Translation initiation factor 1 (IF1) has been shown to be an RNA chaperone. In order to find functional interactions that IF1 may have with rRNA, we have isolated second-site suppressors of a cold-sensitive IF1 mutant. Joining of the ribosomal subunit seems to be affected in the IF1 mutant strain and the suppressive effect is a consequence of decreasing the available pool of mature 50S subunits. The results serve as additional evidence that IF1 is an RNA chaperone and that final maturation of the ribosome takes place during translation initiation. In this study we have also investigated the effect of a cold-sensitive mutant IF1 or kasugamycin addition on gene expression using a 2D gel electrophoresis technique. The effect is much more dramatic when cells are treated with kasugamycin compared to mutant IF1. The ybgF gene is uniquely sensitive to the IF1 mutation as well as the addition of kasugamycin. This effect on the native gene could be connected with some property of the TIR sequence of ybgF and supports the notion that kasugamycin addition and the IF1 cold-sensitive mutation have a similar TIR-specific effect on mRNA translation. Finally we have isolated a suppressor of a temperature-sensitive mutation in ribosomal release factor 1 (RF1) to shed more light on the translation termination process. The suppressor mutation is linked to an IS10 insertion into the cysB gene and results in a Cys- phenotype. Our results suggest that suppression of the thermosensitive growth is a consequence of the mnm5s2U hypomodification of certain tRNA species. The ability of mnm5s2U hypomodified tRNA to induce frameshifting may be responsible for the suppression mechanism and it supports the hypothesis that modified nucleosides in the anticodon of tRNA act in part to prevent frameshifting by the ribosome. / At the time of the doctoral defense, the following paper was unpublished and had a status as follows: Paper 2: Manuscript.

Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

Mitochondrial dysfunction has been implicated as one of the primary causes of Parkinson's disease (PD). The proteins PINK1, a serine-threonine kinase, and Parkin, an E3 ubiquitin ligase, are mutated in many genetic cases of PD. In healthy individuals, Parkin is recruited to damaged mitochondria and leads to autophagic degradation of mitochondria in a process termed mitophagy. Following depolarization of the mitochondrial membrane, PINK1 is stabilized on the outer mitochondrial membrane, and triggers Parkin translocation from the cytosol to mitochondria. Precisely how this phenomenon is regulated is still unclear. We employed RNA interference (RNAi) technology in a 384-well format to identify novel genes that are required for Parkin recruitment to mitochondria. We identified ATPase inhibitory factor 1 (IF1) as the strongest hit required for Parkin recruitment following treatment with the protonophore CCCP. We show that IF1 is upstream of PINK1 and Parkin, and required to sense mitochondrial damage by allowing the loss of membrane potential. In cells treated with CCCP, the absence of IF1 permits the ATP synthase to run freely in reverse, consuming ATP to maintain potential across the inner mitochondrial membrane, thus blocking PINK1 and Parkin activation. Interestingly, Rho0 cells, that lack mitochondrial DNA, have downregulated endogenous expression of IF1 in order to maintain mitochondrial function. Overexpression of IF1 in Rho0 cells results in the depletion of mitochondrial membrane potential and the initiation of mitophagy. These data demonstrate a unique role for IF1 in the regulation of mitochondrial quality control that has not been explored in the etiology of PD.

