During the transformation of the bacterial endosymbiont into current mitochondria the protein import apparatus had to be created de novo. The reduced mitochondria (mitosomes) of the parasitic protist Giardia intestinalis represent unique cellular model for the examination of these fundamental transport processes. As the main objective of this project I will try to characterize the motor complex, which propels the protein transport, and also the translocation channel in the inner mitosomal membrane. To this aim I will exploit the presence of two membrane components Pam16 and Pam18, which were discovered in our laboratory, and which constitute the functional core of the motor complex. Based on the information from the analogous systems of yeast and mammalian mitochondria, these two components should physically interact with so far unknown translocation channel. In all other eukaryotes this channel is formed by a conserved protein Tim23. The absence of this protein in the genome of G. intestinalis suggests presence of completely novel, or maybe the original-bacterial protein. Having in hand this simplified mitochodrial model the project has potential to bring not only new data in parasite biology but also generate new information on the function and evolution of mitochondrial protein import.

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles. / Abstract RNase P is a ribonucleo-protein complex (an RNA enzyme or ribozyme) that cleaves 5’ leader sequences of precursor tRNAs and a few other small RNAs. It occurs in all three domains of life, Bacteria, Archaea and Eukarya, with the latter containing distinct nuclear and organellar (mitochondrial or plastid) activities. In most instances, the complex contains a single, well-conserved RNA subunit that carries the active center of the enzyme. Yet, compare to bacterial and nuclear P RNA, most mtP RNAs are structurally highly reduced. The number of P proteins is highly variable: one in Bacteria, about four in Archaea, and ten in the cytoplasmic form of Eukarya. Much less is known in the case of mitochondria. MtRNase P is usually purified by using numerous separation steps that include unphysiological conditions, leading to complexes having a minimum number of subunits (e.g., in yeast and Aspergillus nidulans), that often loose their activity. Here, using BN PAGE, we have developed an enrichment procedure for A. nidulans mtRNase P that avoids some of the most disruptive conditions. The protein composition of active fractions was identified with LC/MS/MS, indicating that the RNase P holoenzyme is much larger than previously thought. Finally, the question of mtRNase P localization within mitochondria was investigated, by tracing its RNA subunit by RT PCR. We found that mtRNase P of A. nidulans is a predominantly membrane-attached enzyme; it is in part solubilized by detergents such as digitonin and Triton.

Résumé La ribonucléase P (RNase P) est une ribonucléoprotéine omniprésente dans tous les règnes du vivant, elle est responsable de la maturation en 5’ des précurseurs des ARNs de transfert (ARNts) et quelques autres petits ARNs. L’enzyme est composée d'une sous unité catalytique d'ARN (ARN-P) et d'une ou de plusieurs protéines selon les espèces. Chez les eucaryotes, l’activité de la RNase P cytoplasmique est distincte de celles des organelles (mitochondrie et chloroplaste). Chez la plupart des espèces, les ARN-P sont constituées de plusieurs éléments structuraux secondaires critiques conservés au cours de l’évolution. En revanche, au niveau de la structure, une réduction forte été observé dans la plupart des mtARN-Ps. Le nombre de protéines composant la RNase P est extrêmement variable : une chez les bactéries, environ quatre chez les archéobactéries, et dix chez la forme cytoplasmique des eucaryotes. Cet aspect est peu connu pour les formes mitochondriales. Dans la plupart des cas, l’identification de la mtRNase P est le résultat de longues procédures de purification comprenant plusieurs étapes dans le but de réduire au minimum le nombre de protéines requises pour l’activité (exemple de la levure et A. nidulans). Cela mène régulièrement à la perte de l’activité et de l’intégrité des complexes ribonucléo-protéiques natifs. Dans ce travail, par l’utilisation de la technique de BN-PAGE, nous avons développé une procédure d’enrichissement de l’activité RNase P mitochondriale native, donnant un rendement raisonnable. Les fractions enrichies capables de cette activité enzymatique ont été analysées par LC/MS/MS et les résultats montrent que l’holoenzyme de la RNase P de chacune des fractions contient un nombre de protéines beaucoup plus grand que ce qui était connue. Nous suggérons une liste de protéines (principalement hypothétiques) qui accompagnent l’activité de la RNase P. IV De plus, la question de la localisation de la mtRNase P de A. nidulans a été étudiée, selon nos résultats, la majorité de la mtRNase P est attachée á la membrane interne de la mitochondrie. Sa solubilisation se fait par l’utilisation de différents types de détergent. Ces derniers permettent l’obtention d’un spectre de complexes de la RNase P de différentes tailles. / Abstract RNase P is a ribonucleo-protein complex (an RNA enzyme or ribozyme) that cleaves 5’ leader sequences of precursor tRNAs and a few other small RNAs. It occurs in all three domains of life, Bacteria, Archaea and Eukarya, with the latter containing distinct nuclear and organellar (mitochondrial or plastid) activities. In most instances, the complex contains a single, well-conserved RNA subunit that carries the active center of the enzyme. Yet, compare to bacterial and nuclear P RNA, most mtP RNAs are structurally highly reduced. The number of P proteins is highly variable: one in Bacteria, about four in Archaea, and ten in the cytoplasmic form of Eukarya. Much less is known in the case of mitochondria. MtRNase P is usually purified by using numerous separation steps that include unphysiological conditions, leading to complexes having a minimum number of subunits (e.g., in yeast and Aspergillus nidulans), that often loose their activity. Here, using BN PAGE, we have developed an enrichment procedure for A. nidulans mtRNase P that avoids some of the most disruptive conditions. The protein composition of active fractions was identified with LC/MS/MS, indicating that the RNase P holoenzyme is much larger than previously thought. Finally, the question of mtRNase P localization within mitochondria was investigated, by tracing its RNA subunit by RT PCR. We found that mtRNase P of A. nidulans is a predominantly membrane-attached enzyme; it is in part solubilized by detergents such as digitonin and Triton.

