Characterization of mitochondrial C₁-tetrahydrofolate synthase transcript and protein expression in adult and embryonic mammalian tissues and the role of the mitochondrial one-carbon pathway in the cytoplasmic methyl cycle

Abstract

In eukaryotes, folate-dependent one-carbon (1-C) metabolism is composed of two parallel pathways compartmentalized to either the cytoplasm or mitochondria. In each, 1-C units, carried on tetrahydrofolate (THF), are interconverted by four catalytic activities. Serine hydroxymethyltransferase transfers the 3-carbon of serine to THF forming 5,10-methylene-THF which is oxidized in 3 successive steps to formate via the intermediates, 5,10-methenyl-THF and 10-formyl-THF. Because of the redox potential in each compartment, 1-C flux is thought by most authors to be from formate to serine in the cytosol and in the opposite direction in mitochondria. Transport of serine, glycine and formate across the mitochondrial membranes creates a 1-C cycle. All eukaryotes characterized to date contain a cytoplasmic trifunctional C1-THF synthase possessing 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase and 10-formyl-THF synthetase activities which interconvert the catalytic intermediates between 5,10-methylene-THF and formate. However, despite the observation that adult rat liver mitochondria oxidize serine to formate, no known enzymatic activities correlate with those of cytoplasmic C1-THF synthase. In embryos, a bifunctional protein, containing 5,10-methylene-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase, accounts for two of these activities. But the 10-formyl-THF synthetase activity has no associated enzyme in mitochondria. Reported here is the discovery of a monofunctional homolog of C1-THF synthase in mammalian mitochondria. Characterization of the protein confirms mitochondrial localization and 10-formyl-THF synthetase activity. Likewise, the adult human transcript is present and differs in size and tissue distribution from cytosolic C1-THF synthase. In mouse embryos, the temporal expression of the mRNA starts out relatively low and increases as the embryos age. The spatial distribution of the transcript is ubiquitous but with areas of elevated expression corresponding to proliferative regions within the embryo. The temporal expression pattern of the protein and transcript correspond well. However, mitochondrial flux studies and immunoblotting data suggest that mitochondrial C1-THF synthase is not the rate-limiting enzyme in mitochondria, at least during the mid to later stages of embryogenesis. Additionally, studies modulating the expression of mitochondria 1-C proteins demonstrate the likelihood that most cytoplasmic 1-C units are mitochondrially derived.