L’ATP synthase ou ATPase de type F, ancrée aux membranes internes des mitochondries, est un complexe macromoléculaire qui utilise le gradient électrochimique généré par l’oxydation de petites molécules (NADH2, FADH2) dans les différents complexes de la chaîne respiratoire pour former l’ATP, vecteur énergétique universel. Le gradient électrochimique ou pm f est transformé en une énergie mécanique qui se traduit par le mouvement du rotor de l’ATP synthase dans un sens horaire vu depuis la membrane. La rotation de la sous-unité γ déforme successivement les trois sites catalytiques et permet ainsi la synthèse d’ATP. Dans certains cas, comme ceux de l’anoxie ou de l’hypoxie, le gradient électrochimique peut s’effondrer et l’ATP synthase hydrolyse alors l’ATP. Pour éviter cette hydrolyse futile, un petit peptide nommé IF1, régulateur spécifique des ATP synthases mitochondriales, vient s’insérer entre les sous-unités d’une interface catalytique et bloque instantanément le fonctionnement de l’ATPase. Cette inhibition est réversible puisque le peptide se décroche lorsque la membrane interne mitochondriale se réenergise.Dans ce travail de thèse, nous nous sommes intéressés à caractériser le mécanisme d’inhibition de l’ATPase de S.cerevisiae par son peptide endogène IF1 en s’appuyant essentiellement sur les quelques données structurales qui ont été publiées sur le peptide et sur le complexe inhibé IF1-F1ATPase de B.taurus.Constitué de 63 acides aminés chez S.cerevisiae et 84 acides aminés chez B.taurus, IF1 est majoritairement structuré en hélice α . Les études menées par Elena Cabezón ont montré qu’IF1 possédait différentes formes dont la prédominance et l’activité dépendait essentiellement du pH. Chez B.tauru , il existe une forme inhibitrice dimérique prédominante à pH inférieurs à 6,5 et une forme tétramérique dont nous connaissons la structure 3D qui est non inhibitrice et prépondérante à pH supérieurs à 6,5. Chez S.cerevisiae, il existe une forme monomérique inhibitrice prépondérante à pH supérieur à 6,5 et une forme dimérique prédominante à pH inférieurs à 6,5 et dont le caractère inhibiteur ou non n’a pas encore été déterminé. Sur la base de la structure 3D de l’IF1 bovin, nous avons voulu identifier les régions de dimérisation du peptide de levure en utilisant la technique de marquage de spin couplée à de la spectroscopie RPE. En plaçant des marqueurs de spin (MTSL) en partie médiane(E33C) ou en C-terminale(L54C),nous avons pu favoriser l’interface de dimérisation plutôt en partie médiane du peptide. Ce travail est encore au stade embryonnaire et ne nous permet pas, à ce jour, d’identifier la zone exacte de dimérisation.Dans un deuxième volet, nous avons voulu caractériser le mécanisme d’inhibition d’un point de vue dynamique et nous avons pu en préciser les différentes étapes : reconnaissance, verrouillage et stabilisation. Pour cela, nous avons associé la mutagenèse sur le peptide et sur l’enzyme aux cinétiques d’inhibition. Nous avons tout d’abord évalué le rôle de plusieurs résidus situés en C-terminal de la sous-unité β, dans la région de l’interface α/β qui se referme sur le peptide IF1, dans la reconnaissance moléculaire spécifique d’IF1 par l’ATPase mitochondriale. Nous avons ensuite montré que la partie N-terminale d’IF1 joue un rôle mineur dans la reconnaissance moléculaire mais son enroulement autour de la sous-unité γ constitue un loquet important dans la stabilisation du complexe inhibé. Enfin, la fermeture de l’interface catalytique sur IF1 crée une zone de contact entre la "bosse" de la sous-unité γ et la partie C-terminale de la sous-unitéα qui constitue la dernière clef de blocage du peptide au sein de la F1 -ATPase. Ce dernier point de fermeture est le seul qui n’implique aucun résidu du peptide IF1. / The F-type ATPase or ATP synthase, anchored to the inner mitochondrial membrane, is a macromolecular complex using the proton motive force (pmf) generated by the oxydation of small molecules, such as NADH2 and FADH2 , in the different respiratory complexes to form ATP. The pmf is converted into mechanical work by the clockwise rotation of the ATP synthase viewed from the membrane. The γ rotation successively distorts the three catalytic interfaces of the enzyme to allow the synthesis of ATP. Anoxia or hypoxia are cases in which the rotation of ATP synthase proceeds in the direction of ATP hydrolysis. A small peptide named IF1, 63 aminoacids-long in yeast and 84 aminoacids-long in bovine, specifically inhibits the mitochondrial ATP synthase in the