Advertisement

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues*

Open AccessPublished:April 14, 2014DOI:https://doi.org/10.1074/jbc.M114.555573
      Mammalian mitochondria are able to produce formate from one-carbon donors such as serine, glycine, and sarcosine. This pathway relies on the mitochondrial pool of tetrahydrofolate (THF) and several folate-interconverting enzymes in the mitochondrial matrix. We recently identified MTHFD2L as the enzyme that catalyzes the oxidation of 5,10-methylenetetrahydrofolate (CH2-THF) in adult mammalian mitochondria. We show here that the MTHFD2L enzyme is bifunctional, possessing both CH2-THF dehydrogenase and 5,10-methenyl-THF cyclohydrolase activities. The dehydrogenase activity can use either NAD+ or NADP+ but requires both phosphate and Mg2+ when using NAD+. The NADP+-dependent dehydrogenase activity is inhibited by inorganic phosphate. MTHFD2L uses the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies. Expression of the MTHFD2L transcript is low in early mouse embryos but begins to increase at embryonic day 10.5 and remains elevated through birth. In adults, MTHFD2L is expressed in all tissues examined, with the highest levels observed in brain and lung.

      Introduction

      Folate-dependent one-carbon (1C)
      The abbreviations used are:
      1C
      one carbon
      THF
      tetrahydrofolate
      CH+-THF
      5,10-methenyltetrahydrofolate
      CH2-THF
      5,10-methylenetetrahydrofolate
      10-CHO-THF
      10-formyltetrahydrofolate
      MTHFD
      methylenetetrahydrofolate dehydrogenase
      eEF2
      eukaryotic translation elongation factor 2
      TBP
      TATA-box-binding protein.
      metabolism is highly compartmentalized in eukaryotes, and mitochondria play a critical role in cellular 1C metabolism (reviewed in Ref.
      • Tibbetts A.S.
      • Appling D.R.
      Compartmentalization of mammalian folate-mediated one-carbon metabolism.
      ). The cytoplasmic and mitochondrial compartments are metabolically connected by the transport of 1C donors such as serine, glycine, and formate across the mitochondrial membranes in a mostly unidirectional flow (clockwise in Fig. 1). In mitochondria, the 1C units are oxidized to formate and released into the cytoplasm, where the formate is reattached to tetrahydrofolate (THF) for use in de novo purine biosynthesis or further reduced for either thymidylate synthesis or remethylation of homocysteine to methionine. The 1C unit interconverting activities represented in Fig. 1 by reactions 1–3 (1m–3m in mitochondria) are catalyzed by members of the methylenetetrahydrofolate dehydrogenase (MTHFD) family in eukaryotes. The cytoplasmic MTHFD1 protein is a trifunctional enzyme possessing 10-formyl-THF (10-CHO-THF) synthetase, 5,10-methenyl-THF (CH+-THF) cyclohydrolase, and 5,10-methylene-THF (CH2-THF) dehydrogenase activities (Fig. 1, reactions 1–3).
      Figure thumbnail gr1
      FIGURE 1Mammalian one-carbon metabolism. Reactions 1–4 are in both the cytoplasmic and the mitochondrial (m) compartments. Reactions 1, 2, and 3, 10-formyl-THF synthetase, 5,10-methenyl-THF cyclohydrolase, and 5,10-methylene-THF dehydrogenase, respectively, are catalyzed by trifunctional C1-THF synthase in the cytoplasm (MTHFD1). In mammalian mitochondria, reaction 1m is catalyzed by monofunctional MTHFD1L, and reactions 2m and 3m are catalyzed by bifunctional MTHFD2 or MTHFD2L. Reactions 4 and 4m are catalyzed by serine hydroxymethyltransferase and reaction 5 by the glycine cleavage system. Gray ovals represent putative metabolite transporters. Hcy, homocysteine; dTMP, thymidylate.
      In contrast to the single trifunctional enzyme found in the cytoplasm, three distinct MTHFD isozymes, encoded by three distinct genes, are now known to catalyze reactions 1m–3m (Fig. 1) in mammalian mitochondria. The final step in the mammalian mitochondrial pathway to formate (Fig. 1, reaction 1m) is catalyzed by MTHFD1L, a monofunctional 10-CHO-THF synthetase (
      • Walkup A.S.
      • Appling D.R.
      Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase.
