Mammalian MTHFD2L encodes a mitochondrial methylenetetrahydrofolate dehydrogenase isozyme expressed in adult tissues

Abstract

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 (CH(2)-THF) to 10-formyl-THF in adult mammalian mitochondria are currently unknown. A new mitochondrial CH(2)-THF dehydrogenase isozyme, encoded by the MTHFD2L gene, has now been identified. The recombinant protein exhibits robust NADP(+)-dependent CH(2)-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 CH(2)-THF dehydrogenase isozyme thus fills the remaining gap in the pathway from CH(2)-THF to formate in adult mammalian mitochondria.

FIGURE 1.

Mammalian 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.

FIGURE 2.

Sequence alignment of MTHFD2L, MTHFD1L, MTHFD2, and MTHFD1 proteins. Full-length MTHFD2L and MTHFD2 are aligned with the N-terminal dehydrogenase/cyclohydrolase domains of MTHFD1 and MTHFD1L. The leading lowercase letter in each protein name indicates the source (h, human; m, mouse; r, rat). Black boxes denote identity, and white boxes denote conservative substitutions. Residues shown to be critical to dehydrogenase/cyclohydrolase activity (positions 93, 206, 211, and 238 in the alignment) are indicated by asterisks. The alignment was produced using the CLC Sequence Viewer (version 6.3), and the output was generated by the ESPript 2.2 web server.

FIGURE 3.

Subcellular localization of epitope-tagged rat MTHFD2L expressed in CHO cells. Cytoplasmic (cyto) and mitochondrial (mito) fractions from untransfected control CHO cells (lanes 1 and 2) or cells transfected with pcDNA3.1-rMTHFD2L (lanes 3 and 4) were fractionated on a 10% SDS-polyacrylamide gel and immunoblotted with anti-V5 antibodies (1:1000 dilution). Each lane contains 50 μg of total protein. Sizes of molecular mass markers in kDa are shown on the right.

FIGURE 4.

Submitochondrial localization of epitope-tagged rat MTHFD2L expressed in CHO cells. Mitochondria were subfractionated as described under “Experimental Procedures.” Whole mitochondria (MT), outer membrane (OM), intermembrane space (IMS), matrix (Mat), and inner membrane (IM) fractions were resolved on 10% SDS-polyacrylamide gels and subjected to immunoblotting with anti-V5 (1:1500 dilution), anti-Hsp60 (inner membrane/matrix marker; 1:1200 dilution), anti-COX I (inner membrane marker; 1:1000 dilution), or anti-Bcl-x_S/L (outer membrane marker; 1:750 dilution) antibodies. MTHFD2L migrated at an apparent molecular mass of 37 kDa, Hsp60 migrated at 60 kDa, COX I migrated at an apparent molecular mass of 35 kDa, and Bcl-x_S/L migrated at an apparent molecular mass of 32 and 35 kDa.

FIGURE 5.

Alkaline carbonate extraction of CHO cell mitochondria expressing epitope-tagged rat MTHFD2L. CHO cell mitochondria were prepared and subjected to Na₂CO₃ or NaCl or Triton X-100 extraction as described under “Experimental Procedures.” The insoluble integral membrane proteins (pellet; P) were separated from the soluble and peripheral membrane proteins (supernatant; S) by ultracentrifugation. Equal concentrations of pellet and supernatant proteins were fractionated on a 10% SDS-polyacrylamide gel and subjected to immunoblotting with anti-V5 (1:4000 dilution), anti-manganese superoxide dismutase (MnSOD) (peripheral membrane protein marker; 1:2000 dilution), or anti-porin (integral membrane protein marker; 1:2000 dilution) antibodies. MTHFD2L migrated at an apparent molecular mass of 37 kDa, manganese superoxide dismutase migrated at an apparent molecular mass of 25 kDa, and porin migrated at an apparent molecular mass of 31 kDa.

FIGURE 6.

Proteinase K digestion of CHO cell mitochondria expressing epitope-tagged rat MTHFD2L. Mitoplasts were prepared by the digitonin method and treated with 100 μg/ml proteinase K (PK) as described under “Experimental Procedures.” Protease treatment was stopped by the addition of PMSF to 1 mm, and the mitoplast suspensions were centrifuged through sucrose cushions. The resulting pellets were resuspended in equal volumes of HMS without BSA, fractionated on a 10% SDS-polyacrylamide gel, and subjected to immunoblotting with anti-V5 (1:1500 dilution) or anti-Hsp60 (inner membrane/matrix marker; 1:1200 dilution) antibodies. Mitoplasts incubated with 1% Triton X-100 (TX-100) in the presence or absence of proteinase K were used as controls. These Triton X-100-treated controls were analyzed directly without centrifugation through sucrose cushions. MTHFD2L migrated at an apparent molecular mass of 37 kDa, and Hsp60 migrated at an apparent molecular mass of 60 kDa.

FIGURE 7.

Expression of recombinant rat MTHFD2L in yeast. A, S. cerevisiae strain MWY4.4 (ser1 ura3 trp1 leu2 his4 ade3-65 Δmtd1) harboring YEp-rMTHFD2L and untransformed control cells were grown in selective medium, harvested, and lysed as described under “Experimental Procedures.” Lysates were assayed for NAD⁺- and NADP⁺-dependent CH₂-THF dehydrogenase activity before (whole lysate) and after a 30,000 × g centrifugation (cleared lysate). Enzyme activity is expressed as nmol product per gm of wet weight (nmol/gm wet wt) of cells. Each column represents the average ± S.D. of duplicate determinations. B, strain MWY4.5 (ser1 ura3 trp1 leu2 his4 ade3-30/65 Δmtd1) was transformed to uracil prototrophy with either YEp-rMTHFD2L or empty plasmid (Yep24ES). Ura⁺ transformants were streaked onto yeast minimal plates containing serine (left) or serine + adenine (Ser + Ade, right) and incubated at 30 °C for 4 days. Both plates also contained leucine, tryptophan, and histidine to support the other auxotrophic requirements of MWY4.5.

FIGURE 8.

Human MTHFD2L gene structure and alternative transcript splicing. A, gene structure, alternative splicing, and potential protein products. The entire gene spans 145 kbp on chromosome 4 at 4q13.3. Exons are shown as numbered black bars; introns are shown as thin horizontal lines. Exons are not drawn to scale. Three alternative splicing patterns due to skipping of exon 2 and/or 8 are observed in human brain and placenta (see under “Results” for details). B, RT-PCR analysis of exon 2 splicing using primers binding in exons 1 and 3 (hNCRTfor1 and hNCrace2, respectively; Table 1). The expected sizes of the two products are 335 bp (including exon 2) and 301 bp (excluding exon 2). −RT, minus reverse transcriptase control. C, RT-PCR analysis of exon 8 splicing using primers binding in exons 1 and 9 (hJunc1to3f2 and hNCRTend1, respectively; Table 1). The expected sizes of the two products are 980 bp (including exon 8) and 852 bp (excluding exon 8). DNA size markers are indicated.

FIGURE 9.

MTHFD2L expression in adult rat tissues. Total RNA isolated from the indicated adult rat tissues was analyzed by RT-PCR as described under “Experimental Procedures” using primers binding in exons 7 and 9 (rD2Lexon7 and rD2Lexon9, respectively; Table 1). The expected sizes of the two products are 338 bp (including exon 8) and 212 bp (excluding exon 8). Lanes 1 and 9 contain DNA size markers (sizes indicated on left). Lanes 2–8, reverse transcriptase samples (+ RT); lanes 10–16, minus reverse transcriptase controls (− RT); lane 17, minus RNA control (− RNA; contained reverse transcriptase). The image is overexposed to reveal the smaller band.