direction of ATP hydrolysis. This inhibition is reversible since the peptide is released when the inner mitochondrial membrane is re-energized.In this work, we were interested in characterizing the inhibition mechanism of the mitochondrial ATP synthase of S.cerevisiae by its endogenous peptide IF1. To elaborate and strengthen our statements, we mainly used the structures of IF1 and of the inhibited IF1-F1ATPase complex of B. taurus.The data obtained by Elena Cabezón on bovine and yeast IF1 showed that different forms of the peptide coexist and that their pre-eminence depends on the pH. The bovine IF1 mainly adopts a dimeric form at pH below 6.5 and tetrameric one at pH above 6.5. Its inhibitory properties also vary with the pH. The dimeric form is inhibitory and the tetrameric one is not. In yeast, it is known that a monomeric form is predominant at pH above 6.5 and a dimeric form predominant at pH below 6.5. The monomeric form is inhibitory but nothing has been reported about the inhibitory properties of the dimeric form. By using the structural data of the bovine IF1, we tried to determine the dimerization region of the yeast IF1. For this aim, we decided to combine Site-Directed Spin Labeling (SDSL) with electron paramagnetic resonance (EPR) spectroscopy. Thus, we attached labels on the C-ter or the mid-region and we could propose that the dimer of yeast IF1 preferentially forms by the mid-region. This work is currently in the preliminary stage and other experiments would be necessary to confirm the precize region of dimerization. In a second part, we tried to precise the inhibitory mechanism by detailing the different steps of recognition, locking and stabilization of the inhibited complex. This was achieved by combining the mutagenesis of yeast IF1 and F1ATPase with kinetics of inhibition. First, we evaluated the role of some residues located in the C-terminal part of β subunit in the specific molecular recognition of IF1 by the mitochondrial ATPase. These residues belong to the region of the α/β interface that closes up on IF1 peptide. Then, we showed that the N-terminal part of IF1 plays a minor role in the molecular recognition but its winding around the γ subunit constitute an important lock in the inhibited complex. Finally, the closing of the catalytic interface on IF1 creates a contact region between the α and the γ subunit which is the last key that definitively locks the peptide in the cage "F1ATPase". This last locking point is the only one that does not involve any IF1 residue.

<p>Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated.</p><p>In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype.</p><p>The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome.</p><p>In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.</p>

L'ATP synthase ou ATPase de type F, ancrée aux membranes internes des mitochondries, est un complexe macromoléculaire qui utilise le gradient électrochimique généré par l'oxydation de petites molécules (NADH2, FADH2) dans les différents complexes de la chaîne respiratoire pour former l'ATP, vecteur énergétique universel. Le gradient électrochimique ou pmf est transformé en une énergie mécanique qui se traduit par le mouvement du rotor de l'ATP synthase dans un sens horaire vu depuis la membrane. La rotation de la sous-unité déforme successivement les trois sites catalytiques et permet ainsi la synthèse d'ATP. Dans certains cas, comme ceux de l'anoxie ou de l'hypoxie, le gradient électrochimique peut s'effondrer et l'ATP synthase hydrolyse alors l'ATP. Pour éviter cette hydrolyse futile, un petit peptide nommé IF1, régulateur spécifique des ATP synthases mitochondriales, vient s'insérer entre les sous-unités d'une interface catalytique et bloque instantanément le fonctionnement de l'ATPase. Cette inhibition est réversible puisque le peptide se décroche lorsque la membrane interne mitochondriale se réénergise. Dans ce travail de thèse, nous nous sommes intéressés à caractériser le mécanisme d'inhibition de l'ATPase de S.cerevisiae par son peptide endogène IF1 en s'appuyant essentiellement sur les quelques données structurales qui ont été publiées sur le peptide et sur le complexe inhibé IF1-F1-ATPase de B.taurus. Constitué de 63 acides aminés chez S.cerevisiae et 84 acides aminés chez B.taurus, IF1 est majoritairement structuré en hélice α. Les études menées par Elena Cabezón ont montré qu'IF1 possédait différentes formes dont la prédominance et l'activité dépendait essentiellement du pH. Chez B.taurus, il existe une