Mutations in the CYS4 Gene Provide Evidence for Regulation of the Yeast Vacuolar H+-ATPase by Oxidation and Reduction in Vivo
1997; Elsevier BV; Volume: 272; Issue: 44 Linguagem: Inglês
10.1074/jbc.272.44.28149
ISSN1083-351X
AutoresYemisi Oluwatosin, Patricia M. Kane,
Tópico(s)Mitochondrial Function and Pathology
ResumoThe vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H+-ATPase activity in Saccharomyces cerevisiae. The VMA41 gene was cloned and shown to be allelic to theCYS4 gene. The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells. cys4mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidificationin vivo. The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable. Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. TheCYS4 gene was deleted in a strain in which the catalytic site cysteine residue implicated in oxidative inhibition of the yeast V-ATPase has been mutagenized (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P. M., and Forgac, M. (1997) J. Biol. Chem. 272, 11750–11756). This catalytic site point mutation suppresses the effects of the cys4 mutation. The data indicate that the acidification defect of cys4 mutants arises from inactivation of the vacuolar ATPase in the less reducing cytosol resulting from loss of Cys4p activity and provide the first evidence for the modulation of V-ATPase activity by the redox state of the environment in vivo. The vma41-1 mutant was identified in a genetic screen designed to identify novel genes required for vacuolar H+-ATPase activity in Saccharomyces cerevisiae. The VMA41 gene was cloned and shown to be allelic to theCYS4 gene. The CYS4 gene encodes the first enzyme in cysteine biosynthesis, and in addition to cysteine auxotrophy, cys4 mutants have much lower levels of intracellular glutathione than wild-type cells. cys4mutants display the pH-dependent growth phenotypes characteristic of vma mutants and are unable to accumulate quinacrine in the vacuole, indicating loss of vacuolar acidificationin vivo. The vacuolar proton-translocating ATPases (V-ATPase) is synthesized at normal levels and assembled at the vacuolar membrane in cys4 mutants, but its specific activity is reduced (47% of wild type) and the activity is unstable. Addition of reduced glutathione to the growth medium complements the pH-dependent growth phenotype, partially restores vacuolar acidification, and restores wild type levels of ATPase activity. TheCYS4 gene was deleted in a strain in which the catalytic site cysteine residue implicated in oxidative inhibition of the yeast V-ATPase has been mutagenized (Liu, Q., Leng, X.-H., Newman, P., Vasilyeva, E., Kane, P. M., and Forgac, M. (1997) J. Biol. Chem. 272, 11750–11756). This catalytic site point mutation suppresses the effects of the cys4 mutation. The data indicate that the acidification defect of cys4 mutants arises from inactivation of the vacuolar ATPase in the less reducing cytosol resulting from loss of Cys4p activity and provide the first evidence for the modulation of V-ATPase activity by the redox state of the environment in vivo. Many organelles of the vacuolar network of eukaryotic cells, including the vacuoles/lysosomes, Golgi apparatus, endosomes, clathrin-coated vesicles, synaptic membrane vesicles, chromaffin granules, and other secretory vesicles, are acidified by a single class of proton pumps, the vacuolar proton-translocating ATPases (V-ATPases) 1The abbreviations used are: V-ATPase, vacuolar proton-translocating ATPase; V1, peripheral sector of the yeast vacuolar H+-ATPase; V0, integral membrane sector of the yeast vacuolar H+-ATPase; SD-ura (or SD-leu, SD-met), supplemented minimal medium containing 2% dextrose lacking uracil (or leucine or methionine); GSH, reduced glutathione; GSSG, oxidized glutathione; MES, 2-(N-morpholino)ethanesulfonic acid; NEM, N-ethylmaleimide; DTT, dithiothreitol; kb, kilobase(s); MOPS, 