absent (Fig. 42 lane Δatp6 PaATP9-5). In the absence of the Atp9-5 protein, deletion of ATP6 also stimulated the Atp9p synthesis, yet somewhat less efficiently (Fig. 42 lane Δatp6). Although Atp9p can be synthesized in the absence of F 1 , we aimed to know, whether this up-regulation of Atp9p was F 1 -dependent. This was investigated with a null mutation of ATP12. Clearly, all the up-regulations of Atp9p synthesis stimulated by Atp9-5p and lack of Atp6p, disappeared upon deletion of ATP12 (Fig. 42 lanes Δatp12 to Δatp12 Δatp6 PaATP9-5).

As described below in the General Discussion, the data presented in this chapter indicate that Atp9p control its own synthesis, as a means to adjust the production of this protein in relation to its protein partners within ATP synthase.

GENERAL DISCUSSION

analysis revealed that chlorhexidine attenuates the phenotypes observed in the fmc1Δ strain such as lower oxygen consumption, weak energization of the inner mitochondrial membrane, reduced ATP synthase, presence of inclusion bodies in the matrix and almost no cristae in the mitochondria. The transcriptional analysis showed that chlorhexidine probably acts by changing the expression of the genes connected with the respiratory chain and retrograde signaling. Chlorhexidine was also active in the NARP-cybrid model. Altogether, the results of this project establish the usefulness of the yeast model for screening of drugs active against ATP synthase disorders and primary clues of their mode of action.

IV.1.3 Influence of the atp6-"cancer" mutations on the ATP synthase

55 mutations were identified in the mitochondrial MTATP6 gene in various tumors. I introduced in yeast an equivalent of five of these mutations, 8716A>G, 8914C>A, 8932C>T, 8953A>G and 9131T>C, which are in highly conserved positions between humans and yeast.

They resulted in the following amino acid changes in yeast Atp6p: atp6-K90E, atp6-P157T, atp6-P163S, atp6-I170V and atp6-L232P. The respiratory growth of none yeast atp6 mutant was affected, either at 28°C and 36°C. The atp6-P163S, atp6-K90E and atp6-I170V strains were more resistant in vivo to oligomycin, possibly because the mutations compromise the binding of the drug to the ATP synthase. On the contrary, the atp6-P157T and atp6-L232P

strains were more sensitive on the medium supplemented with oligomycin, probably because less drug is needed for the 20% threshold of ATP synthesis below which respiratory growth of yeast is affected [START_REF] Mukhopadhyay | Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media[END_REF][START_REF] Kucharczyk | Consequences of the pathogenic T9176C mutation of human mitochondrial DNA on yeast mitochondrial ATP synthase[END_REF]). The oxygen consumption in the mitochondria of the atp6 mutant strains cultured at 28°C was almost normal. Mitochondria from cultures grown at 36°C had a slightly reduced oxygen consumption in the atp6-P163S and atp6-L232P mutants, whereas respiration in the atp6-K90E strain was a little increased. Proportionate decreases and increases in the rate of ATP synthesis were observed. ATP hydrolysis activity was not changed in the atp6 mutants, with the exception of the atp6-K90E strain, in which it was increased ca. 50%. All mutants efficiently energized in the inner mitochondrial membrane and normally synthesized the proteins encoded by the mitochondrial DNA.

As described in the Introduction, it has been hypothesized that mutations of the mtDNA found in tumors have impact on their development or/and malignancy by compromising the energy-transduction activity of the mitochondria. However, so far, the biochemical consequences of these mutations have not been characterized. My results show that ATP synthase is not drastically affected by the ATP6 mutations found in tumors.

Obtained results does not permit to speculate about the role of these mutations in tumorigenesis.

IV.2 REGULATION OF Atp6p AND Atp9p SYNTHESIS IN RELATION TO THEIR ASSEMBLY IN YEAST ATP SYNTHASE IV.2.1 Atp6p

In the previously analyzed atp6-NARP mutants we have found one, atp6-L183P, where synthesis of the Atp6 protein was 3 -4 times faster than in the wild type. The levels of the transcripts were not changed and Atp6p accumulated less at the steady state levels as compared to the WT. This means that Atp6 was proteolytically degraded in the mitochondria because the L183P mutation impaired the assembly of Atp6p into the ATP synthase. I found that Atp6p synthesis was also up-regulated in another atp6 mutant (atp6-W136R,R179I)

where the assembly of Atp6p was partially compromised. In atp6 mutants where the assembly of Atp6p was normal, the synthesis of this protein was not increased.

It was shown previously that Atp6p translation is activated by the catalytic head [(αβ) 3 ] of the ATP synthase, which is believed to provide a means to avoid formation of free F O particles, i.e. not associated to F 1 , which would otherwise result in proton leak through the mitochondrial inner membrane (Rak and Tzagoloff 2009a;[START_REF] Rak | Modular assembly of yeast mitochondrial ATP synthase[END_REF]. This was quite a surprising discovery. Indeed, we know that non-assembled Atp6p is very rapidly degraded. Therefore it seems that Atp6p is subject to double control, at the level of synthesis and post-translationally. My results suggest that Atp6p synthesis is up-regulated when its assembly is compromised. Such an effect has never been observed before. Only mutations in Atp6p that affect the Atp6p assembly have the potential to increases its rate of synthesis. In current models, Atp6p is believed to be the last subunit to be incorporated into the ATP synthase, after the assembly of all the other subunits. Thus, we hypothesize that this Atp6p-less intermediate, rather than the sole F 1 component, activates the synthesis of Atp6p.

To test this hypothesis, I have combined the atp6-L183P mutation, which causes elevated Atp6p synthesis, with deletions of genes encoding subunits of the ATP synthase or genes encoding proteins that take part in the ATP synthase biogenesis. Consistently with previous reports (Rak and Tzagoloff 2009a), the deletions of the ATP1, ATP11 or ATP12 gene, that prevents formation of F 1 , completely inhibited the Atp6 synthesis. In the fmc1Δ strains there is also a severe, but not total, lack of F 1 catalytic head, but only at an elevated Atp6p synthesis. This can be viewed as a mechanism enabling coupling of Atp6p synthesis to its assembly (Fig. 43).

IV.2.2 Atp9p

In the third part of my work I have studied the regulation of expression of Atp9p.

There are three known proteins that are necessary for ATP9 expression: Aep1p, Aep2p and Atp25p. Normally, the loss of one of these proteins strongly destabilizes the mitochondrial genome. This effect could be overcome with the use of the plasmid encoding nuclear ATP9-5 gene from Podospora anserina, which allowed me to analyze the effects of deletions of AEP1, AEP2 and ATP25 in the strains where ATP9 gene was present or replaced by the ARG8m gene.

Strains aep1Δ and aep2Δ expressing Atp9-5p could grow on glycerol despite their inability to express the native ATP9 gene, which is consistent with previous reports indicating that these proteins are only required for synthesis of Atp9p. It has been reported that Atp25p is cleaved into two pieces of almost equal length. The N-terminal part plays a role in the oligomerization of the Atp9p-ring and the C-terminal part is needed for the ATP9 mRNA stability [START_REF] Zeng | ATP25, a new nuclear gene of Saccharomyces cerevisiae required for expression and assembly of the Atp9p subunit of mitochondrial ATPase[END_REF]. Strain atp25Δ expressing Atp9-5p was thus expected to be unable to grow on glycerol. It turned out that it could, but very slowly, much more slowly compared to strains aep1Δ and aep2Δ expressing Atp9-5p. Growth of atp25Δ expressing Atp9-5p was substantially improved upon addition of the N-terminal domain of Atp25p but not of its C-terminal domain. It can be inferred that the chaperon function of Atp25p is active on Atp9-5p.