      ). The CH2-THF dehydrogenase reaction (Fig. 1, reaction 3m) is catalyzed by two homologous enzymes, MTHFD2 and MTHFD2L. MTHFD2 was initially identified in 1985 as an NAD+-dependent 5,10-methylene-THF dehydrogenase (
      • Mejia N.R.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells.
      ). Upon purification, this protein was found to be a bifunctional enzyme (
      • Mejia N.R.
      • Rios-Orlandi E.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells: purification and properties.
      ), also possessing CH+-THF cyclohydrolase activity (Fig. 1, reaction 2m). This enzyme, now referred to as MTHFD2, has been extensively characterized with respect to kinetics, substrate specificity, and expression profile (
      • Mejia N.R.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells.
      ,
      • Mejia N.R.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme.
      ,
      • Yang X.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
      ,
      • Pelletier J.N.
      • MacKenzie R.E.
      Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
      ,
      • Pawelek P.D.
      • MacKenzie R.E.
      Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10- methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes.
      ,
      • Di Pietro E.
      • Wang X.L.
      • MacKenzie R.E.
      The expression of mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase supports a role in rapid cell growth.
      ,
      • Christensen K.E.
      • Mirza I.A.
      • Berghuis A.M.
      • Mackenzie R.E.
      Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
      ).
      In 2011 we reported the discovery of a new mammalian mitochondrial CH2-THF dehydrogenase, termed MTHFD2L (
      • Bolusani S.
      • Young B.A.
      • Cole N.A.
      • Tibbetts A.S.
      • Momb J.
      • Bryant J.D.
      • Solmonson A.
      • Appling D.R.
      Mammalian MTHFD2L Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.
      ). MTHFD2L is homologous to MTHFD2, sharing 60–65% identity. Recombinant rat MTHFD2L exhibits NADP+-dependent CH2-THF dehydrogenase activity when expressed in yeast (
      • Bolusani S.
      • Young B.A.
      • Cole N.A.
      • Tibbetts A.S.
      • Momb J.
      • Bryant J.D.
      • Solmonson A.
      • Appling D.R.
      Mammalian MTHFD2L Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.
      ), but the enzyme was not purified in that study. In addition, it could not be determined whether MTHFD2L was bifunctional because CH+-THF cyclohydrolase assays are unreliable in crude extracts. This new isozyme is expressed in adult mitochondria (
      • Bolusani S.
      • Young B.A.
      • Cole N.A.
      • Tibbetts A.S.
      • Momb J.
      • Bryant J.D.
      • Solmonson A.
      • Appling D.R.
      Mammalian MTHFD2L Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.
      ), whereas MTHFD2 is expressed only in transformed mammalian cells and embryonic or nondifferentiated tissues (
      • Mejia N.R.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells.
      ).
      The existence of these two CH2-THF dehydrogenases (MTHFD2 and MTHFD2L) in mammalian mitochondria raises several questions. Do the two enzymes differ in their catalytic activity or in their substrate or cofactor specificity? Do they differ in their tissue distribution or expression profiles? To answer these questions, we report here the purification and kinetic characterization of MTHFD2L and its embryonic and adult gene expression profiles. We show that MTHFD2L possesses CH+-THF cyclohydrolase activity and can use either NAD+ or NADP+ in its CH2-THF dehydrogenase activity. MTHFD2L is expressed during mouse embryonic development, and there appears to be a switch from MTHFD2 to MTHFD2L expression at about the time of neural tube closure. A comparison of the enzymatic characteristics and expression profiles of MTHFD2 and MTHFD2L revealed differences that may shed light on the roles of these two isozymes in adult and embryonic 1C metabolism.

      DISUSSION

      The experiments described here demonstrate that the mammalian MTHFD2L isozyme, like MTHFD1 and MTHFD2, possesses both CH2-THF dehydrogenase and CH+-THF cyclohydrolase activities (Fig. 2). The dehydrogenase activity of this bifunctional enzyme can use either NAD+ or NADP+ but requires both phosphate and Mg2+ when using NAD+ (Fig. 3A). The NADP+-dependent dehydrogenase activity of MTHFD2L is inhibited by inorganic phosphate (Fig. 3B). MTHFD2L can use the mono- and polyglutamylated forms of CH2-THF with similar catalytic efficiencies (kcat/Km; Table 2). Expression of the MTHFD2L transcript is low in early mouse embryos, begins to increase at E10.5, and continues through birth (Fig. 5). In adults, MTHFD2L was expressed in all tissues examined, with the highest levels observed in brain and lung (Fig. 7).