forme inhibitrice dimérique prédominante à pH inférieurs à 6,5 et une forme tétramérique dont nous connaissons la structure 3D qui est non inhibitrice et prépondérante à pH supérieurs à 6,5. Chez S.cerevisiae, il existe une forme monomérique inhibitrice prépondérante à pH supérieur à 6,5 et une forme dimérique prédominante à pH inférieurs à 6,5 et dont le caractère inhibiteur ou non n'a pas encore été déterminé. Sur la base de la structure 3D de l'IF1 bovin, nous avons voulu identifier les régions de dimérisation du peptide de levure en utilisant la technique de marquage de spin couplée à de la spectroscopie RPE. En plaçant des marqueurs de spin (MTSL) en partie médiane (E33C) ou en C-terminale (L54C), nous avons pu favoriser l'interface de dimérisation plutôt en partie médiane du peptide. Ce travail est encore au stade embryonnaire et ne nous permet pas, à ce jour, d'identifier la zone exacte de dimérisation. Dans un deuxième volet, nous avons voulu caractériser le mécanisme d'inhibition d'un point de vue dynamique et nous avons pu en préciser les différentes étapes : reconnaissance, verrouillage et stabilisation. Pour cela, nous avons associé la mutagenèse sur le peptide et sur l'enzyme aux cinétiques d'inhibition. Nous avons tout d'abord évalué le rôle de plusieurs résidus situés en Cterminal de la sous-unité β, dans la région de l'interface α/ β qui se referme sur le peptide IF1, dans la reconnaissance moléculaire spécifique d'IF1 par l'ATPase mitochondriale. Nous avons ensuite montré que la partie N-terminale d'IF1 joue un rôle mineur dans la reconnaissance moléculaire mais son enroulement autour de la sous-unité constitue un loquet important dans la stabilisation du complexe inhibé. Enfin, la fermeture de l'interface catalytique sur IF1 crée une zone de contact entre la "bosse" de la sous-unité γ et la partie C-terminale de la sous-unité α qui constitue la dernière clef de blocage du peptide au sein de la F1-ATPase. Ce dernier point de fermeture est le seul qui n'implique aucun résidu du peptide IF1.

Initiation is the first step in protein biosynthesis representing a fundamental event in cell life which determines fidelity, efficiency and regulation of gene expression. In addition to the ribosome and mRNA, three protein factors IF1, IF2 and IF3 are involved in the initiation of translation in prokaryotes. Several minor functions have been attributed to the smallest of these factors, IF1. However, the main function of IF1 remains to be elucidated. In order to investigate the role of this protein in the initiation process we have mutated the corresponding gene infA. Using a high-copy plasmid and site-directed mutagenesis, the six arginine residues of IF1 were separately altered to leucine or aspartate. Another set of plasmid-encoded IF1 mutants with a cold-sensitive phenotype was collected using localized random mutagenesis. This strategy was followed by deletion of the chromosomal infA gene. All variants with a mutated infA gene on a plasmid and a deletion of the chromosomal infA copy were viable, except for an R65D alteration. Several of the mutated infA genes were successfully recombined into the chromosome thereby replacing the wild-type allele. Some of these mutants displayed reduced growth rates and a partial cold-sensitive phenotype. The influence of the leucine group of mutants in IF1 on the expression of two reporter genes with different initiation and/or +2 codons has been investigated. Our results do not indicate any involvement of IF1 in recognition of the +2 codon immediately following the start codon, thus representing the A-site. In addition, this group of mutants has no changed efficiency of decoding at the near-cognate initiation codons UUG and GUG. However, one cold-sensitive IF1 mutant shows a general overexpression of both reporter genes, in particular at low temperatures. Overall, the results do not support the hypothesis that IF1 could possess codon discriminatory functions while blocking the A-site of the ribosome. In this study we also identify that IF1 has RNA chaperone activity both in vitro and in vivo. The chaperone assays are based on splicing of the group I intron in the thymidylate synthase gene (td) from phage T4. Some of the IF1 mutant variants are more active as RNA chaperones than the wild-type. Both wild-type IF1 and mutant variants bind with high affinity to RNA in a band-shift assay. It is suggested that the RNA chaperone activity of IF1 contributes to RNA rearrangements during the early phase of translation initiation.