Strains aep1Δ, aep2Δ and atp25Δ with the atp9::ARG8m mtDNA were unable to grow on plates lacking arginine, showing that the three proteins are absolutely necessary to express ARG8m from the ATP9 locus. Interestingly, arginine prototrophy was restored in the atp25Δ strain after transformation with a plasmid expressing the C-terminal domain of Atp25p, which is consistent with the role of this domain of Atp25 in the expression of the ATP9 locus. Taken together, the results show that Aep1p, Aep2p and Atp25p act only in the synthesis and assembly of atp9p and have no additional functions.

Complementation by the C-terminal and N-terminal domains of Atp25p expressed separately was rather inefficient. I found that this was due to poor accumulation of these polypeptides when expressed separately. It is possible that they are unstable and partially degraded in their way to organelle or inside mitochondrial. Why does a proper expression of Atp25p requires this protein to be synthesized in one block and then cleaved into two fragments is an interesting question.

Interestingly, expression of Atp9-5p in wild type yeast resulted in a dramatic increase of Atp9p synthesis in the mitochondria, whereas synthesis of other proteins encoded by mtDNA remained unchanged. At the steady state, the levels of Atp9p were not increased, meaning that the excess of synthesized Atp9p proteins is degraded. This is an interesting observation indicating that Atp9p is subject to post-translation control like many other subunits of the mitochondrial energy-transducing system. The results suggest also that Atp9p is subject to control at the level of synthesis.

Although synthesis of Atp6p was not modified in the wild type with Atp9-5p, the steady state levels of this protein were substantially decreased (by >50%). We reasoned that this lack of Atp6p could be what provoked the increased synthesis of Atp9p. Consistent with this hypothesis, Atp9p synthesis was also increased in the atp6Δ strain. However, synthesis of Atp9p was further stimulated when the atp6Δ strain expressed Atp9-5p, indicating that Atp9-5 protein by itself has the capacity to enhance Atp9p synthesis.

As already mentioned, previous work has shown that Atp6p synthesis is strongly down-regulated when the catalytic head of F 1 cannot form. We aimed to determine whether the up-regulation of Atp9p observed in strains expressing Atp9-5 and/or lacking Atp6p, was also dependent upon F 1 . Indeed, in strains lacking F 1 , Atp9p synthesis was no longer stimulated. We further observed, that the steady state levels of Atp9p were strongly diminished in strains unable to express F 1 . Thus, when F 1 is absent, Atp9p can still be synthesized but is largely degraded.

Taken together, these observations allow us to propose a scenario in which Atp9p controls its own synthesis, as a means to adjust the production of this protein relative to its protein partners within the ATP synthase, F 1 and Atp6p. When F 1 is absent, Atp9p is synthesized but is mostly degraded, because it cannot assemble with F 1 . An interesting scenario would be that the Atp9p-ring assembles on F 1 . If F 1 is absent, Atp9p remains essentially in a monomeric state and Atp9p monomers are unstable and rapidly degraded.

Atp6p, which is believed to be the last subunit to be incorporated into the ATP synthase, is not required for the assembly of the Atp9p ring. However, a 'nude' Atp9-ring, i.e. not associated to Atp6p, might be unstable and partially dissociates. This results in the accumulation of incomplete Atp9p-rings that are sensed as a signal that more Atp9p proteins are requested to complete the assembly of the ATP synthase (Fig. 43). When the Atp9-ring interacts with Atp6p, it becomes silenced and ceases to activate Atp9p synthesis. This scenario may provide an explanation for the up-regulation of Atp9p by Atp9-5p. Indeed, Atp9-5 possibly interacts less efficiently with Atp6p, which results in an enhanced degradation of Atp6p. As a consequence, incomplete Atp9p-rings accumulate and stimulate Atp9p synthesis. When there is no F 1 , incomplete Atp9p-rings do not accumulate. If the assembly of the ring requires the presence of F 1 : Atp9p synthesis is no longer stimulated.

Obviously it is too difficult to imagine that Atp9p, or incomplete Atp9p-rings, can activate Atp9p synthesis directly. This control probably involves proteins (Aep1, Aep2, the Cterminal domain of Atp25p) that are required to synthesize Atp9p, as well as the N-terminal domain of Atp25p that facilitates the assembly of the Atp9p-ring (Fig. 43). In future work, it will be interesting to determine how these proteins interact with each other and with ATP9 mRNA/Atp9p, to understand how synthesis of Atp9p is coupled to its synthesis. Ma thèse concerne deux sujets :

1. La construction des modèles de levure S. cerevisiae de mutations du gène mitochondrial ATP6 de l'ATP synthase découvertes chez des patients atteints de maladies neurologiques ou dans des tumeurs et analyser les effets biochimiques de ces mutations.

2. L'analyse de la régulation de la synthèse des sous-unités 6 et 9 dans les mitochondries de levure S. cerevisiae.

Ad.1

Dans notre laboratoire, la levure S. cerevisiae était utilisée avec succès pour analyser des effets de mutations du gène mitochondrial ATP6 de l'ATP synthase découvertes chez des patients atteints du syndrome NARP.

Le gène ATP6 code la sous-unite 6 (Atp6p) de l'ATP synthase. L'Atp6p avec l'anneau de sous-unités 9 forment le canal à protons dans la membrane interne de la mitochondrie. Dans la première partie de ce travail, j'ai construit des modèles de levure de deux nouvelles mutations du gène mitochondrial ATP6 découvertes chez des patients atteints du syndrome NARP (9185T>C and 9191T>C) et cinq mutations dans ce gène identifiées dans des tumeurs (8716A>G, 8914C>A, 8932C>T, 8953A>G et 9131T>C).

L'effet majeur de la mutation 9185T>C était une diminution de la vitesse de synthèse Les résultats de la deuxième partie de ma thèse ont permis une proposition de modèle où la synthèse de sous-unités 9 et 6 dépendent de leur assemblage dans le complexe d'ATP synthase.

