RESUMO
Import of carrier proteins from the cytoplasm into the mitochondrial inner membrane of yeast is mediated by a distinct system consisting of two soluble 70-kDa protein complexes in the intermembrane space and a 300-kDa complex in the inner membrane, the TIM22 complex. The TIM22 complex contains the peripheral subunits Tim9p, Tim10p, and Tim12p and the integral membrane subunits Tim22p and Tim54p. We identify here an additional subunit, an 18-kDa integral membrane protein termed Tim18p. This protein is made as a 21.9-kDa precursor which is imported into mitochondria and processed to its mature form. When mitochondria are gently solubilized, Tim18p comigrates with the other subunits of the TIM22 complex on nondenaturing gels and is coimmunoprecipitated with Tim54p and Tim12p. Tim18p does not cofractionate with the TIM23 complex upon immunoprecipitation or nondenaturing gel electrophoresis. Deletion of Tim18p decreases the growth rate of yeast cells by a factor of two and is synthetically lethal with temperature-sensitive mutations in Tim9p or Tim10p. It also impairs the import of several precursor proteins into isolated mitochondria, and lowers the apparent mass of the TIM22 complex. We suggest that Tim18p functions in the assembly and stabilization of the TIM22 complex but does not directly participate in protein insertion into the inner membrane.
Assuntos
Antiporters , Proteínas de Transporte/metabolismo , Proteínas Fúngicas/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Transporte Biológico Ativo , Proteínas de Transporte/química , Proteínas de Transporte/genética , DNA Fúngico/genética , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Deleção de Genes , Genes Fúngicos , Membranas Intracelulares/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/química , Proteínas de Membrana/genética , Mitocôndrias/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Proteínas do Complexo de Importação de Proteína Precursora Mitocondrial , Dados de Sequência Molecular , Estrutura Quaternária de Proteína , Saccharomyces cerevisiae/genéticaRESUMO
Earlier work on the protein import system of yeast mitochondria has identified two soluble 70 kDa protein complexes in the intermembrane space. One complex contains the essential proteins Tim9p and Tim10p and mediates transport of cytosolically-made metabolite carrier proteins from the outer to the inner membrane. The other complex contains the non-essential proteins Tim8p and Tim13p as well as loosely associated Tim9p; its function was unclear, but it interacted structurally or functionally with the Tim9p-Tim10p complex. We now show that the two 70 kDa complexes each mediate the import of a different subset of integral inner membrane proteins and that they can transfer these proteins to one of three different membrane insertion sites: the TIM22 complex, the TIM23 complex or an as yet uncharacterized insertion site. Yeast mitochondria thus use multiple pathways for escorting hydrophobic inner membrane proteins across the aqueous intermembrane space.