Mitochondrial C1-Tetrahydrofolate Synthase (MTHFD1L) Supports the Flow of Mitochondrial One-carbon Units into the Methyl Cycle in Embryos

Abstract

Mitochondrial folate-dependent one-carbon (1-C) metabolism converts 1-C donors such as serine and glycine to formate, which is exported and incorporated into the cytoplasmic tetrahydrofolate (THF) 1-C pool. Developing embryos depend on this mitochondrial pathway to provide 1-C units for cytoplasmic process such as de novo purine biosynthesis and the methyl cycle. This pathway is composed of sequential methylene-THF dehydrogenase, methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase activities. In embryonic mitochondria, the bifunctional MTHFD2 enzyme catalyzes the dehydrogenase and cyclohydrolase reactions, but the enzyme responsible for the mitochondrial synthetase reaction has not been identified in embryos. A monofunctional 10-formyl-THF synthetase (MTHFD1L gene product) functions in adult mitochondria and is a likely candidate for the embryonic activity. Here we show that the MTHFD1L enzyme is present in mitochondria from normal embryonic tissues and embryonic fibroblast cell lines, and embryonic mitochondria possess the ability to synthesize formate from glycine. The MTHFD1L transcript was detected at all stages of mouse embryogenesis examined. In situ hybridizations showed that MTHFD1L was expressed ubiquitously throughout the embryo but with localized regions of higher expression. The spatial pattern of MTHFD1L expression was virtually indistinguishable from that of MTHFD2 and MTHFD1 (cytoplasmic C 1 -THF synthase) in embryonic day 9.5 mouse embryos, suggesting coordinated regulation. Finally, we show using stable isotope labeling that in an embryonic mouse cell line, greater than 75% of 1-C units entering the cytoplasmic methyl cycle are mitochondrially derived. Thus, a complete pathway of enzymes for supplying 1-C units from the mitochondria to the methyl cycle in embryonic tissues is established.

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).…”

Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice

“…Stained embryos were embedded in paraffin, sectioned at the level of the forelimb (4-μm thickness) and counterstained with nuclear fast red. Mthfd1l whole mount in situ hybridization was performed using a riboprobe against the 3′ UTR as previously described (12). Embryos were then embedded in OCT medium and cryosectioned at the level of the forelimb (12-μm thickness).…”

“…The Mthfd1l gene is expressed in most adult tissues, but at higher levels in spleen, thymus, brain, and placenta (9,11). The Mthfd1l gene is also expressed at all stages of mammalian embryogenesis and ubiquitously throughout the embryo but with localized regions of higher expression along the neural tube, the brain, craniofacial structures, limb buds, and the tail bud (12). Moreover, metabolic tracer experiments in mouse embryonic fibroblasts showed that more than 75% of 1C units that enter the cytoplasmic methyl cycle are mitochondrially derived (12).…”

“…The Mthfd1l gene is also expressed at all stages of mammalian embryogenesis and ubiquitously throughout the embryo but with localized regions of higher expression along the neural tube, the brain, craniofacial structures, limb buds, and the tail bud (12). Moreover, metabolic tracer experiments in mouse embryonic fibroblasts showed that more than 75% of 1C units that enter the cytoplasmic methyl cycle are mitochondrially derived (12). Thus, in both embryos and adults, MTHFD1L catalyzes production of formate from 10-formyl-THF, the last step in the flow of 1C units from mitochondria to cytoplasm.…”

Suppression of MTHFD2 in MCF-7 Breast Cancer Cells Increases Glycolysis, Dependency on Exogenous Glycine, and Sensitivity to Folate Depletion

“…It has been estimated through experiments conducted in mouse embryonic fibroblasts (MEF) that approximately 80% of one-carbon units that flow into the methylation cycle originate from the mitochondrial pathway 7 .…”