Polyglutamylation of Folate Coenzymes Is Necessary for Methionine Biosynthesis and Maintenance of Intact Mitochondrial Genome inSaccharomyces cerevisiae
2000; Elsevier BV; Volume: 275; Issue: 19 Linguagem: Inglês
10.1074/jbc.275.19.14056
ISSN1083-351X
AutoresHélène Cherest, Dominique Thomas, Yolande Surdin-Kerjan,
Tópico(s)Enzyme Structure and Function
ResumoOne-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes ofSaccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by theYMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extention of the glutamate chains of the folate coenzymes, is encoded by theMET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase. One-carbon metabolism is essential to provide activated one-carbon units in the biosynthesis of methionine, purines, and thymidylate. The major forms of folates in vivo are polyglutamylated derivatives. In organisms that synthesize folate coenzymes de novo, the addition of the glutamyl side chains is achieved by the action of two enzymes, dihydrofolate synthetase and folylpolyglutamate synthetase. We report here the characterization and molecular analysis of the two glutamate-adding enzymes ofSaccharomyces cerevisiae. We show that dihydrofolate synthetase catalyzing the binding of the first glutamyl side chain to dihydropteroate yielding dihydrofolate is encoded by theYMR113w gene that we propose to rename FOL3. Mutant cells bearing a fol3 mutation require folinic acid for growth and have no dihydrofolate synthetase activity. We show also that folylpolyglutamate synthetase, which catalyzes the extention of the glutamate chains of the folate coenzymes, is encoded by theMET7 gene. Folylpolyglutamate synthetase activity is required for methionine synthesis and for maintenance of mitochondrial DNA. We have tested whether two folylpolyglutamate synthetases could be encoded by the MET7 gene, by the use of alternative initiation codons. Our results show that the loss of mitochondrial functions in met7 mutant cells is not because of the absence of a mitochondrial folylpolyglutamate synthetase. tetrahydrofolate folylpolyglutamate synthetase open reading frame polymerase chain reaction 2-mercaptoethanol dihydrofolate synthetase YNB medium containing all the growth requirements except methionine In contrast to enteric bacteria or fungi such as Neurospora crassa and Aspergillus nidulans, Saccharomyces cerevisiae possesses a complete set of enzyme activities, which allows its growth on a large number of inorganic or organic sulfur sources. Because of the arrangement of the metabolic pathway for sulfur amino acids biosynthesis (1.Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (554) Google Scholar), a mutant strain unable to assimilate sulfate is capable of growing in the presence of either homocysteine, methionine, cysteine, or S-adenosylmethionine. In contrast, mutations impairing the conversion of homocysteine into methionine are the only ones that are expected to lead to strains that cannot grow on homocysteine or cysteine and require strictly methionine orS-adenosylmethionine for growth. In this reaction, catalyzed by homocysteine 5-methyltetrahydrofolate methyltransferase (methionine synthetase), the methyl group is supplied by 5-methyltetrahydrofolate, one of the products of the one-carbon metabolism (1.Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (554) Google Scholar). One-carbon metabolism, in which one-carbon units are carried and donated by tetrahydrofolate derivatives, is essential for providing activated one-carbon groups to the biosynthesis of methionine, but also to those of purines and thymidylate (Fig. 1). There is no simple nomenclature system for naming tetrahydrofolate (H4folate)1 derivatives but it is usually admitted to use H4 folate as an equivalent of tetrahydropteroylmonoglutamate (i.e. bearing one glutamate) (for nomenclature see Ref. 2.Schirch V. Strong W.B. Arch. Biochem. Biophys. 1989; 269: 371-380Crossref PubMed Scopus (132) Google Scholar). In S. cerevisiae, as well as in other eukaryotes, both the cytoplasmic and mitochondrial compartments possess a set of enzymes that catalyzes the interconversion of folate coenzymes, which differ by the oxidation state of their one-carbon unit (3.Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (308) Google Scholar). However, the reactions involving folate coenzymes and folate-dependent enzymes differ from one subcellular compartment to the other (Fig. 1). In the cytoplasm, the folate coenzymes participate in the synthesis of methionine, purines, and thymidylate. In mitochondria, folate coenzymes are required for the formylation of the initiator tRNA for mitochondrial protein synthesis. The interaction of cytoplasmic and mitochondrial one-carbon metabolism is not yet completely understood (3.Appling D.R. FASEB J. 1991; 5: 2645-2651Crossref PubMed Scopus (308) Google Scholar), but it has been shown in yeast that one-carbon donors such as serine, glycine, and formate are able to cross the mitochondrial membrane (4.McNeil J.B. Bognar A.L. Pearlman R.E. Genetics. 1996; 142: 371-381Crossref PubMed Google Scholar) (5.Kastanos E.K. Woldman Y.Y. Appling D.R. Biochemistry. 1997; 36: 14956-14964Crossref PubMed Scopus (85) Google Scholar). The major cellular forms of folate coenzymes contain polyglutamate tails attached to the para-aminobenzoate moiety. Polyglutamylation of folates is catalyzed by the folylpolyglutamate synthetase (FPGS), which appears to exist in virtually all organisms. Accordingly, polyglutamylated folates are the preferred substrates of folate-dependent enzymes (for review, see Ref. 2.Schirch V. Strong W.B. Arch. Biochem. Biophys. 1989; 269: 371-380Crossref PubMed Scopus (132) Google Scholar). The importance of polyglutamylation in one-carbon metabolism is supported by the study of cell lines bearing a mutation-impairing FPGS activity, which exhibit auxotrophies for the products of one-carbon metabolism (2.Schirch V. Strong W.B. Arch. Biochem. Biophys. 1989; 269: 371-380Crossref PubMed Scopus (132) Google Scholar). In yeast, according to the one-carbon metabolism currently accepted, two reactions are specifically dedicated to methionine biosynthesis catalyzed respectively by methylene tetrahydrofolate reductase and by methionine synthetase. A strict requirement for methionine or S-adenosylmethionine is thus expected to arise from mutations inactivating one of the two genes encoding these enzymes (Fig. 1). However mutant strains ofS. cerevisiae unable to grow on homocysteine had been classified into five complementation groups defining genesMET6, MET7, MET13, MET23,and MET24 (6.Masselot M. De Robichon-Szulmajster H. Mol. Gen. Genet. 1975; 139: 121-132Crossref PubMed Scopus (67) Google Scholar). More recent genetic analysis has shown thatmet6 and met24 mutations on the one hand andmet7 and met23 on the other hand were allelic. 2H. Cherest, unpublished results. TheMET13 gene, which corresponds to the ORF YGL125w, has been shown to encode methylene tetrahydrofolate reductase (7.Tizon B. Rodriguez-Torres A.M. Cerdan M.E. Yeast. 1999; 15: 145-154Crossref PubMed Scopus (23) Google Scholar, 8.Raymond R.K. Kastanos E.K. Appling D.R. Arch. Biochem. Biophys. 1999; 372: 300-308Crossref PubMed Scopus (31) Google Scholar). The MET6 gene has already been shown to encode methionine synthetase (9.Mountain H.A. Bystrom A.S. Larsen J.T. Korch C. Yeast. 1991; 7: 781-803Crossref PubMed Scopus (89) Google Scholar). It is moreover noteworthy that S. cerevisiaecells possess only one methionine synthetase, which functions without vitamin B12 as a cofactor. The work presented here shows that the MET7 gene encodes FPGS. In addition, it shows that FPGS activity is absolutely required for methionine biosynthesis and for the maintenance of mitochondrial DNA. We show also that the gene YMR113w, displaying the capacity to specify a protein closely related to Met7p, encodes dihydrofolate synthetase. Escherichia coli strain JM103 was used as the host for plasmid maintenance. Yeast strains used in this work are listed in Table I. Standard yeast media were prepared as described by Cherest and Surdin-Kerjan (10.Cherest H. Surdin-Kerjan Y. Genetics. 