From the Department of Biochemistry, McGill University, Montréal, Quebec H3G 1Y6, Canada
Received for publication, February 19, 2003
, and in revised form, March 11, 2003.
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ABSTRACT |
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INTRODUCTION |
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Methylenetetrahydrofolate dehydrogenase-cyclohydrolase activities are located in both cellular compartments. In yeast, both cytoplasmic and mitochondrial isoenzymes occur as trifunctional NADP-dependent dehydrogenase-cyclohydrolase-synthetase (DCS) proteins (6). The D and C activities interconvert methyleneTHF and 10-formylTHF, and the S converts formate to formylTHF in an ATP-dependent reaction. Dean Appling's group (7, 8, 9) has proposed a rational model wherein mitochondria use the synthetase activity "in reverse" to produce formate, ATP, and THF from formylTHF. The formate is then recaptured as an important source of active one-carbon units by the activities of the cytoplasmic DCS (7, 8, 9, 10). In mammals, the cytoplasmic DCS is ubiquitously expressed in all cells and tissues(11, 12, 13, 14),whereas a mitochondrial version(15), NAD+-dependent DC (NMDMC), is expressed during embryogenesis and is detectable in all immortalized cells (11, 13, 15). This bifunctional enzyme was shown to have Mg2+ and inorganic phosphate-dependent methyleneTHF dehydrogenase activity (11, 16). Based on its kinetic properties, NMDMC was proposed to have derived from an NADP+-dependent precursor (17, 18). More recent evidence comparing the nucleotide sequence of the synthetase domain of the cytoplasmic DCS with the sequence of the 3'-untranslated region of the NMDMC cDNA supported the conclusion that its putative precursor was in fact an NADP+-dependent DCS (19). The metabolic advantage for the mitochondrial NMDMC enzyme to lose its synthetase activity is not obvious. This feature raises the question as to whether mammalian cells expressing the NMDMC enzyme export formate from their mitochondria to the cytoplasm, and if so, what is the mechanism for the generation of formate?
Although the metabolic role of the NMDMC is not well understood, deletion of the gene in mice has been shown to be embryonic lethal beyond E13.5 (20). At E12.5, the embryos appear smaller and pale when compared with their wild type littermates. They also demonstrate a failure of the liver to develop and to take over hematopoiesis from the yolk sac, despite the presence of hematopoietic precursor cells (20). It was suggested earlier that the role of the enzyme was to provide formylTHF for the production of formylmethionyl-tRNA used in mitochondrial protein synthesis or to produce one-carbon units for purine synthesis (15). Gene deletion studies indicated that the role of formylTHF in protein synthesis is not essential in yeast (21), and examination of an NMDMC null mouse cell line indicated that mitochondrial protein synthesis is also not impaired when compared with a wild type control (20). In this report, we establish and characterize immortalized cell lines and examine their nutritional requirements to further characterize the metabolic consequences caused by inactivation of the nmdmc gene.
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EXPERIMENTAL PROCEDURES |
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Rho 0 Cell LineThe 0 cell and the 143B parent cell line from which it was derived were generous gifts from Dr. Eric Shoubridge. The cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 1x penicillin/streptomycin, 1x glutamine, and 50 µg/ml uridine.
Mitochondrial Staining with Mitotracker Mitochondrion-selective ProbesMedium containing Mitotracker Red CMXRos (Molecular Probes) in a range of 10500 nM was added to cells grown on tissue culture chamber slides (Nunc Lab-Tek) and incubated at 37 °C for 30 45 min. After incubation, the medium containing the Mitotracker dye was replaced with fresh medium, and the stained cells were observed by fluorescence microscopy.