4-morpholinepropanesulfonic acid. (1Forgac M. Physiol. Rev. 1989; 69: 765-796Crossref PubMed Scopus (480) Google Scholar). Vacuolar H+-ATPases are multisubunit complexes with an overall structure and subunit composition very similar to the F1F0-ATPases of bacteria, chloroplasts, and the inner mitochondrial membrane (2Nelson N. Taiz L. Trends Biochem. Sci. 1989; 14: 113-116Abstract Full Text PDF PubMed Scopus (244) Google Scholar). The V1 sector of the V-ATPase, which contains the ATP-binding sites, is a cytoplasmically-oriented complex of peripheral subunits, while the V0 sector consists of integral membrane subunits and contains the proton pore (3Kane P.M. Stevens T.H. J. Bioenerg. Biomembr. 1992; 24: 383-393Crossref PubMed Scopus (56) Google Scholar). The electrochemical gradient generated by the V-ATPases is crucial for processes such as protein sorting, zymogen activation, receptor-mediated endocytosis, and the transport of ions, amino acids, and other metabolites (4Anraku Y. Umemoto N. Hirata R. Ohya Y. J. Bioenerg. Biomembr. 1992; 24: 395-405Crossref PubMed Scopus (64) Google Scholar, 5Mellman I. Fuchs R. Helenius A. Annu. Rev. Biochem. 1986; 55: 663-700Crossref PubMed Google Scholar). V-ATPases are highly conserved between fungi, plants, and animals. Thirteen different polypeptides, ranging in molecular mass from 10 to 100 kDa have been identified as subunits of the V-ATPase of the yeastSaccharomyces cerevisiae. The genes encoding all of these subunits have been cloned (Ref. 6Graham L.A. Hill K.J. Stevens T.H. J. Biol. Chem. 1995; 270: 15037-15044Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar and references therein, see also, Refs. 7Supekova L. Supek F. Nelson N. J. Biol. Chem. 1995; 270: 13726-13732Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar and 8Hirata R. Graham L.A. Takatsuki A. Stevens T.H. Anraku Y. J. Biol. Chem. 1997; 272: 4795-4803Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). The products of four other genes (VMA12,VMA21–23) are also required for assembly of the yeast V-ATPase even though they are not part of the final complex (9Hirata R. Umemoto N. Ho M.N. Ohya Y. Stevens T.H. Anraku Y. J. Biol. Chem. 1993; 268: 961-967Abstract Full Text PDF PubMed Google Scholar, 10Hill K.J. Stevens T.H. Mol. Biol. Cell. 1994; 5: 1039-1050Crossref PubMed Scopus (96) Google Scholar, 11Hill K.J. Stevens T.H. J. Biol. Chem. 1995; 270: 22329-22336Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). V-ATPases are present in several distinct locations within a single cell. Very little is known about how enzyme activity is regulatedin vivo to maintain different organelles within a single cell at different specific pH values or to adjust organelle acidification in response to changing extracellular conditions. Several mechanisms have been proposed for the regulation of vacuolar acidification by V-ATPases (see Ref. 12Forgac M. Organellar Ion Channels and Transporters. The Rockefeller University Press, New York1996: 121-132Google Scholar for a recent review). Reversible dissociation of the peripheral V1 and integral V0 domains in response to changes in growth conditions (13Graf R. Harvey W.R. Wieczorek H. J. Biol. Chem. 1996; 271: 20908-20913Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar,14Kane P.M. J. Biol. Chem. 1995; 270: 17025-17032Abstract Full Text Full Text PDF PubMed Google Scholar) has indicated that disassembly and reassembly may be a means of regulating V-ATPase activity in vivo. Regulation of acid secretion by changes in the density of V-ATPase in the apical membrane has been demonstrated in intercalated cells in the kidney (15Gluck S. Cannon C. Al-Awquati Q. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 4327-4331Crossref PubMed Scopus (209) Google Scholar). Reversible disulfide bond interchange (16Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 5817-5822Abstract Full Text PDF PubMed Google Scholar, 17Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 19769-19772Abstract Full Text PDF PubMed Google Scholar, 18Feng Y. Forgac M. J. Biol. Chem. 1994; 269: 13224-13230Abstract Full Text PDF PubMed Google Scholar), changes in the degree of coupling between ATP hydrolysis and proton pumping (19Kibak H. Van Eeckhout D. Cutler T. Taiz S.L. Taiz L. J. Biol. Chem. 1993; 268: 23325-23333Abstract Full Text PDF PubMed Google Scholar, 20Nelson N. Bioenerg. Biomembr. 1992; 24: 407-414Crossref PubMed Scopus (71) Google Scholar), and changes in membrane potential (21Arai H. Pink S. Forgac M. Biochemistry. 1989; 28: 3075-3082Crossref PubMed Scopus (78) Google Scholar, 22Fuch S. Schmid S. Mellman I. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 539-543Crossref PubMed Scopus (161) Google Scholar) have also been suggested as possible means of regulating V-ATPase activity. Biochemical studies on the enzyme isolated from bovine clathrin-coated vesicles have indicated that reversible sulfhydryl-disulfide bond interconversion within the catalytic subunit may play a role in controlling V-ATPase activity in vivo (16Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 5817-5822Abstract Full Text PDF PubMed Google Scholar, 17Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 19769-19772Abstract Full Text PDF PubMed Google Scholar, 18Feng Y. Forgac M. J. Biol. Chem. 1994; 269: 13224-13230Abstract Full Text PDF PubMed Google Scholar). Specifically, these studies show that disulfide bond formation between conserved cysteine residues near the nucleotide-binding site of the catalytic subunit results in inactivation of the V-ATPase and that this inactivation can be reversed by a disulfide interchange within the catalytic subunit. Furthermore, Dschida and Bowman (23Dschida W.J.A. Bowman B.J. J. Biol. Chem. 1995; 270: 1557-1563Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) showed that reducing agents have a stabilizing effect on the V-ATPase fromNeurospora crassa and oxidizing agents are potent inhibitors of the V-ATPase in vitro. These results suggest that the redox state of the immediate environment may be an important regulator of vacuolar ATPase activity, but this has not been demonstrated in vivo. In a genetic screen designed to identify novel genes affecting V-ATPase activity, we isolated a mutation in the CYS4 gene. We report here that the product of the CYS4 gene is required for thein vivo activity, but not the biosynthesis and assembly of the yeast vacuolar H+-ATPase, when cells are grown in rich medium. Mutations in CYS4 lead to a decrease in the cellular concentration of reduced glutathione as a result of impaired cysteine biosynthesis. Our results provide the first in vivo evidence in support of previous results (17Feng Y. Forgac M. J. Biol. Chem. 1992; 267: 19769-19772Abstract Full Text PDF PubMed Google Scholar, 23Dschida W.J.A. Bowman B.J. J. Biol. Chem. 1995; 270: 1557-1563Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar) which suggest that the V-ATPase may be inactivated in a less reducing environment and that this inactivation involves a highly conserved catalytic site cysteine. Restriction endonucleases were purchased from New England Biolabs and Boehringer Mannheim. Taq DNA polymerase was purchased from Boehringer Mannheim. Zymolyase 100T was purchased from ICN. 35S-dATP was purchased from NEN Life Science Products. 1-kb DNA ladder and prestained protein molecular mass standards were obtained from Life Technologies, Inc. Synthetic oligonucleotide primers for polymerase chain reaction and sequencing were obtained from Genosys. Zwittergent 3–14 (ZW3–14) was obtained from Calbiochem. All other reagents were purchased from Sigma. Yeast strains used in this study and their genotypes are listed in TableI. Yeast cells were grown aerobically in media prepared as described by Sherman et al. (24Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1982: 177-186Google Scholar) or Yamashiro et al. (25Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (146) Google Scholar), except that 50 mm MES and 50 mm MOPS were used to buffer YEPD, pH 7.5, containing 50 mm calcium chloride. Sporulation medium (SPIII-22) was prepared as described (26Klapholz S. Esposito R.E. Genetics. 