      How do these cofactor requirements compare with those of the other CH2-THF dehydrogenase/CH+-THF cyclohydrolase found in mammalian mitochondria? The MTFHD2 isozyme has been named NAD+-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase (
      • Yang X.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
      ,
      • Christensen K.E.
      • Mirza I.A.
      • Berghuis A.M.
      • Mackenzie R.E.
      Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
      ) but in fact exhibits dehydrogenase activity with NADP+, albeit with a much higher Km and lower Vmax (
      • Yang X.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
      ). In fact, the redox cofactor requirements of the two isozymes are quite similar: both exhibit lower Km values for NAD+ than for NADP+; their NAD+-dependent activities require phosphate and Mg2+; and their NADP+-dependent activities are inhibited by phosphate. The absolute requirement of the NAD+-dependent activity of MTHFD2 for Mg2+ and Pi has been characterized in great detail by Mackenzie and co-workers (
      • Christensen K.E.
      • Mirza I.A.
      • Berghuis A.M.
      • Mackenzie R.E.
      Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
      ). MTHFD2 uses Mg2+ and Pi to convert an NADP binding site into an NAD binding site. Pi binds in close proximity to the 2′-hydroxyl of NAD and competes with NADP binding. Mg2+ plays a role in positioning Pi and NAD. Mackenzie and co-workers (
      • Christensen K.E.
      • Mirza I.A.
      • Berghuis A.M.
      • Mackenzie R.E.
      Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
      ) identified several amino acid residues in MTHFD2 that are involved in the Pi and Mg2+ binding, and these residues are highly conserved in MTHFD2L in mammals. It is thus likely that Mg2+ and Pi play a mechanistically similar role in the NAD+-dependent dehydrogenase activity of MTHFD2L as well.
      At saturating levels of CH2-THF, using NAD+ as a cofactor, the kcat/Km value for the CH2-THF dehydrogenase reaction of MTHFD2 is 2.9 s−1μm−1 (
      • Pawelek P.D.
      • MacKenzie R.E.
      Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10- methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes.
      ). This value is ∼45-fold higher than the efficiency exhibited by MTHFD2L (kcat/Km = 0.067 s−1μm−1). These differences in kinetic parameters are unlikely to be due to different assay conditions. When we performed the CH2-THF dehydrogenase assay under buffer conditions that were used to characterize MTHFD2 (MOPS (pH 7.3)) rather than the HEPES (pH 8.0) used in this article, we did not observe significant changes in the values for kcat or Km (data not shown).
      The apparent preference of MTHFD2L for NAD+ (Fig. 3A) is most dramatic at nonphysiological levels of phosphate, Mg2+, and folate cofactor. When these experiments were repeated at more physiologically relevant substrate concentrations, MTHFD2L showed much less preference for NAD+ (Fig. 4). In fact, at CH2-THF concentrations below 10 μm, the enzyme is more active with NADP+ than with NAD+ (Fig. 4C). Given that estimates for mitochondrial matrix levels of CH2-THF range from 2.5 to 25 μm (
      • Nijhout H.F.
      • Reed M.C.
      • Lam S.L.
      • Shane B.
      • Gregory 3rd, J.F.
      • Ulrich C.M.
      In silico experimentation with a model of hepatic mitochondrial folate metabolism.
      ,
      • Seither R.L.
      • Trent D.F.
      • Mikulecky D.C.
      • Rape T.J.
      • Goldman I.D.
      Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates: experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamates in antifolate action in cells.
      ,
      • Horne D.W.
      • Patterson D.
      • Cook R.
      Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver.
      ,
      • Eto I.
      • Krumdieck C.L.
      Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver.
      ), it is likely that MTHFD2L exhibits dual redox cofactor specificity in vivo.
      The use of NAD+ versus NADP+ in this step can have a dramatic effect on the rate and direction of flux of one-carbon units through this pathway in mitochondria. The oxidation state of mitochondrial pools of NAD+ and NADP+ is dictated by mitochondrial respiration (
      • Veech R.L.
      Pyridine nucleotides and control of metabolic processes.
      ), which in turn is linked to nutrition, differentiation, and proliferation (
      • Yang H.
      • Yang T.
      • Baur J.A.
      • Perez E.
