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Mammalian MTHFD2L Encodes a Mitochondrial Methylenetetrahydrofolate Dehydrogenase Isozyme Expressed in Adult Tissues*

  • Swetha Bolusani
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Blake A. Young
    Footnotes
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Nicola A. Cole
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Anne S. Tibbetts
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Jessica Momb
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Joshua D. Bryant
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Ashley Solmonson
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Dean R. Appling
    Correspondence
    To whom correspondence should be addressed: Dept. of Chemistry and Biochemistry, The University of Texas, 1 University Station A5300, Austin, TX 78712-0165. Tel.: 512-471-5842;
    Affiliations
    From the Department of Chemistry and Biochemistry and the Institute for Cellular and Molecular Biology, The University of Texas, Austin, Texas 78712
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants DK61425 and GM086856 (to D. R. A.).
    1 Present address: University of Texas Southwestern Medical Center at Dallas, 5323 Harry Hines Blvd., Mailbox 989, Dallas, TX 75390.
Open AccessPublished:December 16, 2010DOI:https://doi.org/10.1074/jbc.M110.196840
      Previous studies in our laboratory showed that isolated, intact adult rat liver mitochondria are able to oxidize the 3-carbon of serine and the N-methyl carbon of sarcosine to formate without the addition of any other cofactors or substrates. Conversion of these 1-carbon units to formate requires several folate-interconverting enzymes in mitochondria. The enzyme(s) responsible for conversion of 5,10-methylene-tetrahydrofolate (CH2-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH2-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP+-dependent CH2-THF dehydrogenase activity when expressed in yeast. The enzyme is localized to mitochondria when expressed in CHO cells and behaves as a peripheral membrane protein, tightly associated with the matrix side of the mitochondrial inner membrane. The MTHFD2L gene is subject to alternative splicing and is expressed in adult tissues in humans and rodents. This CH2-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH2-THF to formate in adult mammalian mitochondria.

      Introduction

      Tetrahydrofolate (THF)
      The abbreviations used are: THF
      tetrahydrofolate
      CH+-THF
      5,10-methenyl-tetrahydrofolate
      CH2-THF
      5,10-methylene-tetrahydrofolate
      10-CHO-THF
      10-formyl-tetrahydrofolate
      MTHFD1
      cytoplasmic C1-tetrahydrofolate synthase
      MTHFD1L
      mitochondrial C1-tetrahydrofolate synthase
      MTHFD2
      NAD+-dependent 5,10-methylene-tetrahydrofolate dehydrogenase/5,10-methenyl-tetrahydrofolate cyclohydrolase
      h
      human
      r
      rat.
      -dependent 1-carbon metabolism is highly compartmentalized in eukaryotes, with THF-dependent enzymes found in mitochondria, cytoplasm, and nuclei (
      • Appling D.R.
      ,
      • Woeller C.F.
      • Anderson D.D.
      • Szebenyi D.M.
      • Stover P.J.
      ). The 3-carbon of serine is the major 1-carbon donor in most organisms, including humans (
      • Davis S.R.
      • Stacpoole P.W.
      • Williamson J.
      • Kick L.S.
      • Quinlivan E.P.
      • Coats B.S.
      • Shane B.
      • Bailey L.B.
      • Gregory 3rd., J.F.
      ), and THF can be charged with this 1-carbon unit in both the cytoplasmic and the mitochondrial compartments via serine hydroxymethyltransferase (Fig. 1, reactions 4 and 4m), resulting in the formation of 5,10-methylene-THF (CH2-THF). Cytoplasmic CH2-THF can be reduced to 5-methyl-THF (CH3-THF) (reaction 6) for entry into the methyl cycle, it can be oxidized to 10-formyl-THF (10-CHO-THF) (reactions 3 and 2) for purine synthesis, or it can be used for nuclear thymidylate (dTMP) synthesis (reaction 10) (
      • Woeller C.F.
      • Anderson D.D.
      • Szebenyi D.M.
      • Stover P.J.
      ). The other product of the serine hydroxymethyltransferase reaction, glycine, can be metabolized by the mitochondrially localized glycine cleavage system (reaction 5), producing CH2-THF from its 2-carbon (
      • Fu T.F.
      • Rife J.P.
      • Schirch V.
      ,
      • Lamers Y.
      • Williamson J.
      • Theriaque D.W.
