Mitochondrial NAD-dependent Methylenetetrahydrofolate Dehydrogenase-methenyltetrahydrofolate Cyclohydrolase is Essential for Embryonic Development

Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA(fmet). One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA(fmet). To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.