The goal of the proposed research is to characterize the mechanisms responsible for protein and metabolite translocations across the mitochondrial membranes of Neurospora crassa. The arginine biosynthetic pathway will be used as a model system for these investigations. Arginine biosynthesis in N. crassa consists of nine enzyme catalyzed reactions distributed between the mitochondrial matrix and the cytoplasm. The enzymes are products of nuclear genes and are synthesized in the cytoplasm. The mitochondrially localized enzymes must be translocated from the cytoplasm to the mitochondrial matrix. Intermediary metabolites (ornithine and citrulline) and the end-product, arginine, must cross the mitochondrial membranes. The structure of the cytoplasmic precursors of ornithine transcarbamylase and carbamyl phosphate synthetase will be determined. The mechanism of translocation will be studied in vitro. Mutations affecting the translocation machinery will be isolated and characterized. The specificity and energetics of amino acid transport across the mitochondrial membranes will be determined. Attempts will be made to identify the components of the transport systems by isolating mutations affecting their function. The properties and submitochondrial localization of the feedback-sensitive enzymes acetylglutamate synthase and acetylglutamate kinase will be determined. The results will be significant in understanding eucaryotic cells.