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J. Biol. Chem., Vol. 278, Issue 21, 19436-19441, May 23, 2003

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Mammalian Fibroblasts Lacking Mitochondrial NAD⁺-dependent Methylenetetrahydrofolate Dehydrogenase-Cyclohydrolase Are Glycine Auxotrophs^*

Harshila Patel  , Erminia Di Pietro  ¶ and Robert E. MacKenzie ||

From the Department of Biochemistry, McGill University, Montréal, Quebec H3G 1Y6, Canada

Primary fibroblasts established from embryos of NAD-dependent mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase (NMDMC) knockout mice were spontaneously immortalized or transformed with SV40 Large T antigen. Mitotracker Red CMXRos staining of the cells indicates the presence of intact mitochondria with a membrane potential. The nmdmc^(-/-) cells are auxotrophic for glycine, demonstrating that NMDMC is the only methylenetetrahydrofolate dehydrogenase normally expressed in the mitochondria of these cell lines. Growth of null mutant but not wild type cells on complete medium with dialyzed serum is stimulated about 2-fold by added formate or hypoxanthine. Radiolabeling experiments demonstrated a 3–10 x enhanced incorporation of radioactivity into DNA from formate relative to serine by nmdmc^(-/-) cells. The generation of one-carbon units by mitochondria in nmdmc^(-/-) cells is completely blocked, and the cytoplasmic folate pathways alone are insufficient for optimal purine synthesis. The results demonstrate a metabolic role for NMDMC in supporting purine biosynthesis. Despite the recognition of these metabolic defects in the mutant cell lines, the phenotype of nmdmc^(-/-) embryos that begin to die at E13.5 is not improved when pregnant dams are given a glycine-rich diet or daily injections of sodium formate.

Received for publication, February 19, 2003 , and in revised form, March 11, 2003.

^|| To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, QC H3G 1Y6, Canada. Tel.: 514-398-7270; Fax; 514-398-7384; Robert.Mackenzie{at}mcgill.ca.

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