Disruption of cytoplasmic and mitochondrial folylpolyglutamate synthetase activity in Saccharomyces cerevisiae.
Similar to other eukaryotes, yeasts have parallel pathways of one-carbon metabolism in the cytoplasm and mitochondria and have folylpolyglutamate synthetase activity in both compartments. The gene encoding folylpolyglutamate synthetase is MET7 (also referred to as MET23) on chromosome XV and appears to encode both the cytoplasmic and mitochondrial forms of the enzyme. In order to determine the metabolic roles of both forms of folylpolyglutamate synthetase, we disrupted the met7 gene and determined that the strain is a methionine auxotroph and an adenine and thymidine auxotroph when grown in the presence of sulfanilamide. The met7 mutant becomes petite under normal growth conditions but can be maintained with a grande phenotype if the strain is tup and all media are supplemented with dTMP. A met7 gly1 strain is auxotrophic for glycine when grown on glucose but prototrophic when grown on glycerol. A met7 ser1 strain cannot use glycine to suppress the serine auxotrophy of the ser1 phenotype. A met7 shm2 strain is nonviable. In order to disrupt just the mitochondrial folylpolyglutamate synthetase activity, we constructed mutants with an inactivated chromosomal MET7 gene complemented by genes that express only cytoplasmic folylpolyglutamate synthetase, including the Lactobacillus casei folC gene and the yeast MET7 gene with its mitochondrial leader sequence deleted (MET7Deltam). All the genes providing cytoplasmic folylpolyglutamate synthetase complemented the methionine auxotrophy as well as the synthetic lethality of the shm2 strain and the synthetic glycine auxotrophy of the gly1 strain. The strains lacking the mitochondrial folylpolyglutamate synthetase had longer doubling times than the isogenic wild-type strains but retained the function of the mitochondrial folate-dependent enzymes to produce formate, serine, and glycine. Mutants complemented by the bacterial folC gene or by the MET7Deltam gene on a 2mu plasmid remained grande without the tup mutation and supplementation and dTMP. Mutants complemented by the MET7Deltam gene integrated in single copy became petites under those conditions, indicating a deficiency in dTMP production but this is likely due to lower expression of cytoplasmic folylpolyglutamate synthetase by the MET7Deltam gene.
DeSouza
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Department of Medical Genetics and Microbiology, Department of Laboratory Medicine and Pathobiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, M5S 1A8, Canada.
Shen
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Bognar
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D000596:Amino Acids / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D003593:Cytoplasm / Q000201:enzymology* / Q000235:genetics; D004272:DNA, Mitochondrial / Q000235:genetics; D000939:Epitopes / Q000276:immunology; D005492:Folic Acid / Q000378:metabolism / Q000494:pharmacology; D018628:Gene Dosage; D020043:Genes, Essential / Q000235:genetics; D005800:Genes, Fungal / Q000235:genetics / Q000502:physiology; D005816:Genetic Complementation Test; D005947:Glucose / Q000378:metabolism; D005990:Glycerol / Q000378:metabolism; D007780:Lacticaseibacillus casei / Q000201:enzymology / Q000235:genetics; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D009154:Mutation / Q000235:genetics; D010453:Peptide Synthases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000276:immunology / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology* / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D000077145:Sulfanilamide; D013424:Sulfanilamides / Q000378:metabolism; D013938:Thymidine Monophosphate / Q000378:metabolism
D000596:Amino Acids; D004272:DNA, Mitochondrial; D000939:Epitopes; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D013424:Sulfanilamides; D000077145:Sulfanilamide; D013938:Thymidine Monophosphate; D005492:Folic Acid; D010453:Peptide Synthases; C023103:folylpolyglutamate synthetase; D005947:Glucose; D005990:Glycerol