      • Matsui T.
      • Carmona J.J.
      • Lamming D.W.
      • Souza-Pinto N.C.
      • Bohr V.A.
      • Rosenzweig A.
      • de Cabo R.
      • Sauve A.A.
      • Sinclair D.A.
      Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival.
      ). Measurements in liver suggest that the redox potential of the NAD+/NADH matrix pool is typically 75–100 mV more positive than that of the NADP+/NADPH matrix pool (
      • Veech R.L.
      Pyridine nucleotides and control of metabolic processes.
      ,
      • Sies H.
      Nicotinamide nucleotide compartmentation.
      ). Thus, the ratio of CH2-THF to 10-CHO-THF in the matrix (reactions 3m and 2m in Fig. 1) will be shifted much further toward 10-CHO-THF with a CH2-THF dehydrogenase linked to the NAD+/NADH pool versus the NADP+/NADPH pool (
      • Yang X.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
      ,
      • Pelletier J.N.
      • MacKenzie R.E.
      Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
      ). A cyclohydrolase/dehydrogenase with dual cofactor specificity, such as MTHFD2L, would be able to adapt immediately to changing metabolic conditions, shifting the equilibrium between CH2-THF and 10-CHO-THF (and formate) depending on the relative levels of oxidized cofactor (NAD+ or NADP+) in the mitochondrial matrix. We do not know whether the MTHFD2 isozyme might also exhibit dual redox cofactor specificity in vivo, as it was not characterized at physiologically relevant substrate concentrations (
      • Yang X.M.
      • MacKenzie R.E.
      NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
      ,
      • Christensen K.E.
      • Mirza I.A.
      • Berghuis A.M.
      • Mackenzie R.E.
      Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
      ).
      We determined expression profiles for the entire MTHFD family of genes during mouse embryogenesis (Fig. 5) and in adult tissues (Fig. 7). The results for MTHFD1 (cytoplasmic reactions 1–3 (Fig. 1)) and MTHFD1L (mitochondrial reaction 1m) are qualitatively similar to previously reported transcript expression patterns in mouse embryos based on a staged Northern blot (
      • Pike S.T.
      • Rajendra R.
      • Artzt K.
      • Appling D.R.
      Mitochondrial C1-THF synthase (MTHFD1L) supports flow of mitochondrial one-carbon units into the methyl cycle in embryos.
      ). Both transcripts are highest in early embryos and decrease during embryonic days 9.5–15.5, only to increase again as the embryo approaches birth. MTHFD2 and MTHFD2L, on the other hand, exhibit very different expression profiles. MTHFD2 expression was low in all embryonic days examined, whereas expression of the MTHFD2L transcript increased beginning at E10.5 and remained elevated through birth (Fig. 5). These data reveal a switch from MTHFD2 to MTHFD2L expression at about the time of neural tube closure in mouse embryos. The spatial expression of MTHFD2L is localized to the neural tube, developing brain, branchial arches, and limb buds (Fig. 6). These regions are also areas where MTHFD2 and MTHFD1L are expressed (
      • Pike S.T.
      • Rajendra R.
      • Artzt K.
      • Appling D.R.
      Mitochondrial C1-THF synthase (MTHFD1L) supports flow of mitochondrial one-carbon units into the methyl cycle in embryos.
      ), suggesting a role for the mitochondrial folate pathway in these embryonic tissues.
      Why might mammals possess these two distinct mitochondrial dehydrogenase/cyclohydrolase isozymes? It appears that under most conditions, the majority of 1C units for cytoplasmic processes are derived from mitochondrial formate (
      • Tibbetts A.S.
      • Appling D.R.
      Compartmentalization of mammalian folate-mediated one-carbon metabolism.
      ). Oxidation of mitochondrial CH2-THF is essential for the production of 10-CHO-THF, which is processed by MTHFD1L to provide formate for cytoplasmic export (Fig. 1). This formate is then reattached to THF for use in de novo purine biosynthesis or further reduced for either thymidylate synthesis or remethylation of homocysteine to methionine. Modeling studies suggest that the oxidation step (reaction 3m (Fig. 1)), catalyzed by either MTHFD2 or MTHFD2L, is a critical control point for mitochondrial 1C metabolism. Using an in silico model, Nijhout et al. (
      • Nijhout H.F.
      • Reed M.C.
      • Lam S.L.
      • Shane B.
      • Gregory 3rd, J.F.