  RESULTS .................................................................................................................................... III.1 Mutations in ATP6 gene and human diseases .............................................................. III.1.1 Analysis of the two newly identified NARP mutations ................................. III.1.2 Use of yeast Saccharomyces cerevisiae as a model organism for drug screening ..................................................................................................... III.1.3 Analysis of five mutations in ATP6 gene identified in human tumors ......... III.1.3.1 Respiratory growth and genetic stability of the yeast atp6 "cancer" mutants ....................................................................... III.1.3.2 Mitochondrial oxygen consumption ............................................. III.1.3.3 Mitochondrial ATP synthesis ....................................................... III.1.3.4 Mitochondrial ATP hydrolysis ..................................................... III.1.3.5 Measurements of inner mitochondrial membrane potential ........... III.1.3.6 Assembly/stability of the ATP synthase ....................................... III.2 Regulation of expression of ATP6 in relation to its assembly into the F 1 F O -ATP synthase .................................................................................................................. III.2.1 Isolation of revertants from the atp6-W136R mutant ................................... III.2.2 Influence of the novel mutations on ATP synthase assembly/stability ......... III.2.3 Hypothesis ................................................................................................. III.2.3.1 Construction of deletion mutant strains ........................................ III.2.3.2 The catalytic head of F 1 domain is necessary for up-regulation of Atp6p synthesis ..................................................................... III.2.3.3 The Atp9p-ring is also necessary for up-regulation of Atp6p synthesis .................................................................................... III.2.3.4 Assembly factors Atp10p and Atp23p also play a role in the regulation of Atp6p synthesis .................................................... III.2.3.5 Influence of the central stalk and the peripheral stalk on up-regulation of Atp6p synthesis ...............................................III.3 Regulation of expression of ATP9

ATP25

  genes, and containing wild type mtDNA ........................................ III.3.1.1 ATP25 .......................................................................................... III.3.1.2 AEP1, AEP2................................................................................. III.3.2 Growth phenotypes of ATP9 relocation strains lacking AEP1, AEP2 and ATP25 genes in which ATP9 is replaced by ARG8m .................................. III.3.2.1 ATP25 ..........................................................................................

  mtDNA backgrounds............................................................................... III.3.4 Atp9-5 protein stimulates expression of mitochondrial ATP9 locus ............ III.3.5 Expression of ATP9-5 in WT yeast dramatically affects the stability of Atp6p ........................................................................................................ III.3.6 Is Atp9p stability F 1 -dependent .................................................................. III.3.7 The up-regulation of Atp9p by Atp9-5p is enhanced by lack of Atp6p and is F 1 -dependent ................................................................................... GENERAL DISCUSSION ........................................................................................................ IV.1 Yeast models of human diseases show the influence of mutations in ATP6 gene on ATP synthase ..................................................................................................... IV.1.1 Consequences of the NARP atp6-S250P and atp6-L252P mutations on ATP synthase .......................................................................................... IV.1.2 Yeast-based assay for screening of drugs active against ATP synthase disorders ................................................................................................. IV.1.3 Influence of the atp6 "cancer" mutations on ATP synthase ........................ IV.2 Regulation of Atp6p and atp9p in relation to their assembly in yeast ATP synthase .................................................................................................................. IV.2.1 Atp6p ......................................................................................................... IV.2.2 Atp9p ......................................................................................................... BIBLIOGRAPHY...................................................................................................................,N,N',N'-tetramethyl-p-phenylenediamine TCAtrichloroacetic acid TIMtranslocase of the inner membrane TOMtranslocase of the outer membrane INTRODUCTION I.1 MITOCHONDRIA

  which brought him a Nobel Prize in 1978. The second Nobel Prize in the mitochondrial area was awarded in 1997 to Paul D. Boyer and John E. Walker for revealing the functioning of F 1 F O -ATP synthase.

Figure 1 -

 1 Figure 1 -Two alternatives scenarios of eukaryogenesis. A schema based on Koonin (Koonin 2010).

Figure 2 -

 2 Figure 2 -Structure of a mitochondrion. 1outer mitochondrial membrane, 2inner mitochondrial membrane, 3intermembrane space, 4 -mitochondrial cristae, 5mitochondria matrix (image from: http://microbewiki.kenyon.edu/images/7/7b/Mito_pic_diagram.gif).

Figure 3 -

 3 Figure 3 -Mitochondrial morphology, structure and dynamics. A: Three-dimensional reconstructions of cells expressing mito-DsRed1. The dashed circles indicate the position of nuclei.

Figure 4 -

 4 Figure 4 -Functions of mitochondria. A schema representing example proteins with various mitochondrial functions. The processes of: heme synthesis, β-oxidation of fatty acids, the urea cycle, synthesis of amino acids, biosynthesis of lipids and steroidogenesis are not shown. Based on[START_REF] Reichert | Mitochondriomics or what makes us breathe[END_REF]

Figure 5 -

 5 Figure 5 -A schema of mitochondrial genomes. A: A map of a human mitochondrial genome. Genes encoding subunits of respiratory chain complexes and ATP synthase are marked with different colors, tRNAs are marked in white, rRNAs in yellow, O H and O Lorigins of replication. (The schema made by Małgorzata Rak) B: A map of S. cerevisiae mitochondrial genome. Brown color represents exons, yellow coding introns and grey non-coding introns. The arrows indicate sites of initiation of transcription (The schema made by François Godard).

Figure 6 -

 6 Figure 6 -Protein import pathways of mitochondria (Harbauer, Zahedi et al. 2014).

Figure 7 -

 7 Figure 7 -A schema of the respiratory chain of mammals (A) and Saccharomyces cerevisiae (B).

Figure 8 -

 8 Figure 8 -A schema of F 1 F O -ATP synthase from Saccharomyces cerevisiae. IMSintermembrane space; IMinner membrane.

Figure 9 -

 9 Figure 9 -A schema illustrating ATP synthesis (A) and hydrolysis (B) by the catalytic head of the ATP synthase. The figure shows a 120° rotation of subunit γ, which corresponds to conformational changes in subunit β and synthesis or hydrolysis of ATP. The catalytic binding sites are marked according to their structural descriptions (β E , β HC , β DP and β TP )[START_REF] Abrahams | Structure at 2.8 A resolution of F1-ATPase from bovine heart mitochondria[END_REF][START_REF] Menz | Structure of bovine mitochondrial F(1)-ATPase with nucleotide bound to all three catalytic sites: implications for the mechanism of rotary catalysis[END_REF]) and indicated by various shapes[START_REF] Braig | Structure of bovine mitochondrial F(1)-ATPase inhibited by Mg(2+) ADP and aluminium fluoride[END_REF]. The sequence of events for the ATP synthesis is shown by red arrows and for ATP hydrolysis by blue arrows. The schema made by Róża Kucharczyk.

Figure 10 -

 10 Figure 10 -A two-channel model of generation of rotation by movement of protons through the F O domain of the ATP synthase proposed by Junge (Junge, Lill et al. 1997). Two half channels across the interface between the subunit a (Atp6p) and the c ring (the Atp9p-ring) are linked by the rotation of the c ring. The red arrow indicates the proton flux from the intermembrane space to the matrix. The schema made by Róża Kucharczyk.

Figure 11 -

 11 Figure 11 -A schema illustrating the origin of ATP synthase subunits and proteins essential for expression of mitochondrially encoded subunits and assembly of the enzyme (the schema made by Jean-Paul di Rago).

Figure 12 -

 12 Figure 12 -Atp11p and Atp12p-mediated F1 assembly. CScatalytic sites, NCSnon-catalytic sites. Based on (Ackerman 2002).

Figure 14 -

 14 Figure 14 -Polycistronic transcript of ATP9, tRNA Ser and VAR1. Transcription initiation sites are marked with horizontal arrows. Cleavage sites of the polycistronic transcripts that produce mature messengers are shown by asterisks. Based on (Rak, Zeng et al. 2009b).