1992; 130: 51-58Crossref PubMed Google Scholar). YPEA medium contains 0.5% yeast extract (Difco), 0.5% bacto-peptone (Difco), 2% ethanol, and 40 mg/l adenine). S. cerevisiaewas transformed after lithium acetate treatment as described by Gietzet al. (11.Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425-1426Crossref PubMed Scopus (2930) Google Scholar). Genetic crosses, sporulation, dissection, and scoring of markers were as described by Sherman et al.(12.$$$$$$ ref data missingGoogle Scholar).Table IYeast strainsStrainsGenotypeOriginW303–1AMATa, his3, leu2, ura3, ade2, trp1R. RothsteinW303–1BMATα, his3, leu2, ura3, ade2, trp1R. RothsteinCC788–2DMATα, his3, leu2, ura3, trp1This studyCC788–2BMATa, his3, leu2, ura3, trp1This studyCC831MATa/α, his3/his3, leu2/leu2, ura3/ura3, trp1/trp1This studyCC704–18DMATa, his3, leu2, ura3, trp1, met7This studyCC827MATa/α, his3/his3, leu2/leu2, ura3/ura3, trp1/trp1, MET7∷URA3 MET7/met7This studyCD180–3AMATa, his3, leu2, ura3, trp1, met7∷TRP1This studyCD180–3BMATα, his3, leu2, ura3, trp1, met7∷TRP1This studyCD180–3DMATa, his3, leu2, ura3, trp1This studyCD181–10DMATa, his3, leu2, ura3, trp1, met7∷TRP1, tup1∷LEU2This studyCD208–2BMATα, his3, leu2, ura3, trp1, ymr113w∷HIS3This studyCD200MATa/α, his3/his3, leu2/leu2, ura3/ura3, trp1/trp1 MET7/met7∷TRP1, YMR113W/ymr113w∷HIS3This studyCD214 1DMATα, his3, leu2, ura3, trp1, ymr113w∷HIS3, tup1∷LEU2This studyCD212–61MATα, his3, leu2, ura3, trp1, met7∷TRP1 ymr113w∷HIS3, tup1∷LEU2This studyCD210–1BMATa, his3, leu2, ura3, trp1, ykl132c∷HIS3This studyCH2–16BMATa, his3, leu2, ura3, trp1, ymr113w∷HIS3 ykl132c∷HIS3This studyCH1–6AMATα, his3, leu2, ura3, trp1, met7∷TRP1 ykl132c∷HIS3This study Open table in a new tab Plasmids pEMBLYe23, -25, and -31 as well as pRS313 and -314 were used as shuttle vectors between S. cerevisiae and E. coli (13.Baldari C. Cesarini G. Gene (Amst.). 1985; 35: 27-32Crossref PubMed Scopus (108) Google Scholar, 14.Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar). The S. cerevisiae genomic library used for the cloning of the geneMET7 was constructed by inserting the product of a partialHindIII digest of chromosomal DNA from the wild type strain X2180–1A in the HindIII site of pEMBLYe23. The disruption of gene MET7 was performed by the one-step gene disruption method (15.Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2191) Google Scholar), as follows: theHindIII-HindIII fragment containing theMET7 region was cloned in the HindIII site of plasmid pBR322. The resulting plasmid was deleted of theBglII-EcoRV fragment containing most of the ORF of gene MET7 that was replaced by gene TRP1 from plasmid pFL35 (Fig. 2). In plasmid pFL35, the HindIII site of the TRP1 gene has been removed allowing the met7::TRP1 construct to be excised using the digestion by HindIII and SalI. The fragment was used to transform the diploid strain CC831 to tryptophan prototrophy yielding strain CD180. Correct integration was verified by Southern blot (data not shown). Disruption of ORFs YMR113w and YKL132c were obtained as described in Ref. 16.Lorenz M.C. Muir R.S. Lim E. McElver J. Weber S.C. Heitman J. Gene (Amst.). 1995; 158: 113-117Crossref PubMed Scopus (260) Google Scholar. In both cases, the HIS3gene from Saccharomyces kluyverii was used to replace the ORF. Cells from diploid CC831 were transformed with the amount of DNA generated by one PCR, and histidine prototrophs were selected. Correct replacement of the targeted gene was verified by analytical PCR using DNA extracted from one transformant for each gene. First, theHindIII-XhoI fragment of pM7–1 was inserted in the EcoRV site of plasmid pRS313, yielding plasmid pM7–2. This eliminates the unique EcoRV site of pRS313. Five modified met7 alleles encoding Met7 derivatives with different translation initiation starts, beginning at methionine residue numbers 1, 17, 63, 86, and 98, respectively, were constructed. In these modified genes, the DNA fragments spanning the nucleotides −190 to −1 (for a beginning at Met residue 1), −190 to +48 (for a beginning at Met residue 17), −190 to +186 (for a beginning at met residue 63) or −190 to 255 (for a beginning at met residue 86), and −190 to 291 (for a beginning at methionine residue 98) of theMET7 region were replaced by the promoter region of geneMET25 following a procedure similar to the one described by Muhlrad et al. (17.Muhlrad D. Hunter R. Parker R. Yeast. 