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 encoded by the ADE3 locus, yet ade3 mutants have low but detectable levels of these enzyme activities. Synthetase, cyclohydrolase, and dehydrogenase activities in an ade3 deletion strain co-purify 4,000-fold to yield a single protein species as seen on sodium dodecyl sulfate-polyacrylamide gels. The native molecular weight of the isozyme (Mr = 200,000 by gel exclusion chromatography) and the size of its subunits (Mr = 100,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) are similar to those of C1-tetrahydrofolate synthase. Cell fractionation experiments show that the isozyme, but not C1-tetrahydrofolate synthase, is localized in the mitochondria. Genetic studies indicate that the isozyme is encoded in the nuclear genome. Peptide mapping experiments show that C1-tetrahydrofolate synthase and the isozyme are not structurally identical. However, immunotitration experiments and amino acid sequence analysis suggest that C1-tetrahydrofolate synthase and the isozyme are structurally related. We propose to call the isozyme "mitochondrial C1-tetrahydrofolate synthase."
Highlights
C1-Tetrahydrofolatesynthaseis a trifunctional poly- ase activities are catalyzed by atrifunctional polypeptide, peptide found in eukaryotic organisms that catalyzes termed CI-THF synthase [4,5,6,7,8,9]
Purification of Mitochondrial C1-THFSynthase-In S. cereuisiae, Cl-THF synthase is encoded by the ADE3 locus
Subcellular Localization of Mitochondrial C1-THF Synthase-The isozyme is present in ade3 mutants, but it cannot satisfy the requirements for adenine and histidine that are characteristic of most d e 3 mutants [38]
Summary
Strain M1614C (a serl leul) and strain3-5281 ( a serl ural ade3) were obtained from E. Respiratory-deficient mutants were isolated from 3-5281as described [21] except that ethidium bromide (20 pg/ml) was used as a mutagen. Respiratory-deficient mutants thatfailed to complement a defined p0 strain Cells were grown aerobically at 30 "C. Cells were grown in YPD medium (1%yeast extract, 2% bactopeptone, 2% glucose) to latelog phase (ODm = 10) unless otherwise indicated. Extracts were prepared by disrupting washed cells with glass beads (0.45-mm diameter) inbuffer containing 50 mM Tris-S04, pH 7.5, 10 mM KC1, 10 mM 2-mercaptoethanol, 1 mM PMSF. The supernatant fractions were used for enzyme and protein assays
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