Figure 15 -

 15 Figure 15 -Polycistronic transcript of COX1, ATP8, ATP6 and ENS2. Transcription initiation site is marked with horizontal arrow. Cleavage sites of the polycistronic transcripts that produce mature messengers are shown by asterisks. Based on (Rak, Zeng et al. 2009b).

Figure 16 -

 16 Figure 16 -Assembly of ATP synthase from S. cerevisiae according to Kucharczyk (Kucharczyk, Zick et al. 2009c).

Figure 17 -

 17 Figure 17 -Modular assembly of ATP synthase. A schema based on Rak and Tzagoloff (Rak, Gokova et al. 2011).

Figure 18 -

 18 Figure 18 -Maturation and assembly of Atp6p. Atp6p is marked in violet, the 10aa pre-sequence in blue (leader peptide).

Figure 19 -

 19 Figure 19 -Schema illustrating deletion of genes in yeast nuclear DNA with theKanMX4 deletion cassette. Positions of primers designed for every gene are indicated with arrows.

  DNA Extraction of yeast DNA was used to obtain templates for PCR. 1.2 10 9 of the cells were centrifuged. The pellet was re-suspended in solution I (1 M sorbitol, 0.1 M EDTA pH 7.5) containing 200 µg of Zymolyase 20T. Cells were incubated for 1 hour at 37°C to have the cell wall digested. Spheroplasts were centrifuged at 6,000 g for 5 minutes and the pellet was re-suspended in solution II (50 mM Tris-HCl, 20 mM EDTA pH 7.4, SDS for the final concentration of 1% m/v). After 30 minutes of incubation at 65°C, potassium acetate was added to final concentration of 1.4 M. The suspension was incubated at 4°C for 30

  lengths were 485 nm for excitation and 525 nm for emission of fluorescence. The obtained values are arbitrary and semi-quantitative.A volume of mitochondria corresponding to 150 µg/ml of mitochondrial proteins suspended in 1 ml of the respiratory buffer (0.65 M mannitol, 0.36 mM EGTA, 5 mM Trisphosphate, 10 mM Trismaleate, pH 6.8) in the presence of 0.5 µg/ml of rhodamine 123, was added to the spectrofluorometer cuvette. 10 mM ethanol was used as a substrate to generate the mitochondrial membrane potential. Ethanol was preferred because auto-fluorescence of rhodamine can be excited by NADH. Presence of a respiratory chain substrate leads to generation of the membrane potential, rhodamine 123 entering the mitochondria and quenching of fluorescence. Maintaining of the mitochondrial membrane was measured in the presence of different compounds: 75 µM ADP, 4 µM oligomycin, 4 µM CCCP, 2 mM KCN, 0.2 mM ATP. KCN was used as an inhibitor of cytochrome c oxidase.

  3 M MOPS. The samples were stocked at -20°C before quantification of the produced ATP. Quantification of the produced ATP was based on bioluminescent reaction of luciferin oxidation to oxyluciferin by the luciferase enzyme. One ATP molecule was used per one molecule of oxidized luciferine. Light emitted during this reaction was measured in a bioluminometer. Bioluminescence Assay Kit CLS II from Roche Applied Science was used. The previously collected samples were thawed on ice and centrifuged for 5 minutes at 4°C, 16,000 g. 1 µl was mixed with 200 µl solution containing: 0.1 M Tris, 0.2 mM EDTA at pH 7.75 and luciferine/luciferase. Emission of the light measured in the bioluminometer was proportional to amount of ATP in the mix. First the calibration curve was determined using 10 µM ATP from 0 to 100 µL. Calculation of ATP in the samples was based on this curve and was expressed in nmol of ATP per milligram of mitochondrial proteins per minute. Values obtained from the samples collected in the presence of oligomycin represent ATP that was not produced by ATP synthase (basal ATP in mitochondria).

(

  NH 4 )Mo 7 O 24 4H 2 O, 28.8 mM FeSO 4 7H 2 O), mixed on vortex and incubated in the dark for 10 minutes. Pi released in the reaction associates with molybdate and forms phosphomolybdate that when reduced by iron (II) sulfate in acidic environment becomes violet. The intensity of the violet color was measured in a spectrophotometer at wave length 610 nm. At the same time the calibration curve was made with growing concentration of 1 mM KH 2 PO 4 (from 0 to 300 nmol of Pi). ATP hydrolyzed by complex V was presented as µmol of Pi produced per minute per milligram of mitochondrial proteins. Measurements made in the presence of oligomycin represent the ATPase activity of free F 1 or other enzymes with ATPase activity. Percentage of oligomycin inhibition corresponds to the complete complex of ATP synthase.

Figure 20 -

 20 Figure 20 -Growth phenotypes of yeast strains bearing the MTATP6 mutations identified in cancer cells from patients. Freshly grown cells of wild type yeast (WT) and atp6 mutants were serially diluted and 5μl of each were spotted onto plates containing glucose, glycerol and glycerol supplemented with oligomycin (0.25 µg/ml and 0.5 µg/ml) media. The plates were incubated at 28°C and 36°C. Photos were made after 3 days of incubation.

Figure 21 -

 21 Figure 21 -Energization of inner mitochondria membrane of WT (MR6), atp6-P157T (RKY60), atp6-P163S (RKY61), atp6-K90E (RKY62), atp6-I170V (AKY1) and atp6-L232P (AKY2) strains.

RESULTS 122 Figure 22 -

 12222 Figure 22 -Analysis of assembly/stability of the ATP synthase complex in the WT (MR6), atp6-P157T (RKY60), atp6-P163S (RKY61), atp6-K90E (RKY62), atp6-I170V (AKY1) and atp6-L232P (AKY2) strains. A and B: BN-PAGE analysis of mitochondrial protein extracted with 2% digitonin from strains cultivated at 28°C (A) and 36°C (B). Fully assembled ATP synthase complexes (V 2dimeric. V-monomeric, F 1free F 1 ) are stained in-gel by Coomassie brilliant blue and their ATPase activity. C and D: BN-PAGE analysis of mitochondrial proteins transferred to a nitrocellulose membrane and stained with antibodies against different subunits of ATP-synthase. Proteins were obtained from mitochondria derived from cultures at 28°C (C) and 36°C (D). E: SDS-PAGE analysis of total protein extracts from cultures at 28°C. Proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp6p, Atp1p, Atp4p and porine.

Figure 23 -

 23 Figure 23 -Mitochondrial protein synthesis. Proteins encoded by mtDNA of the WT (MR6), atp6-P157T (RKY60), atp6-P163S (RKY61), atp6-K90E (RKY62), atp6-I170V (AKY1) and atp6-L232P (AKY2) strains were labeled with [ 35 S]-(methionine + cysteine) for 20 min. at 30°C in the presence of cycloheximide (7.5 mg/ml) to inhibit cytosolic protein synthesis. Total proteins extracts were prepared from cells and separated on 12% urea/glycerol polyacrylamide gel and 17.5% polyacrylamide gel (the same amount of radioactivity per line). After electrophoresis the gels were dried and visualized with a Phosphoimager.

