): The long range goal of this research proposal is to understand how proteins of the mitochondrial inner membrane and intermembrane space mediate two events: translocation of proteins into the mitochondrial matrix, and protein sorting to the inner membrane. Mitochondria are essential organelles, whose function requires the import of hundreds of different proteins that are encoded in the nucleus, synthesized in the cytosol and imported into the organelle. Protein import is a multistep pathway which includes the binding of precursor proteins to surface receptors, translocation across one or both mitochondrial membranes, and folding and assembly of the imported protein inside the mitochondrion. To understand how these processes occur, we have isolated and are analyzing mutants in the yeast, Saccharomyces cerevisiae, that are defective in mitochondrial protein import. Many studies indicate that mitochondria in yeast import proteins into their mitochondria by a process virtually identical to the way mammalian mitochondria import proteins. Hence our studies with yeast will yield valuable information about mitochondria biogenesis in all organisms. Supporting this view, a human disease called Mohr-Tranebjaerg syndrome, a neurodegenerative disease characterized by deafness, dystonia , mental retardation and blindness, has recently been shown to result from a defect in mitochondrial protein import. The human mutation is in a gene encoding a protein called DDP, homologous to the yeast Tim8 protein. We anticipate that our future studies will continue to provide insights into the normal functions of mitochondria and their relationships to human disease. We have recently shown that the mitochondrial inner membrane contains two translocation complexes: the TIM23 complex, which is required to translocate precursors across the inner membrane into the matrix and the TIM22 complex, which is required for the insertion of polytopic proteins into the inner membrane. Tim54p, Tim22p and Tim18p comprise the membrane subunits of the TIM22 complex. In this proposal, we ask several questions aimed at understanding the mechanism by which proteins are inserted into the inner membrane. (1) What other proteins comprise the TIM22 complex? (2) Does Tim18p play a direct role in import? (3) Does Tim54p play a docking role in the insertion process? (4) What is the precise pathway that proteins traverse during their import and insertion into the inner membrane? (5) Can inner membrane protein insertion be reconstituted using purified components?
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