RESUMO
Autophagosome biogenesis requires two ubiquitin-like conjugation systems. One couples ubiquitin-like Atg8 to phosphatidylethanolamine, and the other couples ubiquitin-like Atg12 to Atg5. Atg12~Atg5 then forms a heterodimer with Atg16. Membrane recruitment of the Atg12~Atg5/Atg16 complex defines the Atg8 lipidation site. Lipidation requires a PI3P-containing precursor. How PI3P is sensed and used to coordinate the conjugation systems remained unclear. Here, we show that Atg21, a WD40 ß-propeller, binds via PI3P to the preautophagosomal structure (PAS). Atg21 directly interacts with the coiled-coil domain of Atg16 and with Atg8. This latter interaction requires the conserved F5K6-motif in the N-terminal helical domain of Atg8, but not its AIM-binding site. Accordingly, the Atg8 AIM-binding site remains free to mediate interaction with its E2 enzyme Atg3. Atg21 thus defines PI3P-dependently the lipidation site by linking and organising the E3 ligase complex and Atg8 at the PAS.
Assuntos
Endopeptidases/metabolismo , Fosfatos de Inositol/metabolismo , Lipoilação/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Motivos de Aminoácidos , Família da Proteína 8 Relacionada à Autofagia , Proteínas Relacionadas à Autofagia , Endopeptidases/genética , Fosfatos de Inositol/genética , Proteínas Associadas aos Microtúbulos/genética , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Enzimas de Conjugação de Ubiquitina/genéticaRESUMO
The mitochondrial electron transport chain consists of individual protein complexes arranged into large macromolecular structures, termed respiratory chain supercomplexes or respirasomes. In the yeast Saccharomyces cerevisiae, respiratory chain supercomplexes form by association of the bc1 complex with the cytochrome c oxidase. Formation and maintenance of these assemblies are promoted by specific respiratory supercomplex factors, the Rcf proteins. For these proteins a regulatory function in bridging the electron transfer within supercomplexes has been proposed. Here we report on the maturation of Rcf2 into an N- and C-terminal peptide. We show that the previously uncharacterized Rcf3 (YBR255c-A) is a homolog of the N-terminal Rcf2 peptide, whereas Rcf1 is homologous to the C-terminal portion. Both Rcf3 and the C-terminal fragment of Rcf2 associate with monomeric cytochrome c oxidase and respiratory chain supercomplexes. A lack of Rcf2 and Rcf3 increases oxygen flux through the respiratory chain by up-regulation of the cytochrome c oxidase activity. A double gene deletion of RCF2 and RCF3 affects cellular survival under non-fermentable growth conditions, suggesting an overlapping role for both proteins in the regulation of the OXPHOS activity. Furthermore, our data suggest an association of all three Rcf proteins with the bc1 complex in the absence of a functional cytochrome c oxidase and identify a supercomplex independent interaction network of the Rcf proteins.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Transporte de Elétrons , Complexo IV da Cadeia de Transporte de Elétrons/genética , Deleção de Genes , Mutação , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
The cytochrome c oxidase (COX) is the terminal enzyme of the respiratory chain. The complex accepts electrons from cytochrome c and passes them onto molecular oxygen. This process contributes to energy capture in the form of a membrane potential across the inner membrane. The enzyme complex assembles in a stepwise process from the three mitochondria-encoded core subunits Cox1, Cox2 and Cox3, which associate with nuclear-encoded subunits and cofactors. In the yeast Saccharomyces cerevisiae, the cytochrome c oxidase associates with the bc1-complex into supercomplexes, allowing efficient energy transduction. Here we report on Cox26 as a protein found in respiratory chain supercomplexes containing cytochrome c oxidase. Our analyses reveal Cox26 as a novel stoichiometric structural subunit of the cytochrome c oxidase. A loss of Cox26 affects cytochrome c oxidase activity and respirasome organization.
Assuntos
Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Mitocôndrias/enzimologia , Membranas Mitocondriais/enzimologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Complexo IV da Cadeia de Transporte de Elétrons/genética , Complexo IV da Cadeia de Transporte de Elétrons/isolamento & purificação , Potencial da Membrana Mitocondrial , Consumo de Oxigênio , Subunidades Proteicas , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Fatores de TempoRESUMO
Three mitochondria-encoded subunits form the catalytic core of cytochrome c oxidase, the terminal enzyme of the respiratory chain. COX1 and COX2 contain heme and copper redox centers, which are integrated during assembly of the enzyme. Defects in this process lead to an enzyme deficiency and manifest as mitochondrial disorders in humans. Here we demonstrate that COA6 is specifically required for COX2 biogenesis. Absence of COA6 leads to fast turnover of newly synthesized COX2 and a concomitant reduction in cytochrome c oxidase levels. COA6 interacts transiently with the copper-containing catalytic domain of newly synthesized COX2. Interestingly, similar to the copper metallochaperone SCO2, loss of COA6 causes cardiomyopathy in humans. We show that COA6 and SCO2 interact and that corresponding pathogenic mutations in each protein affect complex formation. Our analyses define COA6 as a constituent of the mitochondrial copper relay system, linking defects in COX2 metallation to cardiac cytochrome c oxidase deficiency.
Assuntos
Cardiomiopatias/metabolismo , Proteínas de Transporte/metabolismo , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Doenças Mitocondriais/metabolismo , Proteínas Mitocondriais/metabolismo , Cardiomiopatias/genética , Proteínas de Transporte/genética , Complexo IV da Cadeia de Transporte de Elétrons/genética , Células HEK293 , Humanos , Doenças Mitocondriais/genética , Proteínas Mitocondriais/genética , Chaperonas Moleculares , Saccharomyces cerevisiaeRESUMO
The mitochondrial respiratory chain is essential for the conversion of energy derived from the oxidation of metabolites into the membrane potential, which drives the synthesis of ATP. The electron transporting complexes bc1 complex and the cytochrome c oxidase assemble into large supercomplexes, allowing efficient energy transduction. Currently, we have only limited information about what determines the structure of the supercomplex. Here, we characterize Aim24 in baker's yeast as a protein, which is integrated in the mitochondrial inner membrane and is required for the structural integrity of the supercomplex. Deletion of AIM24 strongly affects activity of the respiratory chain and induces a growth defect on non-fermentable medium. Our data indicate that Aim24 has a function in stabilizing the respiratory chain supercomplexes.