Cell Growth StudiesThe various cell lines used in these experiments were grown in defined medium that resembled Dulbecco's modified Eagle's medium except that glycine, serine, and methionine were added separately so that one or more of these amino acids could be omitted to determine the nutritional requirements of cell lines. Our standard medium also contained 1x glutamine, 1x penicillin/streptomycin, 50 µg/ml uridine, 100 µM sodium formate, and 1x ITS-S (insulin, transferrin, sodium selenite, and ethanolamine) from Invitrogen. Formate was omitted when required. Another component of the medium, dialyzed fetal bovine serum (Invitrogen), was further dialyzed (50 ml against 2 liters of phosphate-buffered saline, pH 7.2) for 16 h with a change of buffer halfway during the dialysis. The cells were adapted to the redialyzed fetal bovine serum in a four-step process. Cells were plated at a density of 2.5 x 104 cells/well in 6-well plates, and 24 h later, the medium was replaced with medium lacking glycine, serine, methionine, or formate. The cells were counted by the trypan blue exclusion method at 24-h intervals.
Precursor Incorporation into DNACells were plated at a density of 5 x 104 cells/well in 24-well plates and grown for 24 h in defined medium but with glycine, serine, or formate at 10% of the normal concentration. The growth medium was changed with the addition of 300 µl of medium with 2 x 106 cpm of [1-14C]glycine (97 µM, 32 mCi/mmol); [2-14C]glycine, (90 µM, 31mCi/mmol); [3-14C]serine (100 µM, 32 mCi/mmol); or [14C]formate (67 µM, 47.5 mCi/mmol) per well. As a control for DNA synthesis, the cells were incubated with [3H]thymidine (40 µM, 25 Ci/mmol). In all cases, experiments are compared where the difference in uptake of thymidine was 10% to ensure equal rates of cell growth. The cells were allowed to grow for 24 h in the presence of each radiolabeled precursor (Amersham Biosciences and Moravek Biochemicals) and were washed twice with 500 µl of cold phosphate-buffered saline, pH 7.2. They were lysed directly in the 24-well plates upon addition of a proteinase K solution (100 mM Tris-HCl, pH 7.5, 100 mM NaCl, 50 mM EDTA, pH 8.0, 1% SDS, 1% 2-mercaptoethanol, and 200 µg/ml proteinase K) for 1 h with moderate shaking at 37 °C. The lysates were transferred to 1.5-ml Eppendorf tubes and extracted twice with one volume of phenol:chloroform:isoamyl alcohol (25:24:1). The super-natants were precipitated with one volume of isopropanol followed by centrifugation at 4 °C for 15 min. The DNA pellets were resuspended in 100 µl of Tris-EDTA, pH 8.0. The DNA was transferred to a nylon membrane using a Bio-Rad slot-blot apparatus and washed three times with 200 µl of Tris-EDTA, pH 8.0. Each individual sample was cut out from the membrane with a sterile blade and placed into a scintillation vial containing 10 ml of Scintisafe (Fisher). This experiment was done in triplicate per cell line per condition, and the values for the amount of radioactivity incorporated into DNA, determined by liquid scintillation counting, were averaged.
Precursor Incorporation into BasesCells were plated at a density of 1 x 105 cells/well in 6-well plates, grown, and DNA isolated as described for the precursor incorporation into DNA. The cells were labeled in the presence of 2 ml of medium containing 4 x 106 cpm of the respective [1-14C]glycine (58 µM, 17 mCi/mmol); [2-14C]glycine (58 µM, 17 mCi/mmol); [3-14C]serine (58.5 µM, 17 mCi/mmol); or [14C]formate (27.5 µM, 36 mCi/mmol). The DNA pellets were resuspended in 8 µl of Tris-EDTA, pH 8.0, and transferred to 200-µl PCR tubes containing 30 µg of single-stranded salmon sperm DNA. Perchloric acid (2 µl of 7.5N) was added to each sample, and the DNA was subjected to hydrolysis at 105 °C for 40 min. The hydrolyzed DNA samples were separated into their individual bases by ascending paper chromatography, using a solvent consisting of isopropanol:HCl:H2O (130:33:37). An equimolar mixture of the four bases of DNA was included in each chromatogram to serve as markers. The chromatograms were dried in a fume hood and examined under short wave UV light, and each spot (representing the individual bases) was marked with a pencil. The spots were carefully cut out and placed in a scintillation vial containing 10 ml of Scintisafe. The amount of radioactivity incorporated into each base was determined by liquid scintillation counting. This experiment was done in duplicate per cell line per condition, and the values were averaged.