      • Shuster J.J.
      • Gilbert L.R.
      • Keeling C.
      • Stacpoole P.W.
      • Gregory 3rd, J.F.
      ). CH2-THF, from either serine or glycine, can be oxidized to 10-CHO-THF by mitochondrial versions of reactions 3 and 2. 10-CHO-THF can either be converted to formate and THF by 10-formyl-THF synthetase (reaction 1m) or oxidized to form CO2 and THF by 10-formyl-THF dehydrogenase (reaction 11) (
      • Barlowe C.K.
      • Appling D.R.
      ,
      • García-Martínez L.F.
      • Appling D.R.
      ).
      Figure thumbnail gr1
      FIGURE 1Mammalian 1-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. The other reactions are catalyzed by the following: 4 and 4m, serine hydroxymethyltransferase; 5, glycine cleavage system; 6, 5,10-methylene-THF reductase; 7, methionine synthase; 8, dimethylglycine dehydrogenase; 9, sarcosine dehydrogenase; 10, thymidylate synthase; 11, 10-formyl-THF dehydrogenase (only the mitochondrial activity of this enzyme is shown, but it has been reported in both compartments in mammals). All reactions from choline to sarcosine are mitochondrial except the betaine-to-dimethylglycine conversion, which is cytoplasmic. Hcy, homocysteine; AdoHcy, S-adenosylhomocysteine; AdoMet, S-adenosylmethionine.
      The cytoplasmic and mitochondrial compartments are metabolically connected by transport of serine, glycine, and formate across the mitochondrial membranes, supporting a mostly unidirectional flow (clockwise in Fig. 1) of 1-carbon units from serine to formate and on to methionine. In fact, it appears that under most conditions, the majority of 1-carbon units for cytoplasmic processes are derived from mitochondrial formate (
      • Barlowe C.K.
      • Appling D.R.
      ,
      • García-Martínez L.F.
      • Appling D.R.
      ,
      • Barlowe C.K.
      • Appling D.R.
      ,
      • Pasternack L.B.
      • Laude Jr., D.A.
      • Appling D.R.
      ,
      • Pasternack L.B.
      • Littlepage L.E.
      • Laude Jr., D.A.
      • Appling D.R.
      ,
      • Kastanos E.K.
      • Woldman Y.Y.
      • Appling D.R.
      ,
      • Gregory 3rd, J.F.
      • Cuskelly G.J.
      • Shane B.
      • Toth J.P.
      • Baumgartner T.G.
      • Stacpoole P.W.
      ,
      • Herbig K.
      • Chiang E.P.
      • Lee L.R.
      • Hills J.
      • Shane B.
      • Stover P.J.
      ,
      • Patel H.
      • Pietro E.D.
      • MacKenzie R.E.
      ,
      • Quinlivan E.P.
      • Davis S.R.
      • Shelnutt K.P.
      • Henderson G.N.
      • Ghandour H.
      • Shane B.
      • Selhub J.
      • Bailey L.B.
      • Stacpoole P.W.
      • Gregory 3rd., J.F.
      ,
      • Anguera M.C.
      • Field M.S.
      • Perry C.
      • Ghandour H.
      • Chiang E.P.
      • Selhub J.
      • Shane B.
      • Stover P.J.
      ,
      • MacFarlane A.J.
      • Liu X.
      • Perry C.A.
      • Flodby P.
      • Allen R.H.
      • Stabler S.P.
      • Stover P.J.
      ).
      In eukaryotes, the cytoplasmic activities of CH2-THF dehydrogenase, 5,10-methenyl-THF (CH+-THF) cyclohydrolase, and 10-CHO-THF synthetase (Fig. 1, reactions 1–3) are present on a trifunctional enzyme called C1-THF synthase (
      • Paukert J.L.
      • Williams G.R.
      • Rabinowitz J.C.
      ,
      • Schirch L.
      ,
      • Smith G.K.
      • Mueller W.T.
      • Wasserman G.F.
      • Taylor W.D.
      • Benkovic S.J.
      ,
      • Hum D.W.
      • Bell A.W.
      • Rozen R.
      • MacKenzie R.E.
      ,
      • Thigpen A.E.
      • West M.G.
      • Appling D.R.
      ). The mammalian version of this trifunctional enzyme is encoded by the MTHFD1 gene (
      • Howard K.M.
      • Muga S.J.
