The overall goal of this research proposal is to understand the various aspects of proline metabolism in Saccharomyces cerevisiae as a model system for the integration of a complex series of interactions that involves the nuclear, cytoplasmic and mitochondrial compartments within the cell. Proline biosynthesis and proline utilization involve a series of reactions which have a common intermediate and pools of substrates and products that must move between the mitochondrial and the cytosolic compartments. It is our objective to learn what roles the identified regulatory proteins play, the importance of the compartmental separation, how the important metabolites enter and leave the mitochondrion and whether the biosynthetic and degradative pathways communicate with one another via regulatory proteins or small molecules. An appreciation for how the basic metabolic processes work will enable us to understand and treat situations in which aberrations in regulation occur. This proposal addresses issues involved in the regulation of proline degradation and biosynthesis and the role of compartmentation in proline metabolism. The positive regulatory element of the proline utilization pathway, the PUT3 gene, will be cloned and analyzed at the molecular level. This will involve studies to determine the regulation of PUT3 gene expression, the phenotype of a null mutation, and interactions the regulator has with other elements in the pathway. Three types of gene fusions (PUTl-galK, PUTl-lacZ and PUT2-lacZ) will be used to isolate mutations in other positive or negative trans-acting regulators that affect proline utilization. Clones of the three structural genes of the proline biosynthetic pathway will be studied to determine whether they are controlled by proline-specific and/or general amino acid control regulatory elements using Northern analysis of mRNAs from the wild-type, proline auxotrophic, and general control defective strains. Physical interactions (e.g. complex formation) and subcellular localization of the enzymes will be studied by enzymatic assays as well as by immunoprecipitation using antibodies generated against purified proteins. The importance of the cellular compartments in proline metabolism will be studied by perturbing their normal locations, using genetic engineering techniques to move a cytoplasmic proline biosynthetic enzyme into the mitochondrion and the mitochondrial proline degradative enzymes into the cytoplasm. The transport of proline into the mitochondrion will be studied by adapting techniques used in higher cells to measure metabolite flux: osmotic swelling of mitochondria and centrifugal filtration through oil. The effects of stereospecificity, energy uncouplers, proline analogs and protein inhibitors on proline transport will be determined.
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