Carbon-13 NMR analysis of intercompartmental flow of one-carbon units into choline and purines in Saccharomyces cerevisiae

Abstract

In Saccharomyces cerevisiae, the three-carbon of serine is normally the major one-carbon donor, although glycine and formate can substitute for serine. The second carbon of glycine enters via the glycine cleavage system in the mitochondria and can satisfy all cellular one-carbon requirements. It remains unresolved, however, as to the route by which these mitochondrial one-carbon units supply cytosolic anabolic processes. In the present work, we have used yeast mutants blocked at selected sites and 13C NMR to trace the incorporation of glycine-derived mitochondrial 5,10-methylenetetrahydrofolate into nonmitochondrial synthesis of choline and purines. Label incorporation into choline traces the methylation pathway of choline synthesis from production of serine to methylation of phosphatidylethanolamine. The active one-carbon unit of S-adenosylmethionine involved in methylation reactions originates almost solely from C3 of serine. On the other hand, flow of mitochondrial one-carbon units to 10-formyltetrahydrofolate for purine synthesis is shown to occur via both serine and formate. Formate transport accounts for at least 25% of the total, even during growth with sufficient serine to provide for the one-carbon requirements of the cell. This work shows that the synthetase function of the cytosolic C1-tetrahydrofolate synthase plays a critical role in the processing of mitochondrial one-carbon units to 10-formyltetrahydrofolate pools. In addition, this study provides evidence of two pools of glycine within the mitochondria and establishes a system of analyzing flux into the different folate derivatives.

Human Mitochondrial C1-Tetrahydrofolate Synthase

“…A second possibility is that formate production is defective in mitochondria from nonketotic hyperglycinemia patients. As we showed both in vitro with rat liver mitochondria (10,11), and in vivo with yeast (28,52), mitochondrial 5,10-methylene-THF is rapidly converted to formate and transported to the cytosol, where it is activated to 10-formyl-THF via cytoplasmic 10-formyl-THF synthetase (Fig. 1, mitochondrial reactions 3, 2, and 1 and cytoplasmic reaction 1).…”

Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues

“…Evidence from both in vivo (9,10,45,54) and in vitro (6, 7) experiments supports a matrix localization for mitochondrial 1-carbon metabolism. Recently, we found that the MTHFD1L protein is tightly associated with the inner mitochondrial membrane, facing the matrix (37).…”

Identification of a Novel One-carbon Metabolism Regulon in Saccharomyces cerevisiae

“…Coupling expression of purine pathway genes to those of one-carbon metabolism under these conditions may be required to maintain optimal functioning of the system as a whole. Additionally, induction of the one-carbon regulon would result in an increased contribution from mitochondrial one-carbon metabolism, which is metabolically closely linked with purine biosynthesis, because at least 25% of one-carbon units incorporated into purines are derived from mitochondrially derived formate, irrespective of the availability of cytoplasmic one-carbon units (44).…”