1992; 8: 79-82Crossref PubMed Scopus (419) Google Scholar). Briefly, bifunctional primers were designed that comprised 44 nucleotides homologous to the target sequence on the MET7 gene followed by 25 nucleotides homologous to the flanking region of the MET25 promoter region. These primers were used to amplify the MET25promoter region from plasmid pRS-proMET25 (plasmid pRS314 bearing the −600 to −6 promoter region of gene MET25). For each construction, the resulting PCR product was co-transformed with plasmid pM7–2 linearized by EcoRV into strain CD180–3B (met7Δ). Transformants were selected on a medium lacking histidine but containing methionine. For each MET7construct, plasmid DNA was recovered from six His+transformants. Restriction analysis allowed the selection of the plasmid bearing the correct MET7 construction. Cells were grown in minimal medium complemented to meet their auxotrophic requirements and harvested by centrifugation when the cell concentration reached 107cells/ml (exponential phase). They were washed with 50 mmTris-HCl buffer, pH 8.0, suspended in the same buffer containing 1 mm phenylmethylsulfonyl fluoride, and broken by passage through an Eaton Press at 9000 p.s.i. (18.Thomas D. Cherest H. Surdin-Kerjan Y. Mol. Cell. Biol. 1989; 9: 3292-3298Crossref PubMed Scopus (99) Google Scholar). Cell lysates were cleared by centrifugation at 10,000 × g for 15 min and were used for enzymatic assays. The assay for folylpolyglutamate synthetase was essentially as described in Ref. 19.Bognar A.L. Shane B. Methods Enzymol. 1986; 122: 349-359Crossref PubMed Scopus (13) Google Scholar. Assay mixtures contained 200 mm Tris-HCl, pH 8, 5 mm ATP, 10 mm MgCl2, 20 mm KCl, 100 mm 2- mercaptoethanol (2-ME), 0.5 mmaminopterine, l-[14C]glutamate (5 mm, 2 × 106 cpm), and extract (about 2 mg of protein) in a total volume of 0.5 ml. The assays were incubated for 2 h at 37 °C. The reaction was stopped by the addition of 20 μl of 2-ME, the mixture was centrifuged to discard the precipitated proteins, and 400 μl of the supernatant were applied to DE52 columns (20 × 8 mm) equilibrated in 10 mm Tris-HCl buffer, pH 7.5 containing 30 mm 2-ME and 80 mm NaCl. Unreacted glutamate was eluted with the equilibrium buffer (3 × 5 ml), and the labeled folate product was eluted with 0.1 N HCl (2 × 500 μl). Dihydrofolate synthetase was measured essentially as described in Ref. 20.McDonald D. Atkinson I.J. Cossins E.A. Shane B. Phytochemistry. 1995; 38: 327-333Crossref PubMed Scopus (10) Google Scholar. Assay mixtures contained 200 mmTris-HCl, pH 7.5, 8 mm ATP, 10 mmMgCl2, 30 mm KCl, 50 mm 2-ME, 0.2 mm dihydropteroate,l-[14C]glutamate (5 mm, 2 × 106 cpm), and crude extract containing about 2 mg of protein in a total volume of 0.5 ml. The incubation was for 2 h at 37 °C. The reaction was stopped by the addition of 20 μl of 2-ME, and the mixture was centrifuged to discard the precipitated proteins. 400 μl of the supernatant were applied to DE52 columns, and radioactive dihydrofolate was eluted as described for the FPGS assay. Protein concentrations were estimated by the method described by Lowryet al. (21.Lowry O.H. Rosebrough J.H. Farr A.L. Randall J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Dihydropteroate was synthesized by dithionite reduction of pteroic acid as described in Ref. 22.Stover P. Schirch V. Anal. Biochem. 1992; 202: 82-88Crossref PubMed Scopus (37) Google Scholar. Strains bearing a met7 mutation had been described as methionine auxotrophs (6.Masselot M. De Robichon-Szulmajster H. Mol. Gen. Genet. 1975; 139: 121-132Crossref PubMed Scopus (67) Google Scholar, 23.Jones E.W. Fink G.R. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 181-299Google Scholar). To clone theMET7 gene, strain CC704–18D (met7) was transformed by the gene library described under "Materials and Methods." Among 150,000 transformants 15 were able to grow in the absence of methionine. They all beared the same plasmid with a 5-kilobase pair insert (Fig. 2 A). To confirm that we have indeed cloned the MET7 gene, we verified that the cloned insert is capable of targeting integration at the MET7locus. The insert was recloned in a plasmid lacking an autonomous replicating sequence bearing the URA3 gene. The resulting plasmid was linearized by XhoI used to transform strain W303–1B (MET7, ura3), and stable uracile prototroph transformants were selected. One of these transformants was then crossed to CC704–18D (met7, ura3); the resulting diploid (CC827) was sporulated, and 39 tetrads were analyzed. In all cases a 2+/2− segregation of the methionine auxotrophy was observed, and the Met+ spores were also Ura+, indicating that the insert had directed the integration to the MET7 locus. The position of theMET7 gene within the cloned DNA of pM7–1 was determined by subcloning. The subclones were tested for their ability to restore growth of strain CC704–18D (met7) on a medium lacking methionine (Fig. 2 A). The comparison of the restriction map of the insert of pM7–1 (Fig.2 A) with that of the region of chromosome XV where geneMET7 had been mapped (24.Mortimer R.K. Contopoulou C.R. King J.S. Yeast. 1992; 8: 817-902Crossref PubMed Scopus (169) Google Scholar) showed that the original cloned insert beared four ORFs, YOR239w–YOR242w and that pM7–3, which restores the growth of a met7 mutant on a medium lacking methionine, bears only one ORF, YOR241w. The polypeptide deduced from YOR241w shows extensive similarities with folylpolyglutamate synthetases that catalyze the extention of the glutamate chains of folylmonoglutamates in an ATP-dependent reaction. YOR241w was disrupted (Fig. 2 B) in a diploid strain yielding strain CD180, and disruption was verified by Southern blotting (not shown). Diploid CD180 was sporulated, and tetrads were dissected. In the 18 tetrads tested, a 2+/2− segregation of a methionine auxotrophy was observed, and the Met− spores were also Trp+, indicating that the yor241wΔ bearing strains were methionine auxotrophs, as are the met7 mutants. Strain CD180–3B (yor241Δ) was crossed to strain CC704–18D (met7), and analysis of the progeny showed, as expected, that YOR241w and MET7 are the same gene. To confirm that the MET7 gene does encode the yeast FPGS, the activity was assayed in both wild type and met7Δ bearing strains. As shown in Table II, the disruption of the MET7 gene results in an undetectable level of FPGS activity.Table IISpecific activities of the glutamate adding enzymes in different mutant strainsStrainRelevant genotypeSpecific activityDHFSFPGSnmol/min/mg of proteinW303–1A0.180.29CD180–3Amet7∷TRP10.09<0.01CD208–2Bymr113w∷HIS3<0.010.22CD210–1Bykl132∷HIS30.110.29CH2–16Bymr113w∷HIS3<0.010.22ykl132c∷HIS3 Open table in a new tab Taken together, all these results show that the MET7 gene corresponds to ORF YOR241w and encodes the yeast FPGS. The Met7 protein deduced from the nucleotide sequence of MET7has a molecular mass of 62,157 and a calculated isoelectric point of 9.04. In mammalian cells, polyglutamylated folates have been reported to serve as one-carbon unit donors in the synthesis of methionine as well as in that of purines and of thymidylate (dTMP). Moreover, it has been reported that met7 mutant cells require both methionine and adenine for growth (23.Jones E.W. Fink G.R. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 181-299Google Scholar). We found that themet7 mutants from our collection did not display the adenine requirement. To address more precisely the role of polyglutamylation of the folate coenzymes, we wanted to characterize more precisely the growth requirements of strains bearing a met7 null allele. As the MET7 gene was disrupted in a homozygousADE2 diploid, which does not require adenine, growth of strain CD180–3B (met7, ADE2) was studied, and results show that its mean generation time was reduced from 5.5 to 3.25 h when adenine was added to the minimal synthetic medium containing methionine, indicating that the absence of polyglutamylation impairs modestly the biosynthesis of adenine (Fig.3 A). Yeast cells can transport extracellular dTMP provided that they bear a mutation in the TUP1 gene, for instance. To test if the disruption of the MET7 gene resulted in an impairment of dTMP synthesis, we inactivated the TUP1 gene in a strain bearing a met7Δ mutation and analyzed the growth of a double tup1Δ, met7Δ disrupted strain (CD181–10D) in the presence and absence of dTMP. On the contrary to what was observed for adenine, the addition of dTMP up to 100 μg/ml was without significant effect on the growth of tup1Δ,met7Δ cells (Fig. 3 B). Therefore, we concluded that dTMP synthesis can be made with monoglutamylated folate coenzymes. met7 mutant cells had been described as respiration-deficient (23.Jones E.W. Fink G.R. Strathern J.N. Jones E.W. Broach J.R. The Molecular Biology of the Yeast Saccharomyces: Metabolism and Gene Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 181-299Google Scholar), and we have verified that all the strains bearing met7 mutations from our collection were indeed "petites," i.e. were unable to use ethanol as a carbon source. When the diploid strain CC827 (met7/MET7::URA3-MET7) was sporulated (see above), we noted that a high number of methionine auxotroph spores were unable to use ethanol. Among the 78 methionine auxotrophs examined, only 32 spores were capable of using ethanol. In contrast, all the methionine prototroph spores could use ethanol as a carbon source. However, when diploid CD180 (MET7/met7 Δ) was sporulated and tetrads were tested, we observed that two spores grew quickly and two gave small colonies (Fig. 4). The quickly growing spores were methionine prototrophs. The small colonies were methionine auxotrophs and could not use ethanol as a carbon source. These experiments suggested that met7 mutant cells lose progressively their capacity to use ethanol and that met7disrupted cells are respiration-deficient. Plasmid pM7–1 was then used to transform both a haploid met7Δ strain and a heterozygous met7Δ/MET7 diploid strain. In the haploid strain, plasmid pM7–1 corrected the methionine requirement but not the respiration-deficient phenotype. Sporulation of diploid cells bearing the pM7–1 plasmid and analysis of the progeny showed that all the Trp+, Ura+ spores (met7::TRP1, harboring the pM7–1 plasmid) were able to use ethanol as a carbon source, indicating that the presence of the wild type MET7 gene on a plasmid allowed the maintenance of mitochondrial functions when mitochondria are present, provided by the respiration-competent parental strain. In addition, onemet7 null mutant was mated to a ρ0 tester strain. The resulting diploid was unable to grow on ethanol, showing that the respiration-deficient met7 mutant is not complemented by a ρ0 tester and thus indicating a loss of wild type mitochondrial DNA. Taken together, our results strongly suggest that the loss of Met7p function results in an increased rate of loss of mitochondrial DNA. Then, in addition to methionine biosynthesis, FPGS activity in yeast is required for an essential mitochondrial function. Different hypotheses can be made to explain why FPGS activity is required for both methionine biosynthesis and for maintaining the mitochondrial integrity. For example, the dual phenotype of met7Δ strains could be because of the need for mitochondria of a metabolite synthesized in the cytosol and whose formation requires polyglutamylation of folate coenzymes. Alternatively, the MET7 gene could encode both a cytoplasmic and a mitochondrial form of FPGS. A nuclear encoded protein destined to be imported into the mitochondrial matrix is generally synthesized with a amino-terminal extention of 20–30 residues that functions as a targeting signal (25.Schatz G. J. Biol. Chem. 1996; 271: 31763-31766Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar,26.Schatz G. Nature. 