Pseudomonas aeruginosa est un pathogène opportuniste impliqué dans les infections nosocomiales. Sa multi résistance aux antibiotiques s’exerce notamment grâce à l’activation de pompes d’efflux membranaires. Il s’agit de systèmes tripartites composés d’une porine de la famille OMF (Outer Membrane Factor) ancrée dans la membrane externe, d’un transporteur de la famille des RND (Resistance Nodulation Division) localisé dans la membrane interne et d’un adaptateur périplasmique de la famille des MFP (Membrane Fusion Protein) qui consolide l’ensemble. Le travail réalisé au cours de cette thèse apporte une contribution à la compréhension des mécanismes d’assemblage et d’ouverture des pompes d’efflux ainsi qu’à leur régulation grâce au développement de nouveaux outils empruntés à la physique, à la biochimie et à la microbiologie. Une première étude a permis de déterminer la stoechiométrie d’interaction entre MexA et OprM par gel bleu natif (Ferrandez, Monlezun et al. 2012). Une deuxième étude a été consacrée, dans le cadre d’une collaboration avec l’équipe de B. Le Pioufle (ENS Cachan), à la caractérisation par électrophysiologie de l'ouverture de la porine OprM, insérée dans une membrane artificielle reconstituée sur une biopuce (Wang, Monlezun et al. 2012). Puis, afin d’étudier cette fois ci, le mécanisme d’ouverture de la porine OprM in vivo, une étude fonctionnelle par complémentation chez Pseudomonas aeruginosa a été initiée. Enfin, dans le cadre d'une collaboration avec l’équipe de P. Plésiat (Laboratoire de Bactériologie, Besançon), deux analyses de mutants cliniques par modélisation ont été réalisées sur le régulateur MexZ de la pompe MexXY/OprM et de la porine d’influx des carbapénèmes OprD. / Pseudomonas aeruginosa is an opportunistic pathogen involved in nosocomial infections. This bacteria has developed various strategies to resist antibiotics treatments, one of them being the activation of membrane efflux pumps. These tripartite systems consist of an OMF (Outer Membrane Factor) family porin, localized in the outer membrane, an active transporter in the inner membrane, belonging to the RND (Resistance Nodulation Division) family and a periplasmic adaptator protein, member of the MFP (Membrane Fusion Protein) family which consolidates the whole complex. Results obtained during this thesis contribute to a better understanding of efflux pumps’ assembly and opening thanks to the development of new research tools borrowed from physic, biochemistry and microbiology. The first study describes the binding stoechiometry of MexA with its cognate partner OprM by Blue Native Polyacrylamide gel Electrophoresis (Ferrandez, Monlezun et al. 2012). Secondly, a study, in collaboration with B. Le Pioufle’s team (ENS Cachan), was dedicated to the electrophysiologic caracterization of OprM opening using a microfluidic device incorporated with a miniaturized artificial bilayer membrane (Wang, Monlezun et al. 2012). Then, to complete this analysis in vivo, in the third part of this thesis, complementation experiments were initiated in a Pseudomonas aeruginosa strain deleted of its chromosomal oprM gene. Finally, in collaboration with P. Plésiat’s team (Laboratoire de Bactériologie, Besançon), modelling of MexZ, the MexXY/OprM pump’s regulator and modelling of the carbapenems’ porin OprD were made in order to link structural modifications to mutations observed in clinical strains.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

The two related putative cytochrome c oxidase (COX) assembly factors HCC1 and HCC2 from Arabidopsis thaliana are Homologs of the yeast Copper Chaperones Sco1p and Sco2p. The hcc1 null mutation was previously shown to be embryo lethal while the disruption of the HCC2 gene function had no obvious effect on plant development, but increased the expression of stress-responsive genes. Both HCC1 and HCC2 contain a thioredoxin domain, but only HCC1 carries a Cu-binding motif also found in Sco1p and Sco2p. In order to investigate the physiological implications suggested by this difference, various hcc1 and hcc2 mutants were generated and analyzed. The lethality of the hcc1 knockout mutation was rescued by complementation with the HCC1 gene under the control of the embryo-specific promoter ABSCISIC ACID INSENSITIVE 3. However, the complemented seedlings did not grow into mature plants, underscoring the general importance of HCC1 for plant growth. The HCC2 homolog was shown to localize to mitochondria like HCC1, yet the function of HCC2 is evidently different, because two hcc2 knockout lines developed normally and exhibited only mild growth suppression compared with the wild type (WT). However, hcc2 knockouts were more sensitive to UV-B treatment than the WT. Complementation of the hcc2 knockout with HCC2 rescued the UV-B-sensitive phenotype. In agreement with this, exposure of wild-type plants to UV-B led to an increase of HCC2 transcripts. In order to corroborate a function of HCC1 and HCC2 in COX biogenesis, COX activity of hcc1 and hcc2 mutants was compared. While the loss of HCC2 function had no significant effect on COX activity, the disruption of one HCC1 gene copy was enough to suppress respiration by more than half compared with the WT. Therefore, we conclude that HCC1 is essential for COX function, most likely by delivering Cu to the catalytic center. HCC2, on the other hand, seems to be involved directly or indirectly in UV-B-stress responses.

F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...