The proposed studies are directed to understanding the system of mitochondrial compartmentation, whereby mitochondrial protein precursors, encoded in the nucleus and synthesized on cytoplasmic polyribosomes, are posttranslationally recognized via their NH2- terminal leader peptides, translocated across one or both membranes, and proteolytically processed to their active forms. We will continue to use as a model for analysis of this system the human mitochondrial matrix enzyme ornithine transcarbamylase (OTC), which catalyzes the second step of the urea cycle in mammals. The studies proposed here are aimed at: identification and characterization of components of the mitochondria of both Saccharomyces cerevisiae and mammalian cells that are involved with specific recognition, import to the matrix compartment, and proteolytic processing of the OTC precursor; determination of whether such components are shared by other proteins destined for mitochondria; and analysis of the higher-order structure of the OTC leader peptide. In both yeast and mammalian cells, genetic approaches will be taken to isolating genes encoding import components: suppressing mutations will be isolated in both Saccharomyces and HeLa cells that permit mutant OTC precursors to reach the mitochondria; mutations that block import/processing of the wild-type OTC precursor and result in a conditional growth-deficient state will also be isolated in Saccharomyces. Biochemical approaches will be taken to studying components involved with import. The wild-type OTC precursor will be overproduced in E. coli by mutagenizing a plasmid programming production of a fusion protein joining the wild-type OTC leader peptide with galactokinase, and assaying for high- level expression of enzyme activity by a colony color assay. The overproduced precursor will be purified and used in a variety of studies, including mitochondrial binding studies designed to examine kinetics of recognition by the organelles and competition for recognition with other precursors; crosslinking reactions designed to identify specific components that interact with the OTC precursor; and structural studies designed to analyze the higher-order structure of the leader peptide. In vitro synthesized precursors with either reactive amino acid side chains or enzymatically active mature portions will also be used as affinity- labeling reagents, to identify additional interacting mitochondrial components.