1997; 388: 121-122Crossref PubMed Scopus (88) Google Scholar). There are a number of enzymes from S. cerevisiae that are shared by the cytosol and mitochondria and that are encoded by the same gene. In some cases the mechanism ensuring the different intracellular targeting has been clarified, and it was shown that the gene gives rise to two different proteins with the longer one containing the amino-terminal targeting signal for mitochondria. The second protein, lacking the sequence, is shorter and located in the cytoplasm (27.Chatton B. Walter P. Ebel J.-P. Lacroute F. Fasiolo F. J. Biol. Chem. 1988; 263: 52-57Abstract Full Text PDF PubMed Google Scholar, 28.Ellis S.R. Hopper A.K. Martin N.C. Mol. Cell. Biol. 1988; 9: 1611-1620Crossref Scopus (33) Google Scholar, 29.Wu M. Tzagoloff A. J. Biol. Chem. 1988; 262: 12275-12282Abstract Full Text PDF Google Scholar, 30.Chiu M.I. Mason T.L. Fink G.R. Genetics. 1992; 132: 987-1001Crossref PubMed Google Scholar). In the amino-terminal sequence of MET7 five in frame ATG codons are found at positions 49, 164, 187, 256, and 292 corresponding to the methionine residues 17, 55, 63, 86, and 98 (Fig.5). To test if MET7 could encode both a mitochondrial and a cytoplasmic enzyme, we constructed five different Met7p derivatives beginning at methionine residues 1, 17, 63, 86, and 98, hoping that one derivative would lack the targeting signal and would thus encode only a cytoplasmic form of FPGS. We expected that a strain bearing a Met7p derivative without the targeting signal would not require methionine for growth and would not grow on ethanol as a carbon source. The constructions were made as described under "Materials and Methods." The modified genes were placed under the control of the MET25 promoter region using the GAP repair technique and the centromeric plamid pM7–2. Plasmids bearing modified Met7p were used to transform strain CD180–4C (met7Δ) to histidine prototrophy in a medium containing methionine. At least 30 transformants for each met7 allele were then tested for their methionine requirement. Results showed that the modified met7 alleles encoding proteins beginning at Met-1, -17, and -63 complement the methionine requirement of strain CD180–4C (met7Δ) but that the alleles coding for the proteins beginning at Met-86 or -98 do not complement the methionine auxotrophy of the same strain. The transformants bearing all the Met7p derivatives were respiration-deficient, as expected. To test the ability of the mutants bearing each Met7p derivative to maintain the mitochondrial genome, one transformant for each met7 allele was crossed to strain CC788–2D (MET7, respiration-competent), the resulting diploid was sporulated, and in the progeny, the Trp+, His+ strains (i.e. bearing the met7disruption and carrying the modified Met7p) were examined. In the crosses involving the met7 alleles encoding the Met7p beginning at the Met residues 1, 17, and 63, all the Trp+, His+ spores were methionine prototrophs and could use ethanol as a carbon source. In the crosses involving themet7 allele encoding the Met7 protein beginning at Met-86 and Met-98 all the Trp+, His+ spores were methionine auxotrophs and could not use ethanol on YPEA medium. It is noteworthy that YPEA medium meets the methionine requirement of methionine auxotrophs. These results show that all the Met7p derivatives that led to a Met+ phenotype resulted also in the ability to use ethanol as a carbon source and that the derivatives resulting in a Met− phenotype led also to a respiration deficiency. Taken together, these results argue for the existence of only one cytosolic form of FPGS. As a consequence, these results favor the hypothesis that mitochondrial integrity depends on the presence of a metabolite whose synthesis takes place in the cytosol and is strictly dependent on polyglutamylation of folate coenzymes. In organisms that generate folatesde novo, the addition of glutamyl side chains to cellular folates is achieved by two reactions catalyzed by dihydrofolate synthetase (DHFS) and FPGS, respectively. DHFS catalyzes the binding of the first glutamyl side chain to dihydrop
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