The long-term objective of this proposal is to understand the organization and compartmentation of folate-mediated one-carbon metabolism in eukaryotes. Metabolic compartmentation is found universally in eukaryotes, from the highest mammals to the lowest unicellular forms, and is a critical aspect of normal cell function. The organization, compartmentation, and control of folate-mediated one-carbon metabolism is poorly understood. Previous work supported by this grant demonstrated the usefulness of the yeast Saccharomyces cerevisiae as a model system. The applicant proposes to continue the use of biochemical, molecular genetic, and 13C NMR techniques to further dissect the flow of one-carbon units between the cytoplasm, mitochondria, and endoplasmic reticulum. He will use purine biosynthesis, mitochondrial protein synthesis, and methyl group biogenesis, respectively, as indicators of one-carbon metabolism in each compartment.
Four specific aims are proposed: (1) isolate and characterize mutants in the yeast genes encoding AICAR transformylase/IMP cyclohydrolase and 5,10-methylene-THF reductase; (2) investigate the roles of MIS1, DFR1 and ORF YBL03-11 gene products in mitochondrial one-carbon metabolism; (3) investigate the noncatalytic function of the ADE3 gene product, C1-THF synthase, in purine biosynthesis; (4) determine how one-carbon units are distributed into the three major pools (CHO-THF, CH2-THF, CH3-THF) in response to varying growth and nutritional conditions. The experimental design involves: (a) construction of yeast mutants blocked in specific one-carbon reactions; (b) biochemical assays of formylmethionyl-tRNA levels in mitochondria from wild-type and mutant strains; (c) synthetic lethal and two-hybrid screens for additional components of these pathways; and (d) NMR analysis of wild-type and mutant strains labeled in vivo with 13C-enriched substrates to directly follow the intercompartmental flow of one-carbon units.
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