Inhibitory factor 1 (IF1) is one of the major regulators of mitochondrial ATP synthase activity, a key enzyme of energy metabolism. Its inhibitory effects are known in conditions such as hypoxia or starvation, but the hypothesis that IF1 inhibits ATP synthase activity even under physiological conditions is still not entirely accepted. Disorders of ATP synthase regulation can be fatal to the cell and have been described, for example, in carcinogenesis and ischemia. It has also been found that silencing of the IF1 gene in pancreatic β-cells increases insulin secretion, and thus, IF1 may be important in the pathogenesis of type 2 diabetes. The goal of this work is to summarize the current knowledge about the IF1 protein and to obtain new results that will help elucidate the mechanism by which this protein regulates mitochondrial ATP synthase. Specifically, this work deals with the ratio of IF1 protein to ATP synthase in pancreatic β-cells, depending on different culture conditions. It further investigates the occurrence of post-translational modifications of the IF1 protein in pancreatic β-cells (INS- 1E model cells), which may play a role in the regulation of IF1 activity. It also deals with the cellular ATP/ADP ratio, which is one of the key factors for insulin secretion by pancreatic β-cells. An...

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ATP synthase est une protéine essentielle associée à la membrane interne mitochondriale, qui synthétise l'ATP par couplage d’un transport de protons au travers de la membrane, en dissipant un gradient électrochimique de protons créé par la chaîne respiratoire. Cette réaction assure l’alimentation en énergie des processus biologiques cellulaires. Si la membrane mitochondriale se dépolarise, la réaction inverse d’hydrolyse d’ATP est rapidement bloquée par un inhibiteur soluble naturel de l’ATPase mitochondriale, IF1. Cette régulation efficace et réversible évite le gaspillage de l’énergie par la cellule. Chez la levure, IF1 est une petite protéine de 63 amino-acides. Elle se fixe sur l'une des trois interfaces catalytiques de l’ATP synthase et inhibe l’hydrolyse d’ATP. Bien que les structures cristallographiques des complexes F1-ATPase inhibés par IF1 aient été résolus, l'étape initiale de reconnaissance et celle du verrouillage d’IF1 restent peu claires au niveau moléculaire.Pendant ma thèse, nous nous sommes intéressés au mécanisme d’inhibition de l’ATPase par IF1. Par des analyses des structures disponibles et des alignements de séquence, nous avons sélectionné de nombreux résidus localisés dans différentes régions des sous-unités α et β de l'ATP synthase de Saccharomyces cerevisiae et susceptibles de participer au processus de fixation d'IF1. En utilisant le mutagenèse dirigée combinée à des experiences cinétiques, nous avons étudié les effects des mutations sur l’inhibition de l’ATP synthase par IF1 chez Saccharomyces cerevisiae. Dans ce travail, nous avons identifié des résidus ou motifs des sous-unités α et β de l’ATP synthase impliqués dans les étapes de reconaissance et/ou verrouillage d’IF1, ce qui nous permet de compléter les études structurales et d'esquisser un mécanisme de fixation d'IF1. / ATP synthase is an essential protein complex located in the mitochondrial inner membrane, which synthesize ATP by coupling to a rotary proton transport across the membrane at the expense of the electrochemical proton gradient created by the electron transport chain. This reaction guarantees the supply of energy to biological processes in a cell. When mitochondria get deenergized, i.e. the protomotive force across the mitochondrial inner membrane collapses, the ATP synthase switches from ATP synthesis to hydrolysis. This hydrolytic activity is then immediately prevented by a natural soluble mitochondrial ATPase inhibitor, IF1. This efficient reversible inhibition system protects cells from wasting energy. In yeast, IF1 is a small protein consisting of 63 amino acids. It binds to one of the three (αβ) catalytic interfaces of ATP synthase and thereby blocks the rotary catalysis. Although the crystal structure of the dead-end IF1 inhibited F1-ATPase complex has been resolved, IF1 initial binding and locking to ATPase still remain unclear events at the molecular level.During my thesis, we have been interested in the dynamic mechanism of ATPase inhibition by IF1. By means of analyses of published structures and protein sequence alignment, we selected numerous residues located in different regions of Saccharomyces cerevisiae ATP synthase α, β subunits, which might potentially paticipate in IF1 binding process. Using site-directed mutagenesis combined with kinetic experiments, we studied the effect of mutations of the selected candidates on the rate and extent of ATPase inhibition by IF1. In this way we identified residues or motifs in ATP synthase α, β subunits involved in IF1 recognition and/or locking steps, which allows complementing structural studies and drawing an outline of IF1 binding.