      • Ulrich C.M.
      In silico experimentation with a model of hepatic mitochondrial folate metabolism.
      ) observed that the exclusion of CH2-THF dehydrogenase and CH+-THF cyclohydrolase activities from the mitochondrial folate pathway results in loss of formate export and a dramatic increase in mitochondrial serine production for gluconeogenesis. When CH2-THF dehydrogenase and CH+-THF cyclohydrolase activities are included in the model, the mitochondrial folate pathway produces formate for cytosolic export, where it is incorporated into purines, thymidylate, and the methyl cycle (
      • Nijhout H.F.
      • Reed M.C.
      • Lam S.L.
      • Shane B.
      • Gregory 3rd, J.F.
      • Ulrich C.M.
      In silico experimentation with a model of hepatic mitochondrial folate metabolism.
      ). Christensen and MacKenzie (
      • Christensen K.E.
      • MacKenzie R.E.
      Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
      ) have proposed that the level of MTHFD2 expression could act as a metabolic switch to control the balance between serine and formate production.
      We suggest that the existence of two mitochondrial dehydrogenase/cyclohydrolase isozymes in mammals (MTHFD2 and MTHFD2L) reflects the need to tightly regulate flux through this oxidation step in response to changing metabolic conditions and needs. For example, de novo purine biosynthesis is especially important in rapidly dividing cells, such as during embryogenesis. Thus, early embryos express both MTHFD2 and MTHFD2L isozymes, ensuring that mitochondrial formate production is adequate to support de novo purine biosynthesis. Indeed, embryonic growth and neural tube closure requires mitochondrial formate production (
      • Momb J.
      • Lewandowski J.P.
      • Bryant J.D.
      • Fitch R.
      • Surman D.R.
      • Vokes S.A.
      • Appling D.R.
      Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice.
      ). Compared with embryos, however, adult mammals do not have a high demand for de novo purine biosynthesis (
      • Alexiou M.
      • Leese H.J.
      Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos.
      ). The loss of expression of MTHFD2 as the embryos approach birth may reflect the lower demand for de novo purine biosynthesis in neonate and adult mammals.
      In addition to switching between expressing one or two mitochondrial CH2-THF dehydrogenase/CH+-THF cyclohydrolase enzymes, there may be other ways of regulating MTHFD2L expression such as alternative splicing. We have shown previously that an exon 8 deletion is present in adult tissues (
      • Bolusani S.
      • Young B.A.
      • Cole N.A.
      • Tibbetts A.S.
      • Momb J.
      • Bryant J.D.
      • Solmonson A.
      • Appling D.R.
      Mammalian MTHFD2L Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.
      ). We observed significant expression of this same splice variant of MTHFD2L throughout embryogenesis (Fig. 8). However the protein that would be produced from the transcript lacking exon 8 did not show activity in vitro or in vivo (Fig. 9), and the function of this splice variant is unknown.

      Acknowledgments

      We thank Nafee Talukder for assistance with the purification of recombinant MTHFD2L and Jordan Lewandowski for performing the in situ hybridizations.

      REFERENCES

        • Tibbetts A.S.
        • Appling D.R.
        Compartmentalization of mammalian folate-mediated one-carbon metabolism.
        Annu. Rev. Nutr. 2010; 30: 57-81
        • Walkup A.S.
        • Appling D.R.
        Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase.
        Arch. Biochem. Biophys. 2005; 442: 196-205
        • Mejia N.R.
        • MacKenzie R.E.
        NAD-dependent methylenetetrahydrofolate dehydrogenase is expressed by immortal cells.
        J. Biol. Chem. 1985; 260: 14616-14620
        • Mejia N.R.
        • Rios-Orlandi E.M.
        • MacKenzie R.E.
        NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase from ascites tumor cells: purification and properties.
        J. Biol. Chem. 1986; 261: 9509-9513
        • Mejia N.R.
        • MacKenzie R.E.
        NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase in transformed cells is a mitochondrial enzyme.
        Biochem. Biophys. Res. Comm. 1988; 155: 1-6
        • Yang X.M.
        • MacKenzie R.E.
        NAD-dependent methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase is the mammalian homolog of the mitochondrial enzyme encoded by the yeast MIS1 gene.
        Biochemistry. 1993; 32: 11118-11123
        • Pelletier J.N.
        • MacKenzie R.E.
