Dissertations / Theses on the topic 'Photosynthesis; Phosphorylation; ATP synthase'

Ces travaux portent sur l'étude de mutants nucléaires de la levure S. Cerevisiae, modifiés dans la synthèse mitochondriale des sous-unités 8 et 6 de l'ATPsynthase, qui restent respiratoire-compétents. Ces souches sont modifiées dans la régulation de la synthèse de l'ATP par la concentration en phosphate externe. Ceci est dû à la modification de la stoechiométrie relative des sous-unités 8, 6 et 9 dont le résultat est une altération de la perméabilité aux protons de la membrane interne. Une analyse des transcrits mitochondriaux a permis de corréler cette baisse du taux protéique à une modification spécifique du cotranscrit ATP8-ATP6. Une analyse génétique des mutants a mis en évidence deux mutations nucléaires indépendantes toujours associées à une mutation mitochondriale, qui entraîne une sensibilité accrue à la paromomycine, un antibiotique qui augmente le taux d'erreurs au niveau traductionnel mitochondrial. Les séquences nucléotidiques de l'ARN ribosomique 15S et de l'ARN messager VAR1, deux composants des mitoribosomes codés par l'ADN mitochondrial, ont été étudiées : aucune différence n'a pu être mise en évidence entre les souches mutante et sauvage. Les deux gènes nucléaires mutés entraînent un phénotype de cryosensibilité sur substrat respiratoire seulement lorsqu'ils sont présents simultanément. Donc, les deux gènes sauvages ont été recherchés par complémentation fonctionnelle. Deux gènes nucléaires NCA2 et NCA3, impliqués dans l'expression spécifique des sous-unités 8 et 6 de l'ATP synthase, ont été isolés et séquencés. Aucune homologie significative avec des protéines connues n'a été identifiée dans les banques de données. NCA2 et NCA3 sont deux gènes monocopie qui codent respectivement pour des protéines de 70800 et 35400 Da. NCA2 est localisé sur le chromosome 16 et NCA3 sur le chromosome 4. Leur disruption au locus chromosomique ne conduit pas à une incompétence respiratoire<br>These works concerned the study of respiratory-competent nuclear mutants of the yeast S. Cerevisiae, altered in the mitochondrial synthesis of subunits 8 and 6 of the ATPsynthase. These strains are altered in the regulation of the ATP synthesis by the external phosphate concentration. It was due to a modification of the relative stoichiometry of the mt DNA-encoded 8, 6 and 9 subunits which results in an enhanced proton-leakage through the inner membrane. The mitochondrial transcripts has permitted to correlate the decrease in the subunits 6 and 8 ratio with a specific modification of cotranscript ATP8-ATP6. Genetic analysis of these mutants showed the presence of two unlinked mutations always associated with a mitochondrial mutation, which confered a paromomycin sensitivity, an inhibitor of the mitochondrial protein synthesis. The nucleic sequence of 15S rRNA and VAR1 mRNA, two components of mitoribosomes encoded by mitochondrial DNA, were studied : no difference exist between mutant and wild-type strains. The simultaneous presence of the two mutant nuclear genes induced a cryosensitive phenotype on a nonfermentable carbon source. Then, the two wild-type genes were cloned by functional complementation. Two nuclear genes NCA2 and NCA3, involved in the specific expression of subunits 8 and 6 of the ATPsynthase, were isolated and sequenced. No significant homologies with known proteins were identified in data bases. NCA2 and NCA3 are two single-copy genes which encode for proteins of molecular mass of 70800 and 35400 Da respectively. NCA2 is located on chromosome 16 and NCA3 on chromosome 4. A null mutation of each gene did not let to a respiratory-incompetent phenotype