Figure 24 -

 24 Figure 24 -Analysis of WT and atp6-L183P strains. A: Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells with [35S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The mitochondrial membranes were extracted and loaded on the SDS-PAGE gel (100 000 cpm per lane). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager. B: Pulse chase analysis. Proteins encoded by mtDNA were labeled in whole cells with [ 35 S]-(methionine+cysteine) for 5, 20 and 90min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The mitochondrial membranes were extracted and loaded on the SDS-PAGE gel (100 000 cpm per lane). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager. C: Northern blot analysis of mitochondrial transcripts. Total mitochondrial RNAs were isolated from strains grown for 6 -8 generations in liquid YPGalA at 28°C. The RNAs were separated in formaldehyde agarose gel and transferred to a nitrocellulose membrane. The same membrane was hybridized successively with 32 P-radiolabeled DNA probes specific for 15S rRNA, ATP6L and ATP6S transcripts. D: SDS-PAGE analysis of total mitochondrial proteins. Mitochondrial proteins (10 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp6p, Atp9p and porine. E: BN-PAGE analysis of ATP synthase. Mitochondria were solubilized with 2% digitonin. The digitonin-extracted proteins (50 µg) were separated by BN-PAGE, transferred to a nitrocellulose membrane, and hybridized with antibody against Atp9p.

Figure 25 -

 25 Figure 25 -Growth phenotypes of the atp6-W136R revertant strains. Freshly grown strains of WT (MR6) and atp6 mutants: atp6-W136R (RKY39), atp6-R136I (RKY39-1G), atp6-R136G (RKY39-5G), atp6-W136R,R179I (RKY39-6G), atp6-R136K (RKY39-16G) were spotted on rich glycerol medium (YPGA). The plate was incubated at 28°C and photographed after 7 days of incubation.

Figure 26 -

 26 Figure 26 -BN-PAGE, mitochondrial protein synthesis and SDS-PAGE analysis of the WT (MR6) and atp6 mutants: atp6Δ (MR10), atp6-L247P (RKY38), atp6-L183R (MR14) and atp6-L183P (RKY20) from panel A, WT (MR6), atp6-W136R (RKY39), atp6-R136I (RKY39-1G), atp6-R136G (RKY39-5G), atp6-W136R,R179I (RKY39-6G), atp6-R136K (RKY39-16G). A and B: BN-PAGE analysis of ATP synthase. Mitochondria from the indicated strains were solubilized with 2% digitonin. The digitonin-extracted proteins (50 µg) were separated by BN-PAGE, transferred to a nitrocellulose membrane, and hybridized with an antibody against Atp9p. C and D: Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells (panel C) or the mitochondrial membranes (panel D)

Figure 27 -

 27 Figure 27 -The schema illustrating hypothesis of influence of ATP synthase sub-complex on Atp6p synthesis.

  mentioned in the Introduction, ATP synthase is composed of two functional domains: F 1 , formed of the so-called catalytic head and the central stalk, and of F O , formed of the proton channel and the peripheral stalk. I deleted different genes representing each part of the enzyme. To deprive ATP synthase of the catalytic head I deleted ATP1 gene that encodes subunit α, ATP11, ATP12 and FMC1 genes that encode chaperons needed for α/β hexamer assembly. Regarding the central stalk, I deleted the ATP15 gene that encodes subunit ε. To test the influence of the peripheral stalk I deleted the ATP7 gene encoding subunit d. To know if Atp9p is required for the up-regulation of Atp6p, I deleted two genes, AEP1 and AEP2, encoding proteins that take part in ATP9 expression. I also deleted genes ATP10 and ATP23 that encode proteins taking part in Atp6p maturation and assembly. To create these strains I used KanMX4 disruption cassettes (see Materials and Methods). Below I present the schema showing constructed deletion mutants (Fig.28).

Figure 28 -

 28 Figure 28 -The schema presenting constructed deletion mutant strains. Collection of strains with deletions in different nuclear loci of mitochondrial genes in WT and atp6-L183P mtDNA backgrounds to study regulation ofAtp6p synthesis in relation to its assembly into the ATP synthase.

  only at elevated temperature[START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF], hence all analysis aimed to determination of the influence of the lack of Fmc1p were performed on the basis of cells grown at 35°C.

Figure 29 -

 29 Figure 29 -Growth phenotypes of F 1 deletion strains. Freshly grown wild type (MR6), ρ 0 (MR6 ρ 0 ), atp6-L183P (RKY20), atp1Δ (AKY36), atp1Δ [atp6-L183P] (AKY37), atp11Δ (AKY40), atp11Δ [atp6-L183P] (AKY41), atp12Δ (AKY42), atp12Δ [atp6-L183P] (AKY43), fmc1Δ (AKY76), fmc1Δ [atp6-L183P] (AKY77), were spotted on plates with rich glucose (YPDA), rich glucose supplemented with G418 (200 µg/ml), rich glycerol (YPGA) and rich glucose supplemented with ethidium bromide (BET, 40 µg/l). The plates were incubated at 28°C or 35°C and photographed after 6 days of incubation.

Figure 30 -

 30 Figure 30 -Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells from the wild type (MR6), atp6-L183P (RKY20), atp1Δ (AKY36), atp1Δ [atp6-L183P] (AKY37), atp11Δ (AKY40), atp11Δ [atp6-L183P] (AKY41), atp12Δ (AKY42), atp12Δ [atp6-L183P] (AKY43), fmc1Δ (AKY76), fmc1Δ [atp6-L183P] (AKY77) strains with [ 35 S]-(methionine+cysteine)

Figure 31 -

 31 Figure 31 -Analysis of WT (MR6), ρ 0 (MR6 ρ 0 ), atp6-L183P (RKY20), ATP9-5 (AKY65), ATP9-5 [atp6-L183P] (AKY75), aep1Δ ATP9-5 [atp6-L183P] (AKY63) and aep2Δ ATP9-5 [atp6-L183P] (AKY64). A: Growth phenotypes of the aep1 and aep2 deletion strains. Freshly grown strains were spotted on plates with rich glucose (YPDA), rich glycerol (YPGA) and rich glycerol supplemented with doxycycline (DOX, 40 µg/ml). The plates were incubated at 28°C and photographed after 6 days of incubation. B: Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12%

Figure 32 -

 32 Figure 32 -Growth phenotypes of atp10 (panel A) and atp23 (panel B) deletion strains. Freshly grown wild type (MR6), ρ 0 (MR6 ρ 0 ), atp6-L183P (RKY20), atp10Δ (AKY38), atp10Δ [atp6-L183P] (AKY39), atp23Δ (AKY28) and atp23Δ [atp6-L183P] (AKY27) were spotted on plates with rich glucose (YPDA), rich glucose supplemented with G418 (200 µg/ml) and rich glycerol (YPGA). The plates were incubated at 28°C and photographed after 6 days of incubation.

Figure 33 -

 33 Figure 33 -Mitochondrial protein synthesis and SDS-PAGE of wild type (MR6), atp6-L183P (RKY20), atp10Δ (AKY38), atp10Δ [atp6-L183P] (AKY39), atp23Δ (AKY28), atp23Δ [atp6-L183P] (AKY27), atp23-E168Q (AKY28*) and atp23-E168Q [atp6-L183P] (AKY27*). A: Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells from strains with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12% urea/glycerol gel and 17.5% polyacrylamide gel (the same amount of radioactivity per each line).