        Binding and interconversion of tetrahydrofolates at a single site in the bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase.
        Biochemistry. 1995; 34: 12673-12680
        • Pawelek P.D.
        • MacKenzie R.E.
        Methenyltetrahydrofolate cyclohydrolase is rate limiting for the enzymatic conversion of 10-formyltetrahydrofolate to 5,10- methylenetetrahydrofolate in bifunctional dehydrogenase-cyclohydrolase enzymes.
        Biochemistry. 1998; 37: 1109-1115
        • Di Pietro E.
        • Wang X.L.
        • MacKenzie R.E.
        The expression of mitochondrial methylenetetrahydrofolate dehydrogenase-cyclohydrolase supports a role in rapid cell growth.
        Biochim. Biophys. Acta. 2004; 1674: 78-84
        • Christensen K.E.
        • Mirza I.A.
        • Berghuis A.M.
        • Mackenzie R.E.
        Magnesium and phosphate ions enable NAD binding to methylenetetrahydrofolate dehydrogenase-methenyltetrahydrofolate cyclohydrolase.
        J. Biol. Chem. 2005; 280: 34316-34323
        • Bolusani S.
        • Young B.A.
        • Cole N.A.
        • Tibbetts A.S.
        • Momb J.
        • Bryant J.D.
        • Solmonson A.
        • Appling D.R.
        Mammalian MTHFD2L Encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues.
        J. Biol. Chem. 2011; 286: 5166-5174
        • Blakley R.L.
        The interconversion of serine and glycine: preparation and properties of catalytic derivatives of pteroylglutamic acid.
        Biochem. J. 1957; 65: 331-342
        • Curthoys N.P.
        • Rabinowitz J.C.
        Formyltetrahydrofolate synthetase: binding of adenosine triphosphate and related ligands determined by partition equilibrium.
        J. Biol. Chem. 1971; 246: 6942-6952
        • Appling D.R.
        • West M.G.
        Monofunctional NAD-dependent, 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.
        Methods Enzymol. 1997; 281: 178-188
        • Kallen R.G.
        • Jencks W.P.
        The mechanism of the condensation of formaldehyde with tetrahydrofolic acid.
        J. Biol. Chem. 1966; 241: 5851-5863
        • Rabinowitz J.C.
        Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid.
        Methods Enzymol. 1963; 6: 814-815
        • Suliman H.S.
        • Sawyer G.M.
        • Appling D.R.
        • Robertus J.D.
        Purification and properties of cobalamin-independent methionine synthase from Candida albicans and Saccharomyces cerevisiae.
        Arch. Biochem. Biophys. 2005; 441: 56-63
        • Claros M.G.
        • Vincens P.
        Computational method to predict mitochondrially imported proteins and their targeting sequences.
        Eur. J. Biochem. 1996; 241: 779-786
        • Li M.Z.
        • Elledge S.J.
        Harnessing homologous recombination in vitro to generate recombinant DNA via SLIC.
        Nat. Methods. 2007; 4: 251-256
        • West M.G.
        • Barlowe C.K.
        • Appling D.R.
        Cloning and characterization of the Saccharomyces cerevisiae gene encoding NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase.
        J. Biol. Chem. 1993; 268: 153-160
        • Gietz R.D.
        • Woods R.A.
        Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.
        Methods Enzymol. 2002; 350: 87-96
        • Wagner W.
        • Breksa 3rd, A.P.
        • Monzingo A.F.
        • Appling D.R.
        • Robertus J.D.
        Kinetic and structural analysis of active site mutants of monofunctional NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.
        Biochemistry. 2005; 44: 13163-13171
        • Palmer K.F.
        • Williams D.
        Optical properties of water in the near infrared.
        J. Opt. Soc. Am. 1974; 64: 1107-1110
        • Barlowe C.K.
        • Appling D.R.
        Isolation and characterization of a novel eukaryotic monofunctional NAD+-dependent 5,10-methylenetetrahydrofolate dehydrogenase.
        Biochemistry. 1990; 29: 7089-7094
        • Ye J.
        • Coulouris G.
        • Zaretskaya I.
        • Cutcutache I.
        • Rozen S.
        • Madden T.L.
        Primer-BLAST: a tool to design target-specific primers for polymerase chain reaction.
        BMC Bioinformatics. 2012; 13: 134
        • Willems E.
        • Mateizel I.
        • Kemp C.
        • Cauffman G.
