13C NMR detection of folate-mediated serine and glycine synthesis in vivo in Saccharomyces cerevisiae.
Saccharomyces cerevisiae has both cytoplasmic and mitochondrial C1-tetrahydrofolate (THF) synthases. These trifunctional isozymes are central to single-carbon metabolism and are responsible for interconversion of the THF derivatives in the respective compartments. In the present work, we have used 13C NMR to study folate-mediated single-carbon metabolism in these two compartments, using glycine and serine synthesis as metabolic endpoints. The availability of yeast strains carrying deletions of cytoplasmic and/or mitochondrial C1-THF synthase allows a dissection of the role each compartment plays in this metabolism. When yeast are incubated with [13C]formate, 13C NMR spectra establish that production of [3-13C]serine is dependent on C1-THF synthase and occurs primarily in the cytosol. However, in a strain lacking cytoplasmic C1-THF synthase but possessing the mitochondrial isozyme, [13C]formate can be metabolized to [2-13C]glycine and [3-13C]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [13C]CH2-THF via mitochondrial C1-THF synthase, and subsequent glycine synthesis via reversal of the glycine cleavage system. Additional supporting evidence of reversibility of GCV in vivo is the production of [2-13C]glycine and [2,3-13C]serine in yeast strains grown with [3-13C]serine. This metabolism is independent of C1-THF synthase since these products were observed in strains lacking both the cytoplasmic and mitochondrial isozymes. These results suggest that when formate is the one-carbon donor, assimilation is primarily cytoplasmic, whereas when serine serves as one-carbon donor, considerable metabolism occurs via mitochondrial pathways.
Pasternack
L B
LB
Department of Chemistry and Biochemistry, University of Texas, Austin 78712.
Laude
D A
DA
Appling
D R
DR
D000619:Aminohydrolases / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D005492:Folic Acid / Q000378:metabolism*; D005574:Formate-Tetrahydrofolate Ligase / Q000378:metabolism; D005561:Formates / Q000378:metabolism; D005998:Glycine / Q000378:metabolism*; D007527:Isoenzymes / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D009097:Multienzyme Complexes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D012694:Serine / Q000378:metabolism*
D005561:Formates; D007527:Isoenzymes; D009097:Multienzyme Complexes; D012694:Serine; C021836:formyl-methenyl-methylenetetrahydrofolate synthetase; D005492:Folic Acid; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D005574:Formate-Tetrahydrofolate Ligase; D005998:Glycine