Figure 34 -

 34 Figure 34 -Growth phenotypes and mitochondrial protein synthesis of wild type (MR6), ρ 0 (MR6 ρ 0 ), atp6-L183P (RKY20), atp15Δ (AKY32), atp15Δ [atp6-L183P] (AKY33), atp7Δ (AKY34) and atp7Δ [atp6-L183P] (AKY35). A: Growth test. Freshly grown cultures were spotted on plates with rich glucose (YPDA), rich glucose supplemented with G418 (200 µg/ml) and rich glycerol (YPGA). The plates were incubated at 28°C and photographed after 6 days of incubation. B:Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells from strains with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extract from cells were prepared and loaded on 12% urea/glycerol gel and 17.5% polyacrylamide gel (the same amount of radioactivity per each line). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager.

Figure 35 -

 35 Figure 35 -Growth phenotypes of atp25Δ strains in WT mtDNA background. Freshly grown strains WT (MR6), ρ 0 (MR6 ρ 0 ), atp25Δ PaATP9-5 (AKY74), atp25Δ PaATP9-5 pATP25 (AKY74/pRK56), atp25Δ PaATP9-5 pN-ter (AKY74/pG29ST32), atp25Δ PaATP9-5 pC-ter (AKY138) and ), atp25Δ PaATP9-5 pN-ter pC-ter (AKY139) were spotted on rich glycerol medium (YPGA) and rich glycerol supplemented with doxycycline (DOX, 40 µg/ml). The plates were incubated at 28°C and photographed after indicated number of days.

Figure 36 -

 36 Figure 36 -Growth phenotypes of aep1Δ and aep2Δ strains in WT mtDNA background. Freshly grown strains WT (MR6), ρ 0 (MR6 ρ 0 ), aep1Δ PaATP9-5 (AKY60), aep1Δ PaATP9-5 pAEP1 (AKY60/pRK54), aep2Δ PaATP9-5 (AKY61) and aep2Δ PaATP9-5 pAEP2 (AKY61/pRK57) were spotted on rich glycerol medium (YPGA) and rich glycerol supplemented with doxycycline (DOX, 40 µg/ml). The plates were incubated at 28°C and photographed after 6 days.

Figure 37 -

 37 Figure 37 -Growth phenotypes of atp25Δ, aep1Δ and aep2Δ strains in atp9::ARG8m mtDNA background. Freshly grown strains WT (MR6), atp9Δ (RKY26), atp9Δ PaATP9-5 (AMY10), atp25Δ PaATP9-5 atp9Δ (RKY95), atp25Δ PaATP9-5 pATP25 atp9Δ (RKY89), atp25Δ PaATP9-5 pN-ter atp9Δ (RKY95/pG29ST32), atp25Δ PaATP9-5 pC-ter atp9Δ (AKY140), atp25Δ PaATP9-5 pN-ter pC-ter atp9Δ (AKY141), atp9Δ PaATP9-5 pAEP1 (RKY96), atp9Δ PaATP9-5 pAEP1 aep1Δ (RKY92), atp9Δ PaATP9-5 aep1Δ (RKY93), atp9Δ PaATP9-5 pAEP2 (RKY97), atp9Δ PaATP9-5 pAEP2 aep2Δ (AKY52) and atp9Δ PaATP9-5 aep2Δ (RKY94) were spotted on rich glucose (+arginine) and synthetic glucose without arginine (w/o arginine) plates. The plates were incubated at 28°C and photographed after indicated number of days.

Figure 38 -

 38 Figure 38 -Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells from wild type with the pATP9-5 (AKY65), atp25Δ PaATP9-5 (AKY74), atp25Δ PaATP9-5 pATP25 atp9Δ (RKY89), atp25Δ PaATP9-5 atp9Δ (RKY95), aep1Δ PaATP9-5 (AKY60), aep2Δ PaATP9-5 (AKY61), wild type (MR6), atp9Δ PaATP9-5 (AMY10), atp9Δ PaATP9-5 aep1Δ (RKY93) and atp9Δ PaATP9-5 aep2Δ (RKY94) strains with [ 35 S]-(methionine+cysteine) for 20 min.in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12% urea/glycerol gel (to separate Cox3p and Atp6p) and 17.5% SDS polyacrylamide gel (to separate Atp8p and Atp9p). The same amount of radioactivity per each line was loaded. After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager.

Figure 39 -

 39 Figure 39 -SDS-PAGE analysis of total mitochondrial proteins. Total protein extracts were prepared from WT PaATP9-5 (AKY65), atp25Δ PaATP9-5 (AKY74), atp25Δ PaATP9-5 pATP25 (AKY74/pRK56), atp25Δ PaATP9-5 pN-ter (AKY74/pG29ST32), atp25Δ PaATP9-5 pC-ter (AKY138), atp25Δ PaATP9-5 pN-ter pC-ter (AKY139), atp9Δ (RKY26), atp25Δ PaATP9-5 atp9Δ (RKY95), atp25Δ PaATP9-5 pATP25 atp9Δ (RKY89), atp25Δ PaATP9-5 pN-ter atp9Δ (RKY95/pG29ST32), atp25Δ PaATP9-5 pC-ter atp9Δ (AKY140) and atp25Δ PaATP9-5 pN-ter pCter atp9Δ (AKY141). Mitochondrial proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp9p (panel A), Arg8m (panel B) Ade13p, porine (panel A and B) and HA (panel C).

Figure 40 -

 40 Figure 40 -Mitochondrial protein synthesis and SDS-PAGE analysis of wild type (MR6), wild type with PaATP9-5 (AKAK65), atp9Δ (RKY26), atp9Δ PaATP9-5 (AMY10) and ρ 0 (MR6 ρ 0 ). A, B and D: Proteins encoded by mtDNA were labeled in whole cells with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12% urea/glycerol gel and 17.5% SDS polyacrylamide gel. The same amount of radioactivity per each line was loaded. After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager. C, E: SDS-PAGE analysis of total mitochondrial proteins. Mitochondrial proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp9p, Atp6p (panel C), Arg8m (panel E) Ade13p and porine (panel C and E).

Figure 41 -

 41 Figure 41 -SDS-PAGE analysis of total mitochondrial proteins. Total protein extracts were prepared from the wild type (MR6), atp6Δ (MR10) and atp12Δ (AKY42) strains. Mitochondrial proteins (20 µg) were separated by SDS-PAGE, transferred to a nitrocellulose membrane and probed with antibodies against Atp9p and porine.

Figure 42 -

 42 Figure 42 -Mitochondrial protein synthesis. Proteins encoded by mtDNA were labeled in whole cells from wild type (MR6), wild type with pATP9-5 (AKAK65), atp6Δ (MR10), atp6Δ PaTP9-5 (FG146), atp9Δ (RKY26), atp9Δ PaATP9-5 (AMY10), atp12Δ (AKY42), atp12Δ PaATP9-5 (AKY121), atp12Δ atp6Δ (FG151), atp12Δ atp6Δ PaATP9-5 (FG152), atp12Δ atp9Δ (FG153) and atp12Δ atp9Δ PaATP9-5 (FG154) with [ 35 S]-(methionine+cysteine) for 20 min. in the presence of cycloheximide to inhibit cytosolic protein synthesis. The total protein extracts from cells were prepared and loaded on 12% urea/glycerol gel and 17.5% SDS polyacrylamide gel (the same amount of radioactivity per each line). After electrophoresis the gel was dried and the proteins were visualized with a Phosphoimager.