        • Sermon K.
        • Leyns L.
        Selection of reference genes in mouse embryos and in differentiating human and mouse ES cells.
        Int. J. Dev. Biol. 2006; 50: 627-635
        • Kouadjo K.E.
        • Nishida Y.
        • Cadrin-Girard J.F.
        • Yoshioka M.
        • St-Amand J.
        Housekeeping and tissue-specific genes in mouse tissues.
        BMC Genomics. 2007; 8: 127
        • Pike S.T.
        • Rajendra R.
        • Artzt K.
        • Appling D.R.
        Mitochondrial C1-THF synthase (MTHFD1L) supports flow of mitochondrial one-carbon units into the methyl cycle in embryos.
        J. Biol. Chem. 2010; 285: 4612-4620
        • Kawai J.
        • Shinagawa A.
        • Shibata K.
        • Yoshinom M.
        • Itoh M.
        • Ishii Y.
        • Arakawa T.
        • Hara A.
        • Fukunishi Y.
        • Konno H.
        • Adachi J.
        • Fukuda S.
        • Aizawa K.
        • Izawa M.
        • Nishi K.
        • Kiyosawa H.
        • Kondo S.
        • Yamanaka I.
        • Saito T.
        • Okazaki Y.
        • Gojobori T.
        • Bono H.
        • Kasukawa T.
        • Saito R.
        • Kadota K.
        • Matsuda H.
        • Ashburner M.
        • Batalov S.
        • Casavant T.
        • Fleischmann W.
        • Gaasterland T.
        • Gissi C.
        • King B.
        • Kochiwa H.
        • Kuehl P.
        • Lewis S.
        • Matsuo Y.
        • Nikaido I.
        • Pesole G.
        • Quackenbush J.
        • Schriml L.M.
        • Staubli F.
        • Suzuki R.
        • Tomita M.
        • Wagner L.
        • Washio T.
        • Sakai K.
        • Okido T.
        • Furuno M.
        • Aono H.
        • Baldarelli R.
        • Barsh G.
        • Blake J.
        • Boffelli D.
        • Bojunga N.
        • Carninci P.
        • de Bonaldo M.F.
        • Brownstein M.J.
        • Bult C.
        • Fletcher C.
        • Fujita M.
        • Gariboldi M.
        • Gustincich S.
        • Hill D.
        • Hofmann M.
        • Hume D.A.
        • Kamiya M.
        • Lee N.H.
        • Lyons P.
        • Marchionni L.
        • Mashima J.
        • Mazzarelli J.
        • Mombaerts P.
        • Nordone P.
        • Ring B.
        • Ringwald M.
        • Rodriguez I.
        • Sakamoto N.
        • Sasaki H.
        • Sato K.
        • Schönbach C.
        • Seya T.
        • Shibata Y.
        • Storch K.F.
        • Suzuki H.
        • Toyo-oka K.
        • Wang K.H.
        • Weitz C.
        • Whittaker C.
        • Wilming L.
        • Wynshaw-Boris A.
        • Yoshida K.
        • Hasegawa Y.
        • Kawaji H.
        • Kohtsuki S.
        • Hayashizaki Y
        • RIKEN Genome Exploration Research Group Phase II Team, and the FANTOM Consortium
        Functional annotation of a full-length mouse cDNA collection.
        Nature. 2001; 409: 685-690
        • Ho S.N.
        • Hunt H.D.
        • Horton R.M.
        • Pullen J.K.
        • Pease L.R.
        Site-directed mutagenesis by overlap extension using the polymerase chain reaction.
        Gene. 1989; 77: 51-59
        • Zhang L.
        • Joshi A.K.
        • Smith S.
        Cloning, expression, characterization, and interaction of two components of a human mitochondrial fatty acid synthase: malonyltransferase and acyl carrier protein.
        J. Biol. Chem. 2003; 278: 40067-40074
        • Prasannan P.
        • Appling D.R.
        Human mitochondrial C1-tetrahydrofolate synthase: submitochondrial localization of the full-length enzyme and characterization of a short isoform.
        Arch. Biochem. Biophys. 2009; 481: 86-93
        • Dihazi H.
        • Kessler R.
        • Eschrich K.
        One-step purification of recombinant yeast 6-phosphofructo-2-kinase after the identification of contaminants by MALDI-TOF MS.
        Protein Expr. Purif. 2001; 21: 201-209
        • Thigpen A.E.
