The goal of this project is to characterize the function of the vascular compartmentation of amino acids in Neurospora crassa. Arginine, ornithine and a number of other amino acids are sequestered in the vacuoles in this organism. A specific arginine carrier has been identified in the tonoplast. The protein will be purified sufficiently to obtain a partial amino acid sequence. The amino acid sequence will be used to synthesize gene-specific oligonucleotide probes and polyclonal antibodies to be used to isolate the structural gene. Once isolated, the cloned gene will be used to construct mutants defective in the ability to accumulate arginine in the vacuoles. Attempts will also be made to isolate mutants by standard genetic techniques. Such mutants will be characterized for physiological defects. The identity and specificity of other tonoplast carriers will be determined. Genetic, biochemical and biophysical techniques will be used to investigate the mechanisms responsible for regulating the uptake, retention and release of amino acids from the vacuoles. The effect of environmental alterations on vacuolar pH and the association of vacuolar arginine with polyphosphates will be investigated using in situ 15N-nuclear magnetic resonance spectroscopy. The relationship between nitrogen metabolite regulation and mobilization of vacuolar arginine will be investigated using mutants affecting nitrogen metabolite regulation. The physiological metal ion cofactor for the catabolic enzyme arginase will be identified and the kinetic properties of the enzyme will be measured under physiological conditions to test the hypothesis that the availability of arginine to the enzyme is the primary determinant in controlling arginine catabolism. The structural gene will be cloned and used to determine the origin of the multiple immunologically related species detected with antibodies to the purified enzyme. The results may provide insight into the function of amino acid compartment in eukaryotic cells and organisms.