Dans les cellules eucaryotes aerobies, l'energie provient essentiellement de l'atp synthetise par les phosphorylations oxydatives (oxphos) au niveau de l'atpase-atpsynthetase f0-f1. L'atp est alors exporte hors des mitochondries par le translocateur des nucleotides adenyliques (ant). Certaines sous-unites des complexes oxphos sont codees par l'adn mitochondrial (adnmt), les autres par l'adn nucleaire et importees dans les mitochondries par un mecanisme faisant intervenir le potentiel de membrane mitochondrial (pmm) etabli par les oxphos. Nous avons verifie que 2 lignees de cellules rho o humaines sont totalement depourvues d'adnmt, d'arn et de proteines codees par l'adnmt. Par contre, elles importent normalement des proteines d'origine nucleaire dans leurs mitochondries. Comment un pmm suffisant pour cette importation peut s'etablir dans les rho o en l'absence des oxphos ? nous avons montre que les rho o, depourvues des 2 sous-unites du facteur f0 de l'atpase-atpsynthetase codees par l'adnmt, ont un f1 assemble et fonctionnel pour hydrolyser de l'atp. De plus, 2 inhibiteurs specifiques de f1 (aurovertine) ou de l'ant (acide bongkrekique) reduisent la croissance et le pmm des rho o. Ainsi, la survie des rho o necessite un pmm suffisant maintenu par l'importation d'atp 4 cytosolique dans la mitochondrie par l'ant qui l'echange contre de l'adp 3 mitochondrial produit par l'atpase-f1. Nous avons egalement utilise les rho o pour determiner l'origine genetique de pathologies dues a un deficit d'un complexe oxphos. Les cellules de 3 patients atteints du syndrome de leigh avec deficit en cytochrome oxydase (cox) ont ete enucleees et fusionnees avec les rho o. L'activite cox etant restauree dans les cybrides, le deficit est localise sur l'adn nucleaire des patients. Par contre, la fusion a ete inutilisable pour determiner l'origine d'un deficit en complexe iii. En effet, le deficit est rapidement perdu dans les lymphocytes en culture suggerant un defaut heteroplasmique de l'adnmt.

La F1-Fo-ATP synthase est un complexe protéique mitochondrial composé d'au moins vingt sous-unités chez la levure Saccharomyces cerevsiae. Cette enzyme présente des organisations supra-moleculaires correspondant à des formes dimériques. La sous-unité 4(b) est la composante essentielle du second pied de la F1Fo-ATP synthase qui relie le domaine catalytique F1 au domaine protonophorique membranaire Fo. Afin de décrire l'organisation du second pied de l'ATP synthase, l'environnement protéique de la sous-unité 4 a été exploré grâce à des pontages chimiques utilisant un agent pontant hétéro-bifonctionnel. Cette étude topologique a mis en évidence différents rapports de voisinage avec d'autres sous-unités : l'extrémité N-terminale de la sous-unité 4 est à proximité de la sous-unité g, tandis que la région C-terminale est au voisinage des sous-unités h, d, β, et OSCP. Le voisinage entre le résidu C-terminal de la sous-unité 4 et la sous-unité OSCP laissait envisager que l'extrémité C-terminale dela sous-unité 4 constituait une zone d'interaction avec la sous-unité OSCP. Les dix derniers résidus de la sous-unité 4 sont essentiels à l'assemblage entre les secteurs F1 et Fo. Aussi l'environnement protéique de ces dix derniers résidus de la sous-unité 4 a été étudié grâce à des pontages chimiques utilisant des agents pontant homo-et hétéro-bifonctionnels. Cette étude topologique a permis de préciser que cette zone de xconnexion entre les secteurs F1 et Fo serait composée au moins des sous-unités 4, OSCP, h et α. Enfin, nous avons cherché à établir le rôle que pouvait jouer le premier segment transmembranaire des sous-unités 4 (b) mitochondriales qui est absent dans les sous-unités b procaryotiques et chloroplastiques. Nos études ont montré que cette région N-terminale additionnelle n'est pas indispensable auu fonctionnement de la F1Fo ATP synthase et que sa perte n'altère pas non plus les phosphorylations oxydatives. Par contre, la perte du premier segment transmembranaire de la sous-unité 4 aboutit à la perte d'assemblage de la sous-unité g. La sous-unité g étant indispensable à la dimérisation/oligomérisation de l'enzyme, l'ATP synthase n'est alors plus capable de se dimériser ni de s'oligomériser. L'altération de la morphologie des crêtes mitochondriales est une autre conséquence de la perte d'assemblage de la sous-unité g, l'oligomérisation de l'ATP synthase étant essentielle à la formation des crêtes. Ces observations suggèrent que le premier segment transmembranaire de la sous-unité 4 constitue une zone d'interaction avec le segment transmembranaire de la sous-unité g.

