NAD- and NADP-dependent mitochondrially targeted methylenetetrahydrofolate dehydrogenase-cyclohydrolases can rescue mthfd2 null fibroblasts.
Mouse fibroblasts in which the mthfd2 gene encoding mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) was previously inactivated were infected with retroviral expression constructs of dehydrogenase/cyclohydrolase cDNA. Cellular fractionation confirmed that the expressed proteins were properly targeted to the mitochondria. Expression of the NAD-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase enzyme in mitochondria corrected the glycine auxotrophy of the null mutant cells. A construct in which the cyclohydrolase activity of NMDMC was inactivated by point mutation also rescued the glycine auxotrophy, although poorly. This suggests that the cyclohydrolase activity is also required to ensure optimal production of 10-formyltetrahydrofolate. The expression of the NADP-dependent methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase in the mitochondria also reversed the glycine requirement of the null cells demonstrating that the use of the NAD cofactor is not absolutely essential to maintain the flux of one-carbon metabolites. All rescued cells demonstrated a decrease in the ratio of incorporation of exogenous formate to serine in standardized radiolabeling studies. This ratio, which is approximately 2.5 for nmdmc(-/-) cells and 0.3 for the wild type cells under the conditions used, is a qualitative indicator of the ability of the mitochondria of the cells to generate formate.
Patel
Harshila
H
Department of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, Que., Canada H3G 1Y6.
Di Pietro
Erminia
E
Mejia
Narciso
N
MacKenzie
Robert E
RE
D000619:Aminohydrolases / Q000378:metabolism; D000818:Animals; D015153:Blotting, Western; D002250:Carbon Radioisotopes; D002460:Cell Line; D018076:DNA, Complementary / Q000378:metabolism; D047108:Embryonic Development; D005347:Fibroblasts / Q000201:enzymology*; D005998:Glycine / Q000378:metabolism; D007700:Kinetics; D002955:Leucovorin / Q000031:analogs & derivatives / Q000378:metabolism; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP) / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008928:Mitochondria / Q000201:enzymology*; D009154:Mutation; D009243:NAD / Q000378:metabolism*; D009249:NADP / Q000378:metabolism*
D002250:Carbon Radioisotopes; D018076:DNA, Complementary; D009243:NAD; C010161:10-formyltetrahydropteroylglutamic acid; D009249:NADP; D008754:Methylenetetrahydrofolate Dehydrogenase (NADP); D000619:Aminohydrolases; D002955:Leucovorin; D005998:Glycine