Mitochondrial and Cytoplasmic Distribution in Saccharomyces cerevisiae of Enzymes Involved in Folate‐Coenzyme‐Mediated One‐Carbon‐Group Transfer

Abstract

The study of the subcellular distribution of the enzymes involved in folate-coenzyme-mediated transfer of one-carbon groups in Saccharomyces cerevisiae suggests the existence of two parallel enzyme systems, one mitochondrial and the other cytoplasmic. All the enzymes assayed in the present study, serine transhydroxymethylase, dihydrofolate reductase, 5,lO-methylenetetrahydrofolate dehydrogenase, 10-formyltetrahydrofolate synthetase, 5,lO-methylenetetrahydrofolate reductase and thymidylate synthetase were found to be in both cytoplasm and mitochondria. Two of the mitochondrial enzymes, serine transhydroxymethylase and dihydrofolate reductase, were found to be different by several criteria from their cytoplasmic counterparts.By the use of three nuclear gene mutations ade3, tmp3 and serl, and different combinations of these three mutations, the physiological role of the cytoplasmic enzymes for the biosynthesis of adenine, histidine, methionine, thymidylate and serine was studied. The response of auxotrophes to an external source of tetrahydrofolate, i. e. 5-formyl tetrahydrofolic acid, was also examined. The mitochondrial set of enzymes might be responsible for the de novo formation of tetrahydrofolate, since the loss of the three mitochondrial enzyme activities, serine transhydroxymethylase, thymidylate synthetase and dihydrofolate reductase, due to the mutation tmp3, leads to a generalized requirement for thymidylate, methionine, adenine and histidine. The ade3 mutation in which the cytoplasmic activities of the 10-formyltetrahydrofolate synthetase, 5,lO-methenyltetrahydrofolate cyclohydrolase and 5,lO-methylenetetrahydrofolate dehydrogenase are lost, confers only adenine and histidine auxotrophy, showing that the ade3 proteins are synthesized in, and only function in, the cytoplasm. The mitochondrial counterparts of these enzymes may function solely in the formation of the 10-formyl tetrahydrofolate needed for the transformylation of Met-tRNA involved in mitochondrial protein synthesis.A study of a nuclear mutation in Saccharomyces cerevisiae, tmp3, leading concomittantly to multiple nutritional requirements for thymidylate, methionine, adenine and histidine and to the petite phenotype, was found to have a decreased activity of serine transhydroxymethylase, but a wild-type level of two other tetrahydrofolate-linked enzymes, 10-formylte- trahydrofolate-linked enzymes, 10-formyltetrahydrofolate synthetase and 5,1O-methylenetetrahydrofolate dehydrogenase [l]. Further study showed that wildtype strains of yeast have two forms of serine transhydroxymethylase, one cytoplasmic and the other mitochondrial, separable by TEAE-cellulose chromatography. The mutant tmp3 has lost the mitochondrial form [2].Serine transhydroxymethylase may be the major physiological source of 5,lO-methylene tetrahydrofolate needed for the formation of methionine, thymidylate and adenine. Loss of the mitochondrial enzyme, in tmp3, which leads to that multiple requirement, implies that either the mitochondrial serine transhydroxymethylase is the...

Influence of the nuclear gene tmp3 on the loss of mitochondrial genes in Saccharomyces cerevisiae.

“…The tmp3 mutant has lost the mitochondrial counterparts of the three enzymes serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase (25). A study of the location of mitochondrial serine transhydroxymethylase in S. cerevisiae shows that it resides in the mitoplasm (partly in the inner membrane and mostly in the matrix; our preliminary data).…”

“…The experiments summarized here show that the tmp3 mutant, which is defective in mitochondrial biosynthesis of one-carbon-group-tetrahydrofolate coenzymes and deficient in activities of the three mitochondrial enzymes serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase (25), quickly loses is mitochondrial DNA.…”

“…The mutant described as tmp3 (16,24,25) is deficient in one-carbon-groupfolate coenzyme formation. This mutant can utilize 5-formyl tetrahydrofolate (folinic acid) at high concentrations, 100 to 200 mg/liter, to satisfy all the multiple nutritional requirements inherent to this mutation, i.e., dTMP, adenine, histidine, and methionine.…”

“…The remaining type offol mutant, that bearing tmp3 (16,24,25), has the same multiple requirements as the other fol mutants (adenine, histidine, methionine, and thymidylate), which can be replaced by a high concentration of 5-formyl tetrahydrofolate. This mutant belongs to one of the groups of complementation of fol mutants (Luzzati and Zelikson, unpublished data).…”

“…This mutant belongs to one of the groups of complementation of fol mutants (Luzzati and Zelikson, unpublished data). The tmp3 mutant is deficient in the mitochondrial enzymes serine transhydroxymethylase, dihydrofolate reductase, and thymidylate synthetase (25). Until now no stable respiratory-sufficient segregants were obtained, even if tmp3 strains were crossed to [rho'] strains and both sporulation of the diploid and germination of spores were conducted in the presence of 5-formyl tetrahydrofolate (or dTMP).…”