Supplementation of Mouse Diet with GlycineA custom research diet TD 02123 (Harlan Teklad) was formulated that added 6% glycine (60 g/kg) to the standard rodent diet 2018 that contains 0.8% glycine (Harlan Teklad). Both female and male heterozygous mice were started on the custom research diet 1 week before the first attempt at mating. Supplementation was continued until pregnant females were sacrificed. Embryos were isolated from females, phenotypes were recorded, and yolk sacs were collected for genotypic analysis. As a control, embryos from matings of heterozygous mice fed on the standard rodent diet (2018) were also analyzed. Genotypes of embryos were determined as described previously (20).
Injection of Pregnant Females with Sodium FormatePregnant dams on the standard mouse diet were injected daily with 100 µg/g of body weight of sodium formate in 100 µl of phosphate-buffered saline, pH 7.2, from day E4.5 through day E6.5 with the dose increased to 200 µg/g of body weight from E7.5 through E13.5. Mice were euthanized, embryos were isolated, phenotypes were recorded, and yolk sacs were collected for genotypic analysis as described previously (20).
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RESULTS |
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Analysis of MitochondriaWe previously showed that mitochondria of nmdmc(-/-) fibroblasts are structurally intact by staining with the mitochondrion-selective dye, Mitotracker Green FM, which is taken up by mitochondria independent of a membrane potential. As a further measure of mitochondrial function, we stained fibroblasts with Mitotracker Red CMXRos, which requires a mitochondrial membrane potential for uptake. There are no differences in the staining between wild type and null mutant fibroblasts even at the very low (10 nM) concentrations of dye used (Fig. 2). Because of the location of the NMDMC enzyme in mitochondria and the possibility that the nmdmc(-/-) cells would show a mitochondrial defect, we wanted to have a mitochondrially impaired cell line as a comparison for nutritional studies involving serine and glycine. We used 0 cells that lack mitochondrial DNA (22) and thus cannot produce the proteins of the respiratory chain but retain a small mitochondrial membrane potential (23). The mitochondria of these cells stain with Mitotracker Red CMXRos (24), which we also observed in this study (not shown).
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Nutritional Requirements for NMDMC Null Mutant FibroblastsA series of growth experiments were performed on the NMDMC null mutant and wild type immortalized fibroblasts to evaluate their requirement for one-carbon precursors and products of folate-dependent metabolism. These fibroblasts, adapted for growth in dialyzed serum and defined medium, showed methionine dependence in that cell growth was much reduced by the replacement of methionine by homocysteine (not shown). This is not unexpected since many tumor and immortalized mammalian cells show a reduced or absent ability to replace methionine with homocysteine in the growth medium (25, 26). The nmdmc(-/-) cells are able to grow well in the absence of serine (not shown) but are absolute glycine auxotrophs (Fig. 3, A and B). Growth in glycine-free medium was not stimulated by the addition of -aminolevulinic acid, which might partially spare the requirement for glycine used in heme synthesis (Fig. 3B). All nmdmc(-/-) cell lines obtained from mice derived from two independently targeted ES cells required glycine for growth (Table I), which could not be reversed with added hypoxanthine or formate (Fig. 3B). However, in complete medium containing redialyzed serum, formate and hypoxanthine stimulate the growth of nmdmc(-/-) but not wild type cells, although neither is absolutely essential (Fig. 3, C and D). Addition of thymidine had no effect on cell growth.
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We asked whether cells that have another mitochondrial defect are also glycine auxotrophs and examined the 0 cell line. This cell line, as well as its parent cell line, are methionine-dependent as expected, and the replacement of methionine with homocysteine did not support the growth of either. However, despite the fact that
0 cells lack mitochondrial DNA and thus contain non-respiring mitochondria, they grow equally well on complete and glycine-minus media (not shown).