        • West M.G.
        • Appling D.R.
        Rat C1-tetrahydrofolate synthase: cDNA isolation, tissue-specific levels of the mRNA, and expression of the protein in yeast.
        J. Biol. Chem. 1990; 265: 7907-7913
        • West M.G.
        • Horne D.W.
        • Appling D.R.
        Metabolic role of cytoplasmic isozymes of 5,10-methylenetetrahydrofolate dehydrogenase in Saccharomyces cerevisiae.
        Biochemistry. 1996; 35: 3122-3132
        • Barlowe C.K.
        • Appling D.R.
        Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.
        Mol. Cell. Biol. 1990; 10: 5679-5687
        • Nijhout H.F.
        • Reed M.C.
        • Lam S.L.
        • Shane B.
        • Gregory 3rd, J.F.
        • Ulrich C.M.
        In silico experimentation with a model of hepatic mitochondrial folate metabolism.
        Theor. Biol. Med. Model. 2006; 3: 40
        • Seither R.L.
        • Trent D.F.
        • Mikulecky D.C.
        • Rape T.J.
        • Goldman I.D.
        Folate-pool interconversions and inhibition of biosynthetic processes after exposure of L1210 leukemia cells to antifolates: experimental and network thermodynamic analyses of the role of dihydrofolate polyglutamates in antifolate action in cells.
        J. Biol. Chem. 1989; 264: 17016-17023
        • Horne D.W.
        • Patterson D.
        • Cook R.
        Effect of nitrous oxide inactivation of vitamin B12-dependent methionine synthetase on the subcellular distribution of folate coenzymes in rat liver.
        Arch. Biochem. Biophys. 1989; 270: 729-733
        • Eto I.
        • Krumdieck C.L.
        Determination of three different pools of reduced one-carbon-substituted folates. III. Reversed-phase high-performance liquid chromatography of the azo dye derivatives of p-aminobenzoylpoly-γ-glutamates and its application to the study of unlabeled endogenous pteroylpolyglutamates of rat liver.
        Anal. Biochem. 1982; 120: 323-329
        • Veech R.L.
        Pyridine nucleotides and control of metabolic processes.
        in: Dolphin D. Poulson R. Avramovic O. Pyridine Nucleotide Coenzymes: Chemical, Biochemical, and Medical Aspects, Part B. John Wiley & Sons, New York1987: 79-105
        • Yang H.
        • Yang T.
        • Baur J.A.
        • Perez E.
        • Matsui T.
        • Carmona J.J.
        • Lamming D.W.
        • Souza-Pinto N.C.
        • Bohr V.A.
        • Rosenzweig A.
        • de Cabo R.
        • Sauve A.A.
        • Sinclair D.A.
        Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival.
        Cell. 2007; 130: 1095-1107
        • Sies H.
        Nicotinamide nucleotide compartmentation.
        in: Sies H. Metabolic Compartmentation. Academic Press, New York1982: 205-231
        • Christensen K.E.
        • MacKenzie R.E.
        Mitochondrial one-carbon metabolism is adapted to the specific needs of yeast, plants and mammals.
        Bioessays. 2006; 28: 595-605
        • Momb J.
        • Lewandowski J.P.
        • Bryant J.D.
        • Fitch R.
        • Surman D.R.
        • Vokes S.A.
        • Appling D.R.
        Deletion of Mthfd1l causes embryonic lethality and neural tube and craniofacial defects in mice.
        Proc. Natl. Acad. Sci. U.S.A. 2013; 110: 549-554
        • Alexiou M.
        • Leese H.J.
        Purine utilisation, de novo synthesis and degradation in mouse preimplantation embryos.
        Development. 1992; 114: 185-192
        • Albe K.R.
        • Butler M.H.
        • Wright B.E.
        Cellular concentrations of enzymes and their substrates.
        J. Theor. Biol. 1990; 143: 163-195
        • Corkey B.E.
        • Duszynski J.
        • Rich T.L.
        • Matschinsky B.
        • Williamson J.R.
        Regulation of free and bound magnesium in rat hepatocytes and isolated mitochondria.
        J. Biol. Chem. 1986; 261: 2567-2574
        • Saleet Jafri M.
        • Kotulska M.
        Modeling the mechanism of metabolic oscillations in ischemic cardiac myocytes.
        J. Theor. Biol. 2006; 242: 801-817