L'oxydation des acides gras met en jeu plusieurs enzymes qui se répartissent de la face externe a la matrice des mitochondries et constituent autant d'étapes limitantes potentielles. Une première partie du travail montre que l'altération des membranes internes par un peroxydant lipophile (hydropéroxyde de cumène) perturbe la chaine respiratoire au point de diminuer la capacité des mitochondries à oxyder le palmitate et des intermédiaires du cycle de Krebs. Le resvératrol, qui est un antioxydant lipophile puissant, réduit la dégradation des lipides membranaires. Le traitement des mitochondries par un peroxydant hydrophile (AAPH) épargne les acides gras constitutifs des membranes, mais altère spécifiquement et profondément la phosphorylation oxydative au niveau de la F#1F#0aTPase. Une deuxième partie montre que l'enrichissement des membranes en acide éicosapentaénoïque (EPA) d'origine alimentaire peut déterminer un effet hypoglycémiant ou hypotriglycéridémiant selon que l'EPA est ingéré en période de jeune ou à l'état nourri. Les effets observes seraient dus à l'interférence d'intermédiaires béta-oxydatifs de l'EPA dans plusieurs voies métaboliques. Une troisième partie met l'accent sur les perturbations que la carnitine palmitoyltransférase I (CPT I) peut subir en raison de l'activité d'enzymes membranaires voisines. Ainsi, la glycerol-3-phosphate acyltransférase réduit l'activité CPT I, mais est, elle-même, influencée par cette activité ; de même la monoamine oxydase subit des variations d'activité qui retentissent directement sur les catécholamines et secondairement sur l'activité CPT I. Par ailleurs, il est montré que l'activation des acides gras par le coenzyme A, étape indispensable aux devenirs métaboliques des acides gras, est localisée exclusivement dans le reticulum endoplasmique. La thèse souligne que le contrôle de voies métaboliques peut s'exercer par des modifications membranaires.

In higher eukaryotes, approximately 90% of cellular ATP is produced by ATP synthase, a mitochondrial enzymatic complex. Numerous post-translational modifications, particularly phosphorylation, were reported for all ATP synthase subunits, both in physiological and pathological states. Still, the impact of these modifications for ATP synthase regulation or the signaling pathways involved remains ill-defined. Previously, two phosphorylation sites were identified in the catalytic β subunit of the FoF1 ATP synthase (Atp2) of S. cerevisiae, Threonine 124 (Thr) and Thr317. Phosphorylation of these residues resulted in Atp2p stabilization, increased ATP synthase activity and mitochondrial respiration. Yet, the kinases involved in the phosphorylation of these residues, conserved across species, are unknown. Using S. cerevisiae as a model, the aim of this study was thus to identify the kinase(s) involved in the phosphoregulation of the ATP synthase β subunit. Using bioinformatic motif prediction, four kinases were selected as potential Atp2 regulators: Cdc5, Ipl1, Hrr25 and Pkc1. Cdc28 was also evaluated since a genetic interaction with Atp2 was previously observed. Kinase expression was manipulated to evaluate possible effects in Atp2p phosphorylation and Atp2p levels. Zn2+Phos-Tag™ SDS-PAGE was optimized and used to detect Atp2p phosphorylation. The overexpression of Pkc1, Ipl1 and Cdc5 resulted in increased Atp2 phosphorylation. Overexpressing Ipl1 also caused an increase in Atp2p levels, consistent with increased phosphorylation, while a minor increase was observed for Cdc5. The role of Cdc5 on mitochondrial function was further evaluated. Overexpression of Cdc5 resulted in a significant increase in mitochondrial respiration and repressing Cdc5 prevented growth in respiratory media, indicating that Cdc5 plays an important, previously unsuspected role in mitochondrial function. Though overall evidence supports a role for Cdc5 in Atp2 regulation, further studies are required to confirm that Cdc5 is, in fact, a direct phosphoregulator of Atp2. Identification of Atp2p regulatory kinases will improve our understanding of the signalling pathways regulating ATP synthase function. Since many pathologies are associated with dysfunction of this vital complex, identification of its regulatory kinases can provide, in the long run, new therapeutic targets.<br>Em eucariotas superiores, cerca de 90% do ATP celular é produzido pela ATP sintase, um complexo enzimático mitocondrial. Várias modificações pós tradução, especialmente fosforilação, foram descritas em todas as subunidades da ATP sintase, tanto em condições fisiológicas normais como patológicas. No entanto, o impacto destas modificações na regulação da ATP sintase ou as vias de sinalização envolvidas precisam ainda de elucidação. Recentemente, dois locais de fosforilação foram identificados na subunidade β, catalítica, da ATP sintase (Atp2) em S. cerevisiae, Treonina (Thr) 124 e Thr317. A fosforilação destes resíduos promove a estabilização de Atp2p, e o aumento da atividade da ATP sintase e da respiração mitocondrial. Porém as cinases envolvidas na fosforilação destes resíduos, conservados entre espécies, são ainda desconhecidas. Usando S. cerevisiae como modelo, o objetivo deste estudo consistiu em identificar cinase(s) envolvida(s) na fosforegulação da subunidade β da ATP sintase. A utilização de ferramentas bioinformáticas de algoritmo preditivo de locais de fosforilação, permitiu a seleção de 4 cinases como potenciais reguladores de Atp2p: Cdc5, Ipl1, Hrr25 e Pkc1. Foi ainda avaliada a Cdc28, após observação prévia de interação genética com Atp2p. A expressão das cinases foi manipulada de forma a avaliar possíveis efeitos na fosforilação e nos níveis de Atp2p. O método Zn2+Phos-Tag™ SDS-PAGE foi otimizado e utilizado para deteção da fosforilação de Atp2p. A sobrexpressão de Cdc5, Ipl1 e Pkc1 levou a um aumento da fosforilação de Atp2p. A sobrexpressão de Ipl1 aumentou também os níveis de Atp2p, consistentes com aumento de fosforilação, enquanto que um aumento pouco significativo foi observado para Cdc5. O papel da Cdc5 na função mitocondrial foi estudado, sendo que a sua sobrexpressão causou um aumento significativo da respiração mitocondrial e a repressão de Cdc5 inibiu o crescimento em meio respiratório, sugerindo que a Cdc5 possa ter um efeito regulador importante na função mitocondrial. Mais estudos são necessários para determinar se Cdc5 é um fosforegulador direto de Atp2p. A identificação de cinases reguladoras de Atp2p contribuirá para o conhecimento das vias de sinalização que regulam a função da ATP sintase. Visto que várias patologias estão associadas à disfunção deste complexo essencial, a identificação das suas cinases reguladoras pode nomear, a longo prazo, novos alvos terapêuticos.<br>Mestrado em Biologia Molecular e Celular

