Isolation and characterization of the Saccharomyces cerevisiae MIS1 gene encoding mitochondrial C1-tetrahydrofolate synthase

Abstract

C1-Tetrahydrofolate synthase is a trifunctional polypeptide found in eukaryotic organisms that catalyzes 10-formyltetrahydrofolate synthetase (EC 6.3.4.3), 5,10-methenyltetrahydrofolate cyclohydrolase (EC 3.5.4.9), and 5,10-methylenetetrahydrofolate dehydrogenase (EC 1.5.1.5) activities. In Saccharomyces cerevisiae, C1-tetrahydrofolate synthase is found in both the cytoplasm and the mitochondria. The gene encoding yeast mitochondrial C1-tetrahydrofolate synthase was isolated using synthetic oligonucleotide probes based on the amino-terminal sequence of the purified protein. Hybridization analysis shows that the gene (designated MIS1) has a single copy in the yeast genome. The predicted amino acid sequence of mitochondrial C1-tetrahydrofolate synthase shares 71% identity with yeast C1-tetrahydrofolate synthase and shares 39% identity with clostridial 10-formyltetrahydrofolate synthetase. Chromosomal deletions of the mitochondrial C1-tetrahydrofolate synthase gene were generated using the cloned MIS1 gene. Mutant strains which lack a functional MIS1 gene are viable and can grow in medium containing a nonfermentable carbon source. In fact, deletion of the MIS1 locus has no detectable effect on cell growth.