The research proposed in this application reflects a continuing interest in the mechanisms by which nuclearly coded mitochondrial proteins are proteolytically processed post-translocationally to produce mature, active enzymes. This process is central to the maintenance and propagation of mitochondria, and, hence, to cellular metabolism and homeostasis. This pathway must be understood in order to account for certain aspects of human metabolic diseases and to rationally design strategies for their somatic correction.
The specific aims are: 1) to express in E. coli both subunits of yeast mitochondrial processing peptidase (MPP) and reconstitute its activity; and 2) to carry out site-directed mutagenesis of the active site of yeast MPP and its putative substrate binding site. The proposed research will complement and extend that carried out on rat MPP, the homologous protease from a very dissimilar organism. Site- directed mutagenesis, heterologous expression, purification of MPP, and in vitro assay of its proteolytic activity, metal-ion binding, and substrate binding will yield data on the roles of conserved residues and regions in various aspects of the enzymatic mechanism. Because the homology between rat and yeast MPP is relatively high and the general characteristics of the enzymes are similar, it will be possible to compare and contrast the effects of the same mutations in two homologous enzymes to provide additional insights into the mechanism of the overall reaction and the importance of the inter-species variations for the biological role of this essential enzyme.
