The compartmentalization of folate metabolism in mammalian cells /

Folate metabolism is compartmentalized between the cytoplasm and the mitochondria of mammalian cells. Certain folate-dependent enzymes are present in both of these compartments, such as methylenetetrahydrofolate dehydrogenases, which are required to interconvert one-carbon substituted tetra hydrofolates. In the cytoplasm, there is a trifunctional NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase (DCS). Its mitochondrial counterpart is a bifunctional NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC), expressed during embryogenesis and in immortalized cells. A comparison of the 3' untranslated region of the NMDMC cDNA with the synthetase domain of the DCS cDNA and gene among different species have revealed significant regions of homology. This suggests that the mammalian mitochondrial NAD-dependent DC evolved from an NADP-dependent DCS precursor through a change in cofactor specificity of the dehydrogenase from NADP to NAD.Although the folate pathways are compartmentalized, the mitochondrial folate pathway makes an important contribution to total cellular folate metabolism. Mouse fibroblasts that have a completely inactivated NMDMC gene are glycine auxotrophs. Furthermore, growth of these Nmdmc-/- cell lines is stimulated by supplementation with formate or hypoxanthine. These cell lines also show enhanced incorporation of radioactivity into DNA from formate as compared to serine, demonstrating that formate is a preferred one-carbon donor for the Nmdmc-/- cell lines. This indicates that NMDMC is required for optimal purine biosynthesis during periods of rapid cellular proliferation such as embryogenesis and tumorigenesis. The rescue of these Nmdmc-/- cell lines with NMDMC expression reversed the glycine auxotrophy and the formate to serine incorporation ratio reverted toward the wild type ratio. The rescue of these Nmdmc-/- cell lines with the NAD-dependent monofunctional dehydrogenase activity also reversed the glycine auxotrophy but these cell lines did not grow as well as the NMDMC-rescued cell lines. This indicates that although the cyclohydrolase activity is not required in the mitochondria, the rate of 10-formyl-THF production is not optimal in its absence. Furthermore, when these Nmdmc-/- cell lines were rescued with the expression of the NADP-dependent DCS in the mitochondria there was reversal of the glycine auxotrophy as well, indicating that the NAD cofactor specificity of the mitochondrial methylenetetrahydrofolate dehydrogenase is not absolutely essential to maintain the flux of one-carbon metabolites