  Atp23p has a double function: it cleaves the leader peptide of Atp6p and assist the folding of Atp6p. With a proteolytically inactive version of Atp23p still able to fold Atp6p, the synthesis of Atp6p was still up-regulated in the atp6-L183P mutant, indicating that it is the chaperone function of Atp23p that is important for adjustment of the level of Atp6p synthesis. Finally, I have demonstrated that subunits of the central stalk (Atp15p) and the peripheral stalk (Atp7p) are also required for the up-regulation of the Atp6p synthesis in atp6-L183P mutant.Summing up, these results indicate that Atp6p synthesis is modulated by the assembly intermediate that contains all the ATP synthase subunits and into which Atp6p is incorporated. When this intermediate over-accumulates due to a lack of Atp6p, it stimulates

Figure 43 -

 43 Figure 43 -Model of regulation of Atp6p and Atp9p synthesis in relation to their assembly into yeast ATP synthase. See text for description.



  d'ATP d'environ 30%. A cause du dysfonctionnement partiel du domaine F O . La mutation 9191T>C menait à une diminution de la vitesse de synthèse d'ATP d'environ 95%.Dans la souche avec cette mutation, la protéine synthétisée Atp6 était vite dégradée et le domaine F 1 libre était present : ce qui indiquent que la mutation empêche presque entièrement l'incorporation de la sous-unité 6 dans l'ATP synthase.Les effets de ces mutations dans les modèles de levure correspondent aux phénotypes chez les patients où la mutation 9185T>C donnait la forme aténuée du syndrome NARP. Par contre le patient avec la mutation 9191T>C mourait quelques heures aprés la naissance.Encore une fois, des résultats montraient que la levure S. cerevisiae est un bon modèle pour analyser des effets moléculaire et biochimique des mutations du gène mitochondrial ATP6.Les mutations du gène mitochondrial ATP6 identifiées dans des tumeurs introduites dans le génome mitochondrial de la levure, n'ont pas influencé le fonctionnement de la chaîne respiratoire et l'ATP synthase à 28ºC (température optimale pour la croissance de S.cerevisiae).A une température plus élevée (36ºC), une mutation (8932C>T) conduisait à une diminution de la vitesse de synthèse d'ATP d'environ 60%. Dans les souches modèles d'autres mutations, je n'observais pas de changements de fonctionnement de la chaîne respiratoire et l'ATP synthase. Les resultats indiquent que les mutations analysées dans cette étude ont une influence mineur sur l'ATP synthase de la levure S. cerevisiae. Il n'est pas possible de constater clairement si des mutations du gène mitochondrial ATP6 influencent le développement ou la malignité des tumeurs chez les humaines. Des modèles de levure S. cereivsiae des maladies mitochondriales peuvent être utilisées pour chercher des nouvelles molécules actives contre ces maladies parmi celles déjà utilisées comme médicaments. Une molécule active : la chlorhexidine, était analysée. Chez les humains, des mutations dans des gènes nucléaires ATP12 et TMEM70 causent des maladies mitochondriales qui sont modélisées chez la levure S. cerevisiae par la délétion du gène FMC1. Dans cette souche, nous observions  une diminution de la consommation d'oxygène par des complexes de la chaîne respiratoire,  le potentiel faible de la membrane interne de la mitochondrie,  une diminution de la vitesse de synthèse d'ATP,  une présence des corps d'inclusion dans la mitochondrie,  une absence casi-complète de cristae. La présence de chlorhexidine dans le milieu améliore tous ces phénotypes observés. Dans notre laboratoire, sur deux souches modèles de mutation du gène mitochondrial ATP6 (atp6-L183P et atp6-W136R, R179I) nous observions que le niveau de synthèse d'Atp6p était élevé mais que le niveau de transcrit ne changeait pas. Par contre le niveau d'accumulation d'Atp6p dans la mitochondrie était diminué, de plus, l'anneau de sous-unités 9 s'accumulait dans les deux souches : ce qui indique une dysfonction d'assemblage d'ATP synthase. Sachant que la sous-unité 6 est assemblée comme la dernière, il est possible qu'un complexe où il manque la sous-unité 6 soit présent dans la mitochondrie. Ce sous-complexe peut être un signal pour la synthèse d'Atp6p, donc la synthèse d'Atp6p est augmentée dans les deux souches analysées. Pour prouver cette hypothèse, j'ai construit une collection de souches où j'avais deleté les gènes codants les sous-unités d'ATP synthase (ATP1, ATP7, ATP15) et les gènes codants les protéines nécessaires à l'expression des gènes mitochondriaux ou l'assemblage d'ATP synthase (ATP10, ATP11, ATP12, FMC1, ATP23, AEP1, AEP2). J'ai analysé l'influence de ces deletions au niveau de la synthèse d'Atp6p par analyse du niveau de synthèse de protéines mitochondriales dans les souches de ma collection en comparaison avec la souche sauvage et la souche atp6-L183P. Les résultats obtenus ont montré que dans les souches de ma collection il n'y avait pas d'effets d'augmentation de la synthèse d'Atp6p même si la mutation atp6-L183P était présente. Ces résultats ont confirmé l'hypothèse. L'expression du gène mitochondrial ATP9 est régulée par trois protéines : Aep1p, Aep2p et Atp25p. Des données de la littérature montrent que l'Aep1p et l'Aep2p sont nécessaires pour la traduction de mRNA ATP9 par son 5'UTR. L'Aep2p participe aussi en maturation de mRNA ATP9, sa stabilité et/ou l'initiation de traduction. L'Atp25p est coupée dans la mitochondrie en deux morceaux :  La partie C-terminal d'Atp25p est necessaire pour la stabilisation de mRNA ATP9. La partie N-terminal d'Atp25p est responsable d'oligomerisation d'anneau des sous-unitès 9. L'analyse détaillée des fonctions de ces trois protéines dans la regulation d'expression du gène ATP9 était, jusqu'à maintenant, très difficile à cause de l'instabilité du génome mitochondrial dans des souches ou les gènes AEP1, AEP2 et ATP25 étaient deletés. Dans le laboratoire de Jean-Paul di Rago, un plasmide qui code le gène ATP9-5 de Podospora anserina était construit. Ce plasmide a stabilisé le génome mitochondrial dans la souche de S. cerevisiae où le gène ATP9 était remplacé par le gène ARG8m. En conséquence, j'ai deleté les gènes nucleaires AEP1, AEP2 et ATP25 en présence de plasmide avec ATP9-5 dans des souches avec du génome mitochondrial sauvage et avec le gène ATP9 remplacé par ARG8m. L'analyse de ces souches a montré que :  les protéines Aep1, Aep2 et Atp25 sont nécessaires que pour l'expression de locus ATP9,  la presence de la protéine Atp9-5 stimule la synthèse de locus ATP9,  la synthèse d'Atp9p était augmentée dans la souche où le gène ATP6 était deleté. Ces resultas ont suggéré que la protéine Atp9 stimule sa propre synthèse. Des analyses supplémentaires ont montré que la protéine Atp9p est dégradée par la protéolyse, sa synthèse augmentée et sa stabilité sont dependente de la presence de le domaine F 1 .

