The goal of this proposal is to investigate events that occur in the cytosol of the yeast Saccharomyces cerevisiae prior to or during the import of proteins into mitochondria. Emphasis is placed on interactions between mitochondrial precursor proteins and cytosolic components. Such components will be identified and their function in the import process assessed. The influence of cytosolic factors on precursor conformation will be analyzed. Experiments will be performed to determine if precursor proteins fold tightly in the cytosol subsequent to their synthesis in the cell or are either prevented from folding or actively unfolded prior to the import reaction. Experiments will also be conducted to evaluate the overall importance of a cotranslational import mechanism and to determine if precursor synthesis and import are coupled in vivo. To achieve these goals, S. cerevisiae will be used to perform both in vitro and in vivo experiments. A completely homologous yeast in vitro import system that has been recently developed will be a fundamental component of the approach. Since both the cytosolic extract required for in vitro precursor synthesis and mitochondria are isolated from the same type of cell, interactions between these two cellular components can be studied and the results more directly applied to the situation in vivo. The mitochondrion, a vital organellar component of the eukaryotic cell, is made up of proteins from two genomic sources: some of mitochondrial proteins are encoded by the mitochondrion's own DNA and synthesized by its own transcriptional and translational machinery, while others are encoded by the nuclear genome and synthesized by the cytoplasmic translational machinery. The question of how these proteins get from their sites of synthesis to their functional locations in the mitochondrion is an important and widely-studied one. The two aspects of the problem addressed in this proposal, namely (1) whether or not cytosolic factors are involved in the import of nuclear-encoded mitochondrial proteins into the mitochondria, and (2) whether or not some proteins are translocated into the mitochondria co-translationally, that is, while they are still being synthesized, as opposed to post- translationally, that is, after the synthesis of the protein is completed, are important, somewhat controversial, and timely. It is anticipated that these studies will lead to fundamental new insights into the mechanism of mitochondrial biogenesis.
