9604620 Weiss This research project will characterize the structure, assembly and inter-relationships of the feedback sensitive enzymes of arginine biosynthesis in Neurospora crassa. The first seven biosynthetic enzymes, including the feedback-sensitive enzymes, acetylglutamate synthase and acetylglutamate kinase, are localized in the mitochondria. These enzymes are products of the unlinked arg-14 and arg-6 genes. The latter encodes a polyprotein precursor of acetylglutamate kinase and acetylglutamyl-phosphate reductase; the precursor is cleaved into separable proteins upon entry into the mitochondrion. A molecular analysis will be performed to investigate the biological role and evolutionary origin of the polyprotein precursor of acetylglutamate kinase and acetylglutamylphosphate reductase. The structural and catalytic properties of acetylglutamate synthase will be determined. The structural and functional relationships between acetylglutamate synthase, acetylglutamate kinase and acetylglutamyl-phosphate reductase will be investigated to evaluate various models for coordination of feedback inhibition, enzyme synthesis and assembly. Genetic and biochemical analyses will be performed to ascertain the role(s) of acetylglutamate kinase in synthesis, processing, assembly and functioning of acetylglutamate synthase. Mutants with a phenotype consistent with defects in arginine transport from the cytosol to the mitochondrial matrix will be isolated and characterized. The results will be of significance in understanding metabolic regulation and the assembly of mitochondria (and other organelles) in eukaryotic cells. %%% The cells of complex organisms such as man are distinguishable from lower organisms such as bacteria by the presence of machinery for both the synthesis and breakdown of common biological molecules. In order to prevent the simultaneous occurrence of these potentially competing processes, they are often separated by intracellular membranes, with one of the processes confined to a mem brane-enclosed organelle. These differences require mechanisms to move molecules across intracellular membranes and the utilization of novel mechanisms to contol metabolism. Defects in intracellular localization can have serious consequences for the survival of the organism. This project will characterize the mechanism used to control the synthesis of the amino acid arginine and the importance of transport of arginine across an intracellular membrane as a first step in understanding the unique regulatory mechanisms characteristic of complex cells and organisms. ***