Table 1 -List of ATP synthase subunits from Escherichia coli, Saccharomyces cerevisiae and Homo

 1

sapiens. The list is based on

[START_REF] Ackerman | Function, structure, and biogenesis of mitochondrial ATP synthase[END_REF] Kucharczyk, Zick et al. 2009c;[START_REF] Jonckheere | Mitochondrial ATP synthase: architecture, function and pathology[END_REF]

).

Table 2 :

 2

	Name	Alias	Nuclear genotype	Plasmid name	Insert	Ori.	Mitochondrial genotype	References
	MR6	Ø

  Tetaud et al.

								2007b)
	DFS160/60 α	Ø	Matα kar1-1 ade2-101 ura3-52 leu2 arg8::URA3	Ø	Ø	Ø	ρ 0	(Steele, Butler et al. 1996)
	DFS160/60 a	Ø	Mata kar1-1 ade2-101 ura3-52 leu2 arg8::URA3	Ø	Ø	Ø	ρ 0	(Steele, Butler et al. 1996)
	NB40-3C	Ø	Mata lys2 leu2-3,112 ura3-52 his3ΔHindIII arg8::hisG	Ø	Ø	Ø	ρ + cox2-62	(Steele, Butler et al. 1996)
	D273-10b	Ø	Matα ade2-101 met6	Ø	Ø	Ø	ρ 0	(Foury and Tzagoloff 1976)

Table 2 -Table of yeast strains used in the study.

 2

Table 3 -Table of plasmids used in the study

 3

Table 4 -The list of primers used for amplification of deletion cassettes and introduction of mutations into ATP6 gene

 4

Table 5 -Table of the antibodies used in the study.

 5

		Provenance	Dilution	Epitope	Origin
	Anti-α	Rabbit polyclonal	1/50 000	The whole protein	J. Velours
	Anti-Atp4p	Rabbit polyclonal	1/10 000	The whole protein	J. Velours
	Anti-Atp6p	Rabbit polyclonal	1/5 000	The whole protein	J. Velours
	Anti-Atp9p	Rabbit polyclonal	1/7 500	LINGVSRNPSIKDT	J. Velours
	Anti-cyt b	Rabbit polyclonal	1/5 000	The whole protein	J. Velours
	Anti-Cox2	Mouse monoclonal	1/5 000	The whole protein	Molecular Probes
	Anti-Arg8	Rabbit polyclonal	1/ 5 000	The whole protein	T. Fox
	Anti-porine	Mouse monoclonal	1/10 000	The whole protein	Molecular Probes
	Anti-Ade13	Rabbit	1/50 000	The whole protein	B. Pinson
	Anti-rabbit HRP	Goat polyclonal	1/10 000	The whole protein	Promega
	Anti-mouse HRP	Goat polyclonal	1/10 000	The whole protein	Promega

Type of tumor Nucleotide position and change in human mtDNA Amino acid change in human Atp6 protein Amino acid and codons changes in yeast Atp6p

			Lys 90 >Glu
	Thyroid	8716A>G	Lys 64 >Glu
			AAA>GAA
			Pro 157 >Thr
	Thyroid	8914C>A	Pro 130 >Thr
			CCT>ACT
			Pro 163 >Ser
	Prostate	8932C>T	Pro 136 >Thr
			CCA>TCA
			Ileu 170 >Val
	Thyroid	8953A>G	Ileu 143 >Val
			ATT>GTT
			Leu 232 >Pro
	Breast	9131T>C	Leu 202 >Pro
			TTA>CCA

Table 6 -Mutations in ATP6 gene found in tumors and chosen for modeling in yeast.

 6

Table 7 -Influence of the atp6-mutations on yeast mitochondrial respiration, ATP synthesis and hydrolysis activities

 7

. Mitochondria were isolated

Table 8 Percentage of ρ -/ρ 0 cells in constructed deletion strain collection.

 8

  temperature (35-37°C)[START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF]. In this mutant, Atp6p and Atp8p are almost normally synthesized[START_REF] Lefebvre-Legendre | Identification of a nuclear gene (FMC1) required for the assembly/stability of yeast mitochondrial F(1)-ATPase in heat stress conditions[END_REF]). The up-regulation of Atp6p synthesis conferred by the atp6-L183P mutation was no longer observed, indicating that F 1 is required for its occurrence. To test the influence of the Atp9p-ring on the synthesis of Atp6p, I deleted two genes, AEP1 and AEP2, that are necessary for ATP9 expression. Again, the up-regulation of Atp6p was lost, indicating that not only F 1 , but also the Atp9p-ring influences the Atp6p synthesis.I next aimed to know whether two proteins, Atp10p and Atp23p, involved in the Atp6p assembly, which are normally not required for At6p synthesis, participate in the up-regulation of Atp6p synthesis in the atp6-L183P mutant. After deletion of the ATP10 or ATP23 genes, the atp6-L183P mutation no longer increased the rate of the Atp6p synthesis.

IV.1 YEAST MODELS OF HUMAN DISEASES SHOW INFLUENCE OF MUTATIONS IN ATP6 GENE ON ATP SYNTHASE

In the first part of my work I have analyzed the influence of seven mutations in mitochondrial gene ATP6 on the ATP synthase: two found in patients with the NARP syndrome and five that were identified in human tumors.

IV.1.1 Consequences of the NARP atp6-S250P and atp6-L252P mutations on yeast ATP synthase

Mutations 9185T>C and 9191T>C of the human MTATP6 gene found in patients presenting with the NARP syndrome, resulted in a leucine into proline changes at codon positions 220 and 222. These amino acids correspond to serine 250 and leucine 252 into proline changes in yeast Atp6p, respectively. They are located in the last transmembrane segment of Atp6p that is supposed to contact the Atp9p-ring. The atp6-S250P mutation caused a 30% drop in the ATP synthesis and affected neither assembly nor stability of the ATP synthase. There was no leak across the inner mitochondrial membrane. Therefore the main effect of this mutation is a partial impairment of proton transport through F O . Modest impact of the mutation on the enzyme is consistent with the relatively mild clinical phenotype of 9185T>C. The atp6-L252P mutation was much more deleterious: ATP synthesis was decreased by more than 95% as compared to the WT strain due to a failure in the incorporation of Atp6p into the ATP synthase. It is possible that Atp6p with the L252P mutation cannot interact correctly with the Atp9p-ring to form a proton channel. Another explanation is that the atp6-L252P mutation affects the interaction with chaperon Atp10p which supports the assembly of Atp6p. The severe effects on the ATP synthase caused by this mutation are consistent with the severe clinical phenotype of the patients carrying this mutation.

IV.1.2 Yeast-based assay for screening of drugs active against ATP synthase disorders

In the course of preparation of my thesis I have had an opportunity to participate in a project aimed at identification of drugs active against human ATP synthase disorders. In this project we have used yeast models of these diseases. Two positive hits have been identified: chlorhexidine and oleate. For my part, I was testing the ATP synthase stability in the fmc1Δ strain in the presence of chlorhexidine. The BN-PAGE analysis demonstrated that the ATP synthase was more stable in the fmc1Δ strain in the presence of chlorhexidine. Another