One-carbon Donor Incorporation into the DNA of FibroblastsAs a preliminary approach to understanding the folate metabolism of these cells, we performed a series of radiolabeling experiments that measured the incorporation of [14C]-labeled glycine, serine, and formate into the total DNA of exponentially growing nmdmc(-/-) cells, using wild type cells under identical conditions for comparison. Thymidine incorporation was measured in parallel and was equivalent in null and wild type cells. The incorporation of [1-14C]glycine and [2-14C]glycine into DNA was identical for both wild type and null mutant cells (Fig. 4). A major difference is seen in the incorporation of [14C]formate when compared with [3-14C]serine into total DNA of wild type and mutant cell lines. Under the conditions used in these experiments, more radiolabel is incorporated from serine than from formate in the wild type cells, and the mutant cell line incorporates much higher amounts of label from formate than from serine (Fig. 4). This difference, shown as a ratio of radioactivity (formate/serine) in Table I, is consistent with all the wild type and mutant cell lines examined. As shown in Table II, the incorporation of label from serine into adenine and guanine bases in a similar experiment is much higher for the wild type cell than for the mutant, whereas the incorporation of formate is significantly less in the wild type than in the mutant cells (Table II). The radiolabeling of 0 cells under the same conditions does not show this difference, suggesting that its folate-dependent pathways are not affected.
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Supplementation of Pregnant Dams with Glycine and FormateSince nmdmc(-/-) fibroblasts are glycine auxotrophs, we attempted to rescue the lethality of null mutant embryos by supplementing the standard mouse diet of pregnant dams with 6% glycine. Table III shows that out of a total of 73 embryos, dietary glycine supplementation does not reduce the numbers of resorbed fetuses or increase the viability of (-/-) embryos at E16.5 or even at E14.5. As control, we analyzed 74 embryos from pregnant females that received the standard rodent diet (Table III). Fig. 5 shows that supplementation with glycine does not improve the phenotype of E14.5 null embryos. Injection of the pregnant dams with sodium formate in the embryonic period beginning prior to liver development also did not enhance the phenotype of null mutants at E13.5 (Table III and Fig. 5).
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DISCUSSION |
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Metabolic studies with deuterated substrates indicate that much of the serine that is used as a one-carbon donor passes through the mitochondria and is released to the cytosol as formate (28, 29, 30). Fig. 6 illustrates that the NMDMC null cells can use mitochondrial serine hydroxymethyltransferase to convert the available mitochondrial THF to methyleneTHF while producing glycine from serine. However, the inability to oxidize the methyleneTHF to formylTHF due to lack of the D* and C* activities prevents regeneration of the THF required to maintain the production of glycine from serine and explains the glycine auxotrophy of these cells. Both methyleneTHF from the conversion of serine to glycine, and that produced from the glycine cleavage pathway would act as an intramitochondrial "methylene trap" since folates do not exit to the cytoplasm to a significant extent (31, 32, 33, 34). Although the cell lines used in this study incorporate [1-14C]glycine and [2-14C]glycine equally into DNA, indicating that neither cell line carries out significant glycine cleavage activity, this defect could be very significant in tissues that require the pathway in vivo. Since the nmdmc(-/-) cell lines cannot provide mitochondrially derived one-carbon units, they are entirely dependent on the cytoplasmic folate pathways. Unlike wild type cells, growth of the nmdmc(-/-) cell lines is stimulated by added formate that can be substituted by supplementation with hypoxanthine but not by thymidine. These results demonstrate that the nmdmc(-/-) cells cannot provide sufficient one-carbon units via cytoplasmic pathways to allow for optimal purine synthesis. This conclusion is also supported by studies with radiolabeled precursors that show a 310-fold enhancement of formate over serine incorporation by the nmdmc(-/-) cells relative to the wild type cells. Since the mitochondria of nmdmc(-/-) cells cannot produce intracellular formate, then the radiolabeled formate added to the medium is preferentially incorporated into nucleotides.
