RESUMO
The gamma-subunit is required for the assembly of ATP synthases and plays a crucial role in their catalytic activity. We stepwise shortened the N-terminus and the C-terminus of the gamma-subunit in the mitochondrial ATP synthase of yeast and investigated the relevance of these segments in the assembly of the enzyme and in the growth of the cells. We found that a deletion of 9 residues at the N-terminus or 20 residues at the C-terminus still allowed efficient import of the subunit into mitochondria; however, the assembly of both monomeric and dimeric holoenzymes was partially impaired. gamma-Subunits lacking 13 N-terminal residues or 30 C-terminal residues were not assembled. Yeast strains expressing either of the truncated gamma-subunits did not grow on non-fermentable carbon sources, indicating that non-assembled parts of the ATP synthase accumulated and impaired essential mitochondrial functions.
Assuntos
Proteínas Mitocondriais/genética , ATPases Mitocondriais Próton-Translocadoras/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Sequência de Aminoácidos , Eletroforese em Gel de Poliacrilamida , Proteínas Mitocondriais/química , Proteínas Mitocondriais/metabolismo , ATPases Mitocondriais Próton-Translocadoras/química , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Dados de Sequência Molecular , Subunidades Proteicas/química , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Deleção de SequênciaRESUMO
A family of related carrier proteins mediates the exchange of metabolites across the mitochondrial inner membrane. The carrier signature Px[D/E]xx[K/R] is a highly conserved sequence motif in all members of this family. To determine its function in the biogenesis of carrier proteins, we used the dicarboxylate carrier (DIC) of yeast as a model protein. We found that the carrier signature was dispensable in binding of the newly synthesized protein to the import receptor Tom70, but that it was specifically required for efficient translocation across the mitochondrial outer membrane. To determine the relevance of individual amino acid residues of the carrier signature in the transport activity of the protein, we exchanged defined residues with alanine and reconstituted the mutant proteins in vitro. Substitution of the carrier signature in helix H1 reduced the transport activity for [(33)P]-phosphate by approximately 90% and an additional substitution of the carrier signature in helix H5 blocked the transport activity completely. We conclude that the carrier signature of the dicarboxylate carrier is involved both in the biogenesis and in the transport activity of the functional protein.