The goal of this research project is to characterize the role of compartmentation in the regulation of amino acid metabolism in Neurospora crassa. The organellar localization of metabolic pathways requires considerable expenditure of metabolic energy for protein targeting, organelle assembly, and movement of substrates and products across intracellular membranes. Such expenditures must result in biological efficiencies commensurate with the investment of biological resources. However, it is not always clear what advantages are conferred by such compartmentation. Some general hypotheses have been advanced to explain this compartmentation phenomenon. These include the achievement of high localized concentrations of intermediary metabolites, the isolation of potentially reactive intermediates, the separation of potentially competing reactions and the conservation of solvent capacity. In N. crassa, the biosynthesis of arginine originates in the mitochondria but culminates in the cytosol. Intermediary metabolites (ornithine and citrulline) and the end product (arginine) cross both mitochondrial membranes: ornithine to support polyamine synthesis in the cytosol; citrulline to complete arginine synthesis in the cytosol; and arginine to support mitochondrial protein synthesis. In order to evaluate various hypotheses for the role of metabolic compartmentation, the metabolic and physiological consequences of relocating the arginine biosynthetic enzymes from the mitochondrial matrix to the cytosol will be investigated. Genes for the arginine biosynthetic enzymes leading to citrulline synthesis will be cloned and characterized. Amino acid sequences responsible for targeting the cytosolic precursors to the mitochondria will be identified. The cloned genes will be altered in vitro so that the catalytically active enzymes that they encode will be confined to the cytosol. Where possible, the functionality of the altered genes will be tested by inserting them downstream of a yeast promoter and testing their ability to complement mutations in the homologous yeast genes whose products normally function in the cytosol. The altered genes, arg*, or the homologous yeast (or bacterial) genes inserted downstream of a Neurospora promoter, will be introduced into the appropriate neurospora mutants. Strains carrying multiple altered genes will be constructed by genetic recombination. The consequences of enzyme relocation will be determined by examining the effects of enzyme mislocation on the efficiency of arginine biosynthesis, the channeling of carbamyl-phosphate and ornithine, and the operation of the potentially futile """"""""urea cycle"""""""". The results will contribute to our understanding of the function of metabolic compartmentation in eukaryotic cells. Impairment or alterations in compartmentation are the basis of a variety of human disorders.
