Control of protein synthesis in yeast mitochondria: The concept of translational activators

AbstractMitochondria contain their own genome which codes for a small number of proteins. Most mitochondrial translation products are part of the membrane-embedded reaction centers of the respiratory chain complexes. In the yeast Saccharomyces cerevisiae, the expression of these proteins is regulated by translational activators that bind mitochondrial mRNAs, in most cases to their 5′-untranslated regions, and each mitochondrial mRNA appears to have its own translational activator(s). Recent studies showed that these translational activators can be part of feedback control loops which only permit translation if the downstream assembly of nascent translation products can occur. In several cases, the accumulation of a non-assembled protein prevents further synthesis of this protein but not translation in general. These control loops prevent the synthesis of potentially harmful assembly intermediates of the reaction centers of mitochondrial enzymes. Since such regulatory feedback loops only work if translation occurs in the compartment in which the complexes of the respiratory chain are assembled, these control mechanisms require the presence of a translation machinery in mitochondria. This might explain why eukaryotic cells maintained DNA in mitochondria during the last two billion years of evolution. This review gives an overview of the mitochondrial translation system and summarizes the current knowledge on translational activators and their role in the regulation of mitochondrial protein synthesis. This article is part of a Special Issue entitled: Protein import and quality control in mitochondria and plastids

The Saccharomyces cerevisiae OXA1 gene is required for the correct assembly of cytochrome c oxidase and oligomycin-sensitive ATP synthase

AbstractThe nuclear gene OXA1 was first isolated in Saccharomyces cerevisiae and found to be required at a post-translational step in cytochrome c oxidase biogenesis, probably at the level of assembly. Mutations in OXA1 lead to a complete respiratory deficiency. The protein Oxa1p is conserved through evolution and a human homolog has been isolated by functional complementation of a yeast oxa1− mutant. In order to further our understanding of the role of Oxa1p, we have constructed two yeast strains in which the OXA1 open reading frame was almost totally deleted. Cytochrome spectra and enzymatic activity measurements show the absence of heme aa3 and of a cytochrome c oxido-reductase activity and dramatic decrease of the oligomycin sensitive ATPase activity. Analysis of the respiratory complexes in non-denaturing gels reveals that Oxa1p is necessary for the correct assembly of the cytochrome c oxidase and the ATP synthase complex

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µ-Calpain Conversion of Antiapoptotic Bfl-1 (BCL2A1) into a Prodeath Factor Reveals Two Distinct alpha-Helices Inducing Mitochondria-Mediated Apoptosis

Anti-apoptotic Bfl-1 and pro-apoptotic Bax, two members of the Bcl-2 family sharing a similar structural fold, are classically viewed as antagonist regulators of apoptosis. However, both proteins were reported to be death inducers following cleavage by the cysteine protease µ-calpain. Here we demonstrate that calpain-mediated cleavage of full-length Bfl-1 induces the release of C-terminal membrane active α-helices that are responsible for its conversion into a pro-apoptotic factor. A careful comparison of the different membrane-active regions present in the Bfl-1 truncated fragments with homologous domains of Bax show that helix α5, but not α6, of Bfl-1 induces cell death and cytochrome c release from purified mitochondria through a Bax/Bak-dependent mechanism. In contrast, both helices α5 and α6 of Bax permeabilize mitochondria regardless of the presence of Bax or Bak. Moreover, we provide evidence that the α9 helix of Bfl-1 promotes cytochrome c release and apoptosis through a unique membrane-destabilizing action whereas Bax-α9 does not display such activities. Hence, despite a common 3D-structure, C-terminal toxic domains present on Bfl-1 and Bax function in a dissimilar manner to permeabilize mitochondria and induce apoptosis. These findings provide insights for designing therapeutic approaches that could exploit the cleavage of endogenous Bcl-2 family proteins or the use of Bfl-1/Bax-derived peptides to promote tumor cell clearance

Feu vert pour la construction et l’exploitation d’une unité de traitement des nitrates (TDN) à Malvési

Le site New AREVA de Malvési, situé dans l’Aude, a pour activité la première étape de la conversion de l’uranium naturel, qui se situe entre l’extraction des minerais en provenance des différents sites miniers et l’enrichissement de l’uranium. Il est aujourd’hui le seul site de conversion de l’uranium dans le monde à avoir investi massivement dans un nouvel outil industriel aux plus hauts standards de sûreté et de sécurité, mais également à avoir lancé un programme d’envergure de gestion durable de son cycle de vie industriel. Après 50 ans de fonctionnement, 500 millions d’euros ont ainsi été investis en 10 ans pour renouveler l’équipement industriel et réduire l’empreinte environnementale du site. Le projet de traitement des nitrates (TDN), objet d’une autorisation administrative de mise en oeuvre en novembre 2017, en est un exemple concret