This thesis primarily focuses on the FoF1-ATP synthase/ATPase complex in the parasitic protist, Trypanosoma brucei. Instead of its normal aerobic function to synthesize ATP, it is required to hydrolyze ATP to maintain the m in the infective bloodstream stage of T. brucei and the related parasite, T. b. evansi. To better understand the composition, structure and function of this druggable target, my work focused on deciphering the function of three of the unique Euglenozoa specific subunits that comprise this complex molecular machine. Furthermore, the ADP/ATP carrier, which provides substrates for the FoF1-ATP synthase/ATPase, was functionally characterized and evaluated if it is physically associated with the complexes of the oxidative phosphorylation pathway.

ATP synthase represents the key enzyme of cellular energy provision and ATP synthase disorders belong to the most deleterious mitochondrial diseases affecting pediatric population. The aim of this thesis was to identify nuclear genetic defects and describe the pathogenic mechanism of altered biosynthesis of ATP synthase that leads to isolated deficiency of this enzyme manifesting as an early onset mitochondrial encephalo-cardiomyopathy. Studies in the group of 25 patients enabled identification of two new disease-causing nuclear genes responsible for ATP synthase deficiency. The first affected gene was TMEM70 that encodes an unknown mitochondrial protein. This protein was identified as a novel assembly factor of ATP synthase, first one specific for higher eukaryotes. TMEM70 protein of 21 kDa is located in mitochondrial inner membrane and it is absent in patient tissues. TMEM70 mutation was found in 23 patients and turned to be the most frequent cause of ATP synthase deficiency. Cell culture studies also revealed that enzyme defect leads to compensatory-adaptive upregulation of respiratory chain complexes III and IV due to posttranscriptional events. The second affected gene was ATP5E that encodes small structural epsilon subunit of ATP synthase. Replacement of conserved Tyr12 with Cys caused...