1982; 100: 387-412PubMed Google Scholar) except that p-aminobenzoic acid was omitted from the supplement mixture. cys4 mutant cells were grown on supplemented minimal medium (SD) or YEPD media to which 30 μg/ml reduced glutathione (GSH) was added to give 0.1 mm GSH final. For glutathione depletion, cells were grown in YEPD, pH 7.5, containing 50 μm1-chloro-2,4-dinitrobenzene.Table IYeast strains and genotypesNameGenotypeRef.SF838–5AαMATα ura3–52 leu2–3,112 his4–419 ade657Li Z.-S. Szczypka M. Lu Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (382) Google ScholarYOY14–4BaMATa ura3–52 leu2–3,112 his4–419 ade6pep4–3 vma41–1—1-aFootnote 2.SF838–5Aα cys4-Δ1MATαura3–52 leu2–3,112 his4–419 ade6 cys4Δ::LEU2This studyNO11–2MATα ura3–52 leu2–3,112 his4–419 ade6 vma1Δ::LEU2 URA3::VMA148Taiz L. Nelson H. Maggert K. Morgan L. Brad Y. Taiz S.L. Rubinstein B. Nelson N. Biochim. Biophys. Acta. 1994; 1194: 329-334Crossref PubMed Scopus (35) Google ScholarPNY1MATα ura3–52 leu2–3,112 his4–419 ade6 vma1Δ::LEU2 URA3::vma1–5132Nasmyth K.A. Reed S.I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2119-2123Crossref PubMed Scopus (308) Google ScholarYOY13–2CaMATa ura3–52 leu2–3,112 his4–419 ade6 vma1Δ::URA3 URA3::vma1–51 cys4Δ::LEU2This studyYOY12YOY14–4Ba X SF838–5Aαcys4-Δ1This study1-a Footnote 2. Open table in a new tab Yeast strain YOY14-4Bawas transformed with a yeast genomic library as described (27Randolph E. Biotechniques. 1992; 13: 18-20PubMed Google Scholar). The yeast genomic library, constructed by cloning a yeast partialSau3A genomic DNA into the BamHI site of the CEN plasmid, YCp50, was a kind gift from Dr. Saul Honigberg at Syracuse University. Transformants carrying a URA3-containing plasmid capable of complementing the Vma− growth phenotype of YOY14 were selected directly on SD-ura (supplemented minimal medium lacking uracil) plates buffered to pH 7.5. Plasmids were isolated from transformants as described (28Strathern J.N. Higgins D.R. Methods Enzymol. 1991; 194: 319-329Crossref PubMed Scopus (124) Google Scholar) and retransformed into YOY14-4Ba to confirm the phenotype. Various fragments (Fig.1) of the complementing DNA were subcloned into pRS316 (29Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) and tested for complementation. All DNA manipulations were done as described by Sambrook et al. (30Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). A null cys4 strain was constructed by the one-step allele replacement method (31Rothstein R.J. Methods Enzymol. 1983; 101: 202-211Crossref PubMed Scopus (2033) Google Scholar). The 870-base pair AgeI fragment within the CYS4 open reading frame in plasmid pYO49 was replaced by a 2.1-kb HpaI fragment containing the LEU2 gene. The resulting plasmid (pYO50) was digested with ApaI and SacII to release the LEU2-disrupted allele from the vector and the linear DNA fragment generated was used to transform yeast strain SF838-5Aa. Leu+ transformants were selected and disruption of the CYS4 locus was confirmed by polymerase chain reaction from chromosomal DNA using synthetic oligonucleotides 5′-GGTAGAATTCATCCTTCCAG-3′ and 5′-GATAACATCAGTGACCTTAGC-3′. Isolation of yeast genomic DNA for polymerase chain reaction analysis was carried out as described by Nasmyth and Reed (32Nasmyth K.A. Reed S.I. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 2119-2123Crossref PubMed Scopus (308) Google Scholar) except that DNA was treated with RNase A for 25 min at 37 °C and 5 min at 65