Etude de la traduction mitochondriale chez la levure Schizosaccharomyces pombe

La mitochondrie, organite spécialisé dans la production d énergie, possède son propre génome et son propre système d expression. Le système de traduction mitochondriale est dérivé du système procaryotique et présente une conservation des facteurs impliqués. Ces facteurs sont plus conservés entre les eucaryotes supérieurs et S. pombe, alors que leur étude est classiquement menée chez S. cerevisiae. Nous avons donc étudié la traduction mitochondriale chez S. pombe.L étape d élongation de la traduction mitochondriale implique trois facteurs chez les eucaryotes supérieurs et S. pombe , et seulement deux de ces facteurs sont conservés chez S. cerevisiae. Les facteurs conservés, EF-G et EF-Tu, sont des GTPases et le facteur absent chez S. cerevisiae, EF-Ts, est le facteur d échange des nucléotides de EF-Tu. Nous avons montré que l absence de EF-Ts chez S. pombe conduit à une diminution drastique de la traduction et une absence de respiration. De plus, nous avons montré que la traduction mitochondriale est essentielle à la viabilité chez S. pombe. En présence de la mutation nucléaire ptp1-1, qui permet la viabilité en absence d ADNmt, l abolition de la traduction mitochondriale conduit à une déplétion de l ADNmt. Nous avons aussi démontré que le facteur EF-Tu de S. cerevisiae est bien indépendant d un facteur d échange, contrairement au facteur de S. pombe. Enfin, nous avons isolé des variants du facteur EF-Tu de S. pombe ne nécessitant plus de facteur d échange.De plus, nous avons débuté l analyse de l étape d initiation chez cette levure par l étude de l homologue au facteur d initiation IF3 bactérien qui ne semble pas être conservé chez S. cerevisiae.Mitochondria, the organelle that produces energy, have is own genome and is own system of expression. Mitochondrial translation is derived from prokaryotes and shows conservation of factors implied. These factors are more conserved between higher eukaryotes and fission yeast, whereas studies are mostly investigated in budding yeast. So we have started studying mitochondrial translation in the fission yeast.The elongation step of mitochondrial translation implies three factors in higher eukaryotes and fission yeast, and only two of them are conserved in budding yeast. The conserved factors, EF-Tu and EF-G, are GTPases and the factor absent in budding yeast is the nucleotide exchange factor of EF-Tu; EF-Ts. We have shown that EF-Ts absence in fission yeast leads to a drastic decrease of mitochondrial translation and leads to respiratory deficiency. We have also shown that mitochondrial translation is essential in fission yeast. In a ptp1-1 genetic background, which leads to viability in absence of mtDNA, abolition of mitochondrial translation leads to a mtDNA depletion. Moreover, we have shown that budding yeast EF-Tu is independent of exchange factor, in contrast with fission yeast EF-Tu. And we have isolated several variants of fission yeast EF-Tu independent of exchange factor.We have also started studying the initiation step of translation in this yeast with the study of the bacterial IF3 homolog which seems to be absent in the budding yeast.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Bfl-1, une cible thérapeutique dans les hémopathies malignes1 (étude du mécanisme d action et de l inhibition de la protéine anti-apoptotiques Bfl-)

La protéine Bfl-1 est un membre anti-apoptotique de la famille Bcl-2. Plusieurs études montrent qu une expression élevée de Bfl-1 est retrouvée dans différents cancers, leucémies, et lymphomes B. Ces données suggèrent que, en plus de Bcl-2, Bcl-xL et Mcl-1, Bfl-1 peut être une cible thérapeutique potentielle relevante dans la recherche de nouvelles stratégies anti-cancéreuses. Dans le cadre de ce travail de thèse, nous avons montré in vitro que l inhibition de l expression de Bfl-1 par l interférence ARN ou de sa fonction anti-apoptotique par des aptamères peptidiques sensibilise plusieurs lignées de lymphomes B à la mort induite par des drogues utilisées actuellement en cancérologie. En parallèle, afin de mieux comprendre le mécanisme d action de Bfl-1, nous avons montré que Bfl-1 est constitutivement insérée au niveau des membranes mitochondriale et que son extrémité C-terminale amphiphile est nécessaire pour sa localisation mitochondriale mais pas pour sa fonction anti-apoptotique.Bfl-1 protein is an antiapoptotic member of Bcl-2 family. Elevated expression of Bfl-1 has been reported in several cancer, leukemia and lymphoma. These data suggest that, in addition to Bcl-2, Bcl-xL and Mcl-1, Bfl-1 may be a relevant target for the design of new molecules for cancer therapy. As part of this PhD thesis, we have shown in vitro that inhibition of Bfl-1 expression using shRNA interference or inhibition of Bfl-1 antiapoptotic function using peptide aptamers sensitizes several B lymphoma cell lines to cell death induced by chemotherapeutic molecules. In parallel, in order to understand the mechanism of action of Bfl-1, we have shown that Bfl-1 is preferentially anchored at the mitochondria and that its C-terminal tail is required for its anchorage to the mitochondria but not for its antiapoptotic function.LYON-ENS Sciences (693872304) / SudocSudocFranceF