The broad, long-term objective of this proposal is to develop the yeast Saccharomyces cerevisiae as a eukaryotic model to study the compartmentation of folate-mediated one-carbon metabolism. Metabolic compartmentation is found universally in eukaryotes, from the highest mammals to the lowest unicellular forms. Metabolic compartmentation is a critical aspect of normal cell function: a great number of human diseases, including I-cell disease, pseudo-Hurler polydistrophy, sarcosinemia, and nonketotic hyperglycinemia, are characterized by miscompartmentalization of an enzyme or substrate. Cellular compartmentation also impacts the treatment of disease by drugs. One-carbon transfers mediated by folate coenzymes play essential roles in several major cellular processes, including nucleic acid biosynthesis, mitochondrial and chloroplast protein biosynthesis, amino acid metabolism, methyl group biogenesis, and vitamin metabolism. However, very little is known about the compartmentation of folate-mediated one-carbon metabolism. This fundamental metabolic problem will be addressed using a combination of yeast molecular genetics and 13C NMR.
The specific aims of this proposal are to: (1) complete the set of metabolic blocks in the intercompartmental path way by isolating yeast mutants in the genes encoding the two serine hydroxymethyltransferase isozymes, and the glycine cleavage system; (2) apply 13C-NMR analysis in wild-type and mutant strains of yeast to characterize the flux of one- carbon units between cytoplasm and mitochondria and the reactions that control the flux; and (3) study the role of the MTD1 gene, encoding an NAD-dependent monofunctional methylenetetrahydrofolate dehydrogenase, in the compartmentation of folate-mediated one-carbon units. The experimental design involves (a) classical genetic and molecular genetic methods for the isolation of yeast mutants blocked in specific reactions of the intercompartmental pathway; and (b) NMR analysis of in vivo labeled wild- type and mutant strains incubated with 13C-enriched substrates. These experiments should allow us to determine the role of each of the enzymes in the cytoplasmic and mitochondrial compartments; the role that mitochondrial one-carbon metabolism plays in cytoplasmic processes; and the roles of the two cytoplasmic methylenetetrahydrofolate dehydrogenases with differing coenzyme specificity (NAD vs. NADP).
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