IN-VIVO ANALYSIS OF FOLATE COENZYMES AND THEIR COMPARTMENTATION IN SACCHAROMYCES-CEREVISIAE

In eukaryotes, enzymes responsible for the interconversion of one-carb on units exist in parallel in both mitochondria and the cytoplasm. Str ains of Saccharomyces cerevisiae were constructed that possess combina tions of gene disruptions at the SHM1 [mitochondrial serine hydroxymet hyltransferase (SHMTm)], SHM2 [cytoplasmic SHMT (SHMTc)], MIS1 [mitoch ondrial C-1-tetrahydrofolate synthase (C-1-THFSm)], ADE3 [cytoplasmic C-1-THF synthase (C-1=THFSc)], GCV1 [glycine cleavage system (GCV) pro tein T], and the GLY1 (involved in glycine synthesis) loci. Analysis o f the in vivo growth characteristics and phenotypes was used to determ ine the contribution to cytoplasmic nucleic acid and amino acid anabol ism by the mitochondrial enzymes involved in the interconversion of fo late coenzymes. The data indicate that mitochondria transport formate to the cytoplasmic compartment and mitochondrial synthesis of formate appears to rely primarily on SHMTm rather than the glycine cleavage sy stem. The glycine cleavage system and SHMTm cooperate to specifically synthesize serine. With the inactivation of SHM1, however, the glycine cleavage system can make an observable contribution to the level of m itochondrial formate. Inactivation of SHM1, SHM2 and ADE3 is required to render yeast auxotrophic for TMP and methionine, suggesting that TM P synthesized in mitochondria may be available to the cytoplasmic comp artment.