      • Zhang L.
      • Thigpen A.E.
      • Appling D.R.
      ,
      • Christensen K.E.
      • Patel H.
      • Kuzmanov U.
      • Mejia N.R.
      • MacKenzie R.E.
      ,
      • MacFarlane A.J.
      • Perry C.A.
      • Girnary H.H.
      • Gao D.
      • Allen R.H.
      • Stabler S.P.
      • Shane B.
      • Stover P.J.
      ), and its cytoplasmic protein product will herein be designated as MTHFD1.
      The enzymes catalyzing reactions 1m–3m (Fig. 1) in mammalian mitochondria are much less clear. MacKenzie and co-workers (
      • Mejia N.R.
      • MacKenzie R.E.
      ,
      • Mejia N.R.
      • Rios-Orlandi E.M.
      • MacKenzie R.E.
      ) characterized a bifunctional NAD+-dependent CH2-THF dehydrogenase/CH+-THF cyclohydrolase (reactions 3m and 2m), originally isolated from ascites tumor cells. This enzyme was later shown to be a mitochondrial protein (
      • Mejia N.R.
      • MacKenzie R.E.
      ,
      • Bélanger C.
      • MacKenzie R.E.
      ), encoded by the nuclear MTHFD2 gene. Notably, this enzyme (MTHFD2 protein) is found only in transformed mammalian cells and embryonic or non-differentiated tissues (
      • Mejia N.R.
      • MacKenzie R.E.
      ) but is essential during embryonic development (
      • Di Pietro E.
      • Sirois J.
      • Tremblay M.L.
      • MacKenzie R.E.
      ,
      • Di Pietro E.
      • Wang X.L.
      • MacKenzie R.E.
      ). The final step in the mammalian mitochondrial pathway to formate (10-CHO-THF synthetase; reaction 1m) is catalyzed by mitochondrial C1-THF synthase, encoded by the MTHFD1L gene (
      • Prasannan P.
      • Pike S.
      • Peng K.
      • Shane B.
      • Appling D.R.
      ). This isozyme, herein referred to as MTHFD1L, is a homolog of the cytoplasmic MTHFD1. Unlike MTHFD1, however, MTHFD1L is a monofunctional enzyme, containing only the 10-CHO-THF synthetase activity (
      • Walkup A.S.
      • Appling D.R.
      ). The lack of CH2-THF dehydrogenase/CH+-THF cyclohydrolase activities (reactions 3m and 2m) in MTHFD1L thus leaves a gap in this pathway in adult mammalian mitochondria.
      Here we report the identification and characterization of a new mitochondrial CH2-THF dehydrogenase isozyme, encoded by the MTHFD2L gene. The MTHFD2L gene is expressed in adult tissues, thus completing the pathway from CH2-THF to formate in adult mammalian mitochondria.

      DISCUSSION

      The experiments described here confirm that adult mammals express a CH2-THF dehydrogenase isozyme in mitochondria, encoded by the MTHFD2L gene. This isozyme fills the gap in the adult mammalian mitochondrial 1-carbon pathway left by the lack of CH2-THF dehydrogenase activity in the monofunctional MTHFD1L isozyme (Fig. 1). The MTHFD2L gene encodes a protein of about 340 amino acids that is homologous to the mitochondrial bifunctional dehydrogenase/cyclohydrolase (MTHFD2 gene product) and the N-terminal dehydrogenase/cyclohydrolase domains of cytoplasmic and mitochondrial C1-THF synthase (MTHFD1 and MTHFD1L gene products) (Fig. 2). The recombinant rat protein exhibits robust NADP+-dependent CH2-THF dehydrogenase activity in vitro and can replace the yeast cytoplasmic CH2-THF dehydrogenase activities in vivo (Fig. 7). 5,10-Methenyl-THF cyclohydrolase assays were not performed. The high backgrounds in the cyclohydrolase spectrophotometric assay make it unreliable in crude extracts. Thus, we will have to await purification of the enzyme to determine whether it possesses cyclohydrolase activity. The MTHFD2L proteins have a predicted N-terminal mitochondrial targeting sequence, and when the full-length rat cDNA was expressed in CHO cells, the targeting sequence directed the protein exclusively to mitochondria (Fig. 3).
      Evidence from both in vivo (
      • Pasternack L.B.
      • Laude Jr., D.A.
