Mitochondrial genetic systems have diverged enormously from their bacterial ancestors during the course of eukaryotic evolution, and must be studied directly if we are to understand them. Our work focuses on critical translational and posttranslational steps in the expression of mitochondrial genes specifying the core subunits of cytochrome c oxidase, Coxip, Cox2p and Cox3p. In Saccharomyces cerevisiae we can employ genetic tools, including reporter genes inserted into the mtDNA of living cells, to study translation, protein targeting, protein folding and assembly. One of the important goals of this project is to elucidate the mechanisms that regulate translation and localize it to the surface of the inner membrane. We will seek to understand the roles played here by the mRNA-specific translational activators of yeast mitochondria, by studying their interactions with the general translation apparatus and with each other. We will also attempt to study cytologically the distribution of activator proteins and mitochondrial mRNAs within the organellar reticulum. Another important goal is to understand how the hydrophilic Cox2p N-tail and C-tail domains are translocated through the inner membrane to its outer surface. Our work to date, employing a reporter-based genetic screen, has demonstrated that they are exported by distinct mechanisms, and that C-tail export involves a specific complex of at least three proteins. Our recent findings suggest that the C-tail domain may be recognized and translocated posttranslationally as a folded domain, and that its export may require, surprisingly, prior cleavage of the pre-Cox2p leader peptide from the exported N-tail. We will test these ideas by phenotypic analysis of mutants and studies employing reporter proteins coded by existing synthetic mitochondrial genes, and new one to be constructed. Localization of translation to sites of membrane insertion and cytochrome oxidase assembly could permit control of synthesis by the need for new subunits in assembling complexes, similar to assembly feedback controls observed in other systems. Coxip accumulation is strongly reduced in cells with certain cytochrome oxidase assembly defects, and pulse-chase labeling experiments suggest that synthesis is inhibited. We will explore this phenomenon using reporter gene fusions to monitor COX1 mRNA translation independently of Coxip stability, and to select mutations affecting the feedback process. The results of these studies will help to provide a coherrent picture of mitochondrial gene expression in yeast that will inform research on mitochondrial systems in humans and other organisms.