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The glycine auxotrophy demonstrates that the immortalized or transformed cells do not express a second mitochondrial DC or DCS activity. If a second enzyme were expressed to regenerate THF, we would not observe the glycine auxotrophy. Two types of evidence have shed some light on the origin of the NMDMC protein. First, kinetic properties demonstrated a residual Mg2+-dependent ability to use NADP and suggested that NMDMC evolved from an NADP+-dependent DC by using inorganic phosphate (Pi) and Mg2+ to bind the NAD+ cofactor (17, 18). Second, our recent demonstration that the long 3'-untranslated region of the NMDMC cDNA contains 5 sequences averaging more than 100 nucleotides each with significant homologies to the synthetase domain in both the mouse and human DCS enzymes and two such sequences in the Dro-sophila (19) support the proposal for evolution from a trifunctional precursor through loss of the S domain. Yeast contains two nuclear-encoded NADP+-dependent trifunctional DCS proteins, one located in mitochondria and the other in the cytoplasm (6). It now seems likely that the NMDMC protein evolved from a mammalian mitochondrial DCS through a change of cofactor requirement and the loss of the synthetase domain. The change of cofactor from NADP+ to NAD+ has been estimated to shift the equilibrium between methyleneTHF and formylTHF in mitochondria 60200-fold toward the latter (35) and thus would strongly benefit the ultimate production of formate from serine.
If NMDMC is the only DC expressed in the mitochondria of these cell lines, then there is no synthetase domain available to remove the formate from formylTHF as is proposed in the model of the yeast system (7, 8, 9, 10). It is possible that there is a separate mitochondrial synthetase enzyme in mammalian cells, but it is not clear as to how this would be beneficial over a synthetase domain attached to the DC domain. Moreover, having the synthetase operate in the "reverse direction" in the presence of significant concentrations of ATP is probably a less efficient system to release formate than, for example, the expression of a putative formylTHF hydrolase. Since mitochondrial protein synthesis does not seem to require formylTHF, such a hydrolase activity could be localized safely within the mitochondria. Transport of formylTHF out of the mitochondria for use in the cytoplasm would achieve the same goal and provide formylTHF directly to cytoplasmic enzymes, but this is believed to be too slow a process to support metabolic activity (31, 32, 33, 34). Several metabolic studies have used immortalized mammalian cell lines to investigate fluxes of folate-related intermediates, and all support the concept of formate release by mitochondria. If all immortalized mammalian cell lines express only this single mitochondrial DC, then the mechanism of this formate production requires further elucidation.
Studies with the nmdmc(-/-) cell lines indicate that a metabolic role for NMDMC is to provide one-carbon units for purine synthesis. Despite recognition of the metabolic consequences of the gene deletion at a cellular level, we cannot explain the embryonic lethality. The ability of heterozygous dams to synthesize glycine, the abundance of glycine in the diet, and the failure of glycine to improve the phenotype of (-/-) embryos make it unlikely that embryonic lethality is due to the lack of glycine. Although formate stimulates the growth of null mutant fibroblasts, injection of sodium formate into pregnant females also did not improve the phenotype of embryos at E13.5. How the nmdmc(-/-) block in mitochondrial folate metabolism causes the apparently rather specific inability to establish hematopoiesis in the developing liver is still not clear.
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FOOTNOTES |
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* This work was supported by grant MGP 4479 from the Canadian Institutes of Health Research. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Both authors contributed equally to this work.
Supported by studentships from the Faculty of Medicine and Faculty of Graduate Studies and Research, McGill University.
¶ A recipient of a studentship from Fonds de la recherche en santé du Québec.
|| To whom correspondence should be addressed: Dept. of Biochemistry, McGill University, 3655 Promenade Sir William Osler, Montréal, QC H3G 1Y6, Canada. Tel.: 514-398-7270; Fax; 514-398-7384; Robert.Mackenzie{at}mcgill.ca.
1 The abbreviations used are: THF, tetrahydrofolate; DC, methylenetetrahydrofolate dehydrogenase-cyclohydrolase; DCS, methylenetetrahydrofolate dehydrogenase-cyclohydrolase-synthetase;
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ACKNOWLEDGMENTS |
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REFERENCES |
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