C-13 NMR DETECTION OF FOLATE-MEDIATED SERINE AND GLYCINE SYNTHESIS INVIVO 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 C-13 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 [C-13]formate, C-13 NMR spectra establish that production of [3-C-13]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, [C-13]formate can be metabolized to [2-C-13]glycine and [3-C-13]serine. This provides in vivo evidence for the mitochondrial assimilation of formate, activation and conversion to [C-13]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-C-13]glycine and [2,3-C-13]serine in yeast strains grown with [3-C-13]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.