Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is essential for embryonic development - PubMed (original) (raw)
Mitochondrial NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is essential for embryonic development
E Di Pietro et al. Mol Cell Biol. 2002 Jun.
Abstract
Folate-dependent enzymes are compartmentalized between the cytoplasm and mitochondria of eukaryotes. The role of mitochondrial folate-dependent metabolism and the extent of its contribution to cytoplasmic processes are areas of active investigation. NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (NMDMC) catalyzes the interconversion of 5,10-methylenetetrahydrofolate and 10-formyltetrahydrofolate in mitochondria of mammalian cells, but its metabolic role is not yet clear. Its expression in embryonic tissues but not in most adult tissues as well as its stringent transcriptional regulation led us to postulate that it may play a role in embryonic development. To investigate the metabolic role of NMDMC, we used a knockout approach to delete the nmdmc gene in mice. Heterozygous mice appear healthy, but homozygous NMDMC knockout mice die in utero. At embryonic day 12.5 (E12.5), homozygous null embryos exhibit no obvious developmental defects but are smaller and pale and die soon thereafter. Mutant fetal livers contain fewer nucleated cells and lack the characteristic redness of wild-type or heterozygous livers. The frequencies of CFU-erythroid (CFU-E) and burst-forming unit-erythroid (BFU-E) from fetal livers of E12.5 null mutants were not reduced compared with those of wild-type or heterozygous embryos. It has been assumed that initiation of protein synthesis in mitochondria requires a formylated methionyl-tRNA(fmet). One role postulated for NMDMC is to provide 10-formyltetrahydrofolate as a formyl group donor for the synthesis of this formylmethionyl-tRNA(fmet). To determine if the loss of NMDMC impairs protein synthesis and thus could be a cause of embryonic lethality, mitochondrial translation products were examined in cells in culture. Mitochondrial protein synthesis was unaffected in NMDMC-null mutant cell lines compared with the wild type. These results show that NMDMC is not required to support initiation of protein synthesis in mitochondria in isolated cells but instead demonstrate an essential role for mitochondrial folate metabolism during embryonic development.
Figures
FIG. 1.
Compartmentalization of folate-dependent activities in mammalian cells. Abbreviations are SHMT, serine hydroxymethyltransferase; D, methylene-THF dehydrogenase; C, methenyl-THF cyclohydrolase; S, 10-formyl-THF synthetase; and GCS, glycine cleavage system. Adapted from Yang and MacKenzie (31).
FIG. 2.
Disruption of NMDMC in ES cells. (A) Gene-targeting strategy. A partial restriction map of the nmdmc gene is displayed (top). Exons are numbered and indicated by shaded boxes, and thin lines represent introns. The location of probes for hybridization (E2/161, E8/504, and neo) and primers for PCR (P1, P2, and P3) are indicated. The targeting vector pMC1neoΔ3-8/TK is shown, and the predicted targeted allele is indicated. The vector contains a 5′ arm, including exon 2 and part of exon 3, and a 3′ arm including part of exon 8. The E2/161 probe is specific for _Bam_HI fragments containing exon 2 and detects a 5-kb fragment from the wild-type allele and an 11-kb fragment from the targeted allele generated by _Bam_HI digestion. The E8/504 probe is specific for _Bam_HI fragments containing exon 8 and hybridizes to an 8.5-kb fragment from the wild-type allele and an 11-kb fragment from the targeted allele. The neo probe hybridizes to the neomycin cassette. Abbreviations used: B, _Bam_HI; E, _Eco_RI; H, _Hin_dIII; X, _Xba_I; NEO, neomycin resistance gene; and TK, herpes simplex virus thymidine kinase gene. (B) Southern blot analysis of DNA isolated from ES cell clones. _Bam_HI-digested ES cell DNA was hybridized with E2/161, E8/504, or neo probes. The size (in kilobases) of the NMDMC-specific _Bam_HI fragments is indicated on the left. KO, knockout; wt, wild type.
FIG. 3.
Analysis of DNA isolated from embryos of +/− intercrosses. (A) PCR of yolk sac DNA from 9.5-day-old embryos with PCR primers P1 and either P2 or P3 amplified a 916-bp wild-type fragment or 1,306-bp targeted fragment. L, 100-bp DNA Ladder Plus (MBI Fermentas). (B) Southern blot analysis of yolk sac DNA digested with _Bam_HI and hybridized with the flanking probe E2/161. The 5-kb band indicates transmission of the wild-type (wt) allele, and the 11-kb band indicates transmission of the targeted (KO) allele.
FIG. 4.
Phenotypic comparison of E12.5 littermates from a mating between mice heterozygous for NMDMC. A mutant embryo is shown on the right, and a wild-type embryo is shown on the left. The mutant embryo appears to be developmentally normal in comparison with its littermate except for its paleness and smaller size.
FIG. 5.
Hematopoietic progenitor cell colony assays with livers from E12.5 wild-type, heterozygous, and _nmdmc_−/− embryos. (A) The average number of nucleated cells in wild-type (WT), heterozygous (HET), and _nmdmc_−/− (KO) fetal livers. (B) Numbers of CFU-E detected after 3 days of postplating in methylcellulose medium supplemented with erythropoietin. (C) Numbers of BFU-E detected after 7 to 10 days of postplating in methylcellulose medium supplemented with erythropoietin, stem cell factor, IL-3, and IL-6. The numbers at the top of each error bar indicate the number of embryos analyzed. CFU-E and BFU-E numbers are expressed per 1 × 104 and 2 × 104 nucleated liver cells, respectively. The height of each bar represents the average, and the error bars represent 1 standard deviation.
FIG. 6.
Southern blot analysis of DNA isolated from mouse embryonic fibroblast cell lines and ES cell lines. _Bam_HI-digested DNA isolated from mouse embryonic fibroblast (EF) cell lines and ES cell lines was hybridized with the flanking probe E2/161. The 5-kb fragment indicates the wild-type (wt) allele, and the 11-kb band indicates the targeted (KO) allele.
FIG. 7.
Staining of mitochondria from mouse embryonic fibroblast cells with Mitotracker Green FM. Mouse embryonic fibroblast cells were incubated with 500 nM Mitotracker Green FM at 37°C for 45 min. (A) wild type; (B) heterozygous; and (C) homozygous null mutant cells.
FIG. 8.
Analysis of mitochondrial translation products. Exponentially growing cells were labeled with [35S]methionine in the presence of emetine for 60 min, and labeled polypeptides were separated on a 12 to 20% polyacrylamide linear gradient gel. An equal amount of protein from whole-cell extracts was loaded in each lane. Translation products are assigned according to Chomyn et al. (4) and are abbreviated as follows: ND1 to ND6, subunits of NADH dehydrogenase complex; COI to COIII, subunits of cytochrome oxidase complex; Cytb, cytochrome b; and ATP6 and ATP8, subunits of ATPase complex. CAP indicates an experiment conducted in the presence of chloramphenicol in addition to emetine.
References
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