Mammalian Fibroblasts Lacking Mitochondrial NAD+-dependent Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase Are Glycine Auxotrophs

Abstract

Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3-10 x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.

Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway

“…Absence of both results in a severe enough 1C deficiency to impair growth. Similar to serine, and consistent with prior reports in mouse embryonic fibroblasts and Chinese hamster ovary cells (Patel et al, 2003;Stover et al, 1997), glycine was required to support growth of MTHFD2 and SHMT2, but not SHMT1 knockout cells (Figure 5e and S5c). Interestingly, formate did not rescue but rather worsened growth ( Figure S5d).…”

Integrated Stress Response Modulates Cellular Redox State via Induction of Cystathionine γ-Lyase

“…repressed in ATF4 Ϫ/Ϫ MEFs, including the glycine transporter, which supports the synthesis of GSH; methylenetetrahydrofolate dehydrogenase, an important enzyme in embryonic glycine synthesis (49); and CSE, the final enzyme for the synthesis of cysteine in the transsulfuration pathway (23). Further, the expression of the light chain, xCT, of the cystine/glutamate antiporter system mediating cystine transport into the cell was found to be down-regulated (supplemental Fig.…”

Disruption of the Mthfd1 Gene Reveals a Monofunctional 10-Formyltetrahydrofolate Synthetase in Mammalian Mitochondria

“…However, until recently the only mitochondrial homologue of Saccharomyces cerevisiae mitochondrial DCS to be identified in mammals was the NADdependent methyleneTHF dehydrogenase-methenylTHF cyclohydrolase (NMDMC) (9). Metabolic studies of transformed fibroblasts derived from murine NMDMC knockouts revealed that they are glycine auxotrophs (10). The glycine auxotrophy is most likely because of the formation of a type of folate trap; the available THF in the mitochondria is trapped as methyleneTHF.…”

“…The E11.5 chimeric embryos were used to generate primary embryonic fibroblast cell lines as described previously (10). Disrupted embryos were incubated in Dulbecco's modified Eagle's medium containing 15% fetal bovine serum supplemented with 1ϫ non-essential amino acids, 1ϫ glutamine, 1ϫ penicillin/streptomycin (all Invitrogen), 30 M thymidine, and 30 M hypoxanthine (both Sigma) with 0.3 mg/ml G418 (Geneticin, Invitrogen) to select for null mutant cells.…”