      • Appling D.R.
      ,
      • Pasternack L.B.
      • Littlepage L.E.
      • Laude Jr., D.A.
      • Appling D.R.
      ,
      • West M.G.
      • Horne D.W.
      • Appling D.R.
      ,
      • Pike S.T.
      • Rajendra R.
      • Artzt K.
      • Appling D.R.
      ) and in vitro (
      • Barlowe C.K.
      • Appling D.R.
      ,
      • García-Martínez L.F.
      • Appling D.R.
      ) experiments supports a matrix localization for mitochondrial 1-carbon metabolism. Recently, we found that the MTHFD1L protein is tightly associated with the inner mitochondrial membrane, facing the matrix (
      • Prasannan P.
      • Appling D.R.
      ). The sublocalization experiments described here indicated that MTHFD2L also behaves as a peripheral membrane protein tightly associated with the matrix side of the inner mitochondrial membrane (FIGURE 4, FIGURE 5, FIGURE 6). Given the membrane association of MTHFD1L and MTHFD2L, it is likely that folate-dependent 1-carbon metabolism occurs at the inner mitochondrial membrane. Mitochondrial serine hydroxymethyltransferase, glycine cleavage system, sarcosine dehydrogenase, and dimethylglycine dehydrogenase have all been reported to be associated with the inner membrane in rat liver mitochondria (
      • Motokawa Y.
      • Kikuchi G.
      ,
      • Da Cruz S.
      • Xenarios I.
      • Langridge J.
      • Vilbois F.
      • Parone P.A.
      • Martinou J.C.
      ,
      • Bergeron F.
      • Otto A.
      • Blache P.
      • Day R.
      • Denoroy L.
      • Brandsch R.
      • Bataille D.
      ). These enzymes each produce CH2-THF from their respective substrates, which can be oxidized by the CH2-THF dehydrogenase activity of MTHFD2L. In addition, human mitochondrial folylpolyglutamate synthetase, which adds glutamate residues to the mitochondrial folate pool, has also been reported to be tightly associated with the inner membrane (
      • Nair J.R.
      • McGuire J.J.
      ). It was suggested that the inner membrane localization of mitochondrial folylpolyglutamate synthetase would enable efficient polyglutamylation of folates by promoting substrate channeling between the inner membrane folate carrier and folylpolyglutamate synthetase. It is thus tempting to hypothesize the existence of a large folate-dependent 1-carbon-metabolizing complex at the inner mitochondrial membrane, perhaps including membrane carriers for substrates (e.g. serine, glycine, formate, folate) and/or components of the mitochondrial respiratory chain where the redox cofactors are reoxidized.
      The MTHFD2L gene structure is conserved among human, mouse, and rat, consisting of nine exons (Fig. 8A). RT-PCR analysis revealed three classes of MTHFD2L transcripts in adult human brain and placental RNA, potentially encoding three different protein products (Fig. 8A). Although all three transcript classes are approximately equally represented, at least in human brain and placenta, we do not know whether all three are translated into stable proteins in vivo. RT-PCR analysis of rat RNAs also revealed evidence of alternative splicing of both exon 2 and exon 8.
      RT-PCR analysis of RNA isolated from adult rat tissues revealed that the MTHFD2L gene is widely expressed, with the highest signals observed in brain, heart, lung, spleen, and testes and lower signals in liver and kidney (Fig. 9). We also detected MTHFD2L expression in mouse embryos as early as 9.5 days after fertilization.
      J. Bryant, J. Lewandowski, and S. Vokes, unpublished data.
      The MTHFD2 gene, encoding a mitochondrial NAD+-dependent CH2-THF dehydrogenase, is also expressed early in mouse embryos, but its expression decreases as the embryos approach birth (
      • Di Pietro E.
      • Wang X.L.
      • MacKenzie R.E.
      ,
      • Pike S.T.
      • Rajendra R.
      • Artzt K.
      • Appling D.R.
      ). In adults, MTHFD2 expression is restricted to tissues that contain differentiating cells such as bone marrow (
      • Mejia N.R.
      • MacKenzie R.E.
      ). Thus, after birth, the NADP+-dependent MTHFD2L appears to be the only mitochondrial CH2-THF dehydrogenase expressed in differentiated tissues. Experiments to determine when the switch between MTHFD2 and MTHFD2L occurs, and why, are currently underway.

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