Mitochondrial ATP synthase represents the final complex of oxidative phosphorylation (OXPHOS) system located in the inner mitochondrial membrane. Its primary role is to utilize mitochondrial membrane potential (Δψm) generated by respiratory chain complexes to produce energy in the form of ATP. Mammalian ATP synthase comprises of 17 different subunits organized into membranous Fo and matrix-oriented F1 domains. Defects of complex V and their manifestation have been studied on mitochondrial, cellular, tissue and organism levels using different models, including human cell lines and cell lines derived from patient tissues. In many cases mitochondrial diseases display threshold behaviour, when genetic defect is phenotypically manifested only bellow certain threshold in particular enzyme complex activity and/or content. This work was aimed at elucidation of functional consequences of ATP synthase deficiency in HEK293 cell lines with suppressed gene expression of γ, δ or ε subunits of ATP synthase central stalk. We have analysed range of clones with respective subunits knockdown and found varying decrease in assembled ATP synthase content, which was mirrored by the decrease in individual ATP synthase subunits. The only exception was subunit Fo-c, whose levels remained unchanged or even increased. ATP...

The complexes of the oxidative phosphorylation (OXPHOS) system in the inner mitochondrial membrane are organised into structural and functional super-assemblies, so-called supercomplexes. This type of organisation enables substrate channelling and hence improves the overall OXPHOS efficiency. ATP synthase associates into dimers and higher oligomers. Within the supercomplex of ATP synthasome, it interacts with ADP/ATP translocase (ANT), which exchanges synthesised ATP for cytosolic ADP, and inorganic phosphate carrier (PiC), which imports phosphate into the mitochondrial matrix. The existence of this supercomplex is generally accepted. Experimental evidence is however still lacking. In this thesis, structural interactions between ATP synthase, ANT and PiC were studied in detail. In addition, the interdependence of their expression was examined either under physiological conditions in rat tissues or using model cell lines with ATP synthase deficiencies of different origin. Specifically, they included mutations in the nuclear genes ATP5E and TMEM70 that code for subunit ε and the ancillary factor of ATP synthase biogenesis TMEM70, respectively, and a microdeletion at the interface of genes MT-ATP6 and MT-COX3 that impairs the mitochondrial translation of both subunit a of ATP synthase and subunit Cox3...

In this thesis, phylogenomic and comparative structural analyses of several widespread energy converting enzymes were performed. The focus was on the major subfamilies of the enzymes that process nucleoside triphosphates (ATP and GTP) and on some key enzymes of the electron transfer chains. First, we analyzed the P-loop GTPases, RadA/RecA recombinases, chaperone GroEL, branched-chain α-ketoacid dehydrogenase kinases, chaperone Hsc70, actins, and membrane pyrophosphatases. In the each inspected family we could identify (1) members which were potassium-dependent and/or contained K+ ions in the active site, and (2) potassium-independent enzymes with lysine or arginine residues as catalytic groups that occupy the positions of potassium ions in the homologous, K+-dependent enzymes. Based on the results of our analyses, we suggest that the appearance of the K+-binding sites could precede in evolution the recruitment of positively charged residues (lysine or arginine "fingers") with the latter providing more possibilities to control the enzyme reactions. Second, we have described the distinctive features of a phylogenetically separated subfamily of rotary membrane ATPases which we named N-ATPases. The N-ATPases have a specific operon organization with two additional subunits, absent in other rotary ATPases, and a complete set of Na+-binding ligands in the membrane c-subunits. We made a prediction, which was later confirmed, that these enzymes are capable of Na+ translocation across the membrane and may confer salt tolerance on marine prokaryotes. Third, phylogenomic analysis of the cytochrome bc complexes suggests that these enzyme complexes initially emerged within the bacteria and were then transferred to archaea via lateral gene transfer on several independent occasions. Our analysis indicates that the ancestral form of the cytochrome bc complex was a b6f-type complex; the fusion of the cytochrome b6 and the subunit IV to a "long" cytochrome b of the cytochrome bc1 complexes could have happened in different lineages independently. Fourth, our phylogenomic and comparative structural analyses of the cytochrome bc1 complex and of cytochrome c allowed us to trace how these enzymes became involved in triggering of apoptosis in Metazoa. We could trace the emergence of a specific cardiolipin-binding site within the cytochrome bc complex and the evolution of structural traits that account for the involvement of the cytochrome c as a trigger of apoptosis in vertebrates.