Nuclear enrichment of folate cofactors and methylenetetrahydrofolate dehydrogenase 1 (MTHFD1) protect de novo thymidylate biosynthesis during folate deficiency

Abstract

Folate-mediated one-carbon metabolism is a metabolic network of interconnected pathways that is required for the de novo synthesis of three of the four DNA bases and the remethylation of homocysteine to methionine. Previous studies have indicated that the thymidylate synthesis and homocysteine remethylation pathways compete for a limiting pool of methylenetetrahydrofolate cofactors and that thymidylate biosynthesis is preserved in folate deficiency at the expense of homocysteine remethylation, but the mechanisms are unknown. Recently, it was shown that thymidylate synthesis occurs in the nucleus, whereas homocysteine remethylation occurs in the cytosol. In this study we demonstrate that methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), an enzyme that generates methylenetetrahydrofolate from formate, ATP, and NADPH, functions in the nucleus to support de novo thymidylate biosynthesis. MTHFD1 translocates to the nucleus in S-phase MCF-7 and HeLa cells. During folate deficiency mouse liver MTHFD1 levels are enriched in the nucleus >2-fold at the expense of levels in the cytosol. Furthermore, nuclear folate levels are resistant to folate depletion when total cellular folate levels are reduced by >50% in mouse liver. The enrichment of folate cofactors and MTHFD1 protein in the nucleus during folate deficiency in mouse liver and human cell lines accounts for previous metabolic studies that indicated 5,10-methylenetetrahydrofolate is preferentially directed toward de novo thymidylate biosynthesis at the expense of homocysteine remethylation during folate deficiency.

Keywords: Cell Compartmentalization; DNA Synthesis; Folate; Homocysteine; MTHFD1; Nucleoside/Nucleotide Metabolism; One-carbon Metabolism; Uracil.

FIGURE 1.

One-carbon metabolism is required for the synthesis of purines, thymidylate (dTMP), and methionine. Mitochondrial-derived formate is a major source of 1C units and can enter the cytoplasm to function as a one-carbon unit for folate metabolism. The synthesis of dTMP occurs in the nucleus and mitochondria. At S phase, the enzymes of the thymidylate (dTMP) synthesis pathway undergo SUMO-dependent translocation to the nucleus. The remethylation of homocysteine to methionine by MTR requires vitamin B12. The one carbon is labeled in bold. The inset shows the thymidylate synthesis cycle, which involves the enzymes, SHMT1, SHMT2a, TYMS, and dihydrofolate reductase (DHFR). DHF, dihydrofolate; MTR, methionine synthase; GNMT, glycine N-methyltransferase; MTHFR, 5-methenylenetetrahydrofolate reductase.

FIGURE 2.

MTHFD1 protein levels in liver nuclei from wild type and Mthfd1^gt/+ knockdown mice after 22 ± 2 weeks on diet. A, nuclear tissue lysate was probed with rabbit anti-human Lamin A/C antibody as the loading control. The same membrane was probed with mouse anti-human glyceraldehyde-3-phosphate as a control for nuclei purity. B, immunoblot analysis of total liver extract from wild type and Mthfd1^gt mice. Total cell lysate was probed with mouse anti-human glyceraldehyde 3-phosphate as the loading control. Both membranes were treated with polyclonal sheep anti-mouse MTHFD1 antibody to detect MTHFD1 protein.

FIGURE 3.

MTHFD1 traffics to the nucleus in cell cycle-dependent manner. A, MTHFD1-GFP fusion protein localization within cell cycle-synchronized HeLa cells. HeLa cells were transfected with a plasmid encoding an MTHFD1-GFP fusion protein and arrested in G₁, S, and G₂/M phase. Two representative images for cells for the indicated phases of the cell cycle are shown. MTHFD1-GFP is colored green in the left panels with the nuclear stain Draq5 colored red on the middle panels; the merged images are in the right panels. B, quantitation of nuclear and cytoplasmic localization of MTHFD-GFP wt (black bars) and G1958A polymorphic variant (white bars) at each stage of the cell cycle, as described under “Experimental Procedures.” The ratios of fluorescence intensity in the nucleus to the cytosol were calculated for at least 20 individual cells per condition and are graphed as the mean ± S.E. The statistical significance p was assessed as a t test with Bonferroni correction for multiple comparisons and is represented as follows: NS (non-significant), ***, p < 0.001. Noc, nocodazole; Lpmb, leptomycin B. C, immunoblot analysis of MTHFD1 partitioning between the cytosolic and nuclear fractions in asynchronous HeLa cells, and in HeLa cells arrested in S phase and released into fresh medium for the indicated time points. S phase cell cycle arrest and release were confirmed by FACS analysis.

FIGURE 4.

Formate is utilized for thymidylate biosynthesis in purified nuclei. A, nuclei (2 × 10⁷ per condition) were isolated from HeLa cells arrested in S phase with 1 mm HU and released into fresh media for 3 h. The capacity to convert dUMP and [³H]formate to [³H]dTMP was determined in reactions that contained 1) intact nuclei with all the components of the reaction, 2) intact nuclei with the components of the reaction lacking ATP, and 3) intact nuclei with the components of the reaction lacking dUMP as described under “Experimental Procedures.” All reactions were performed in duplicate, and the experiment was repeated two times. Variation is expressed as the S.D. B, nuclei preparation in A was verified by a Western blot with anti-lamin B antibody. Cyt, cytosol; Nuc, nucleus.

FIGURE 5.

Plasma formate levels as a result of decreased MTHFD1 and SHMT1 expression in mice. A, formate levels from wild-type and Mthfd1^gt/+ knockdown mice after 6 weeks consuming control (white bars) or folate-deficient diet (black bars). B, formate levels from wild-type, Shmt1^+/−, and Shmt1^−/− mice after 6 weeks on control (white bars) or folate-deficient diet (black bars). Data were analyzed using a two-way analysis of variance with a Scheffe post-hoc test for multiple comparisons; groups not connected by the same letter are significantly different (p < 0.05, n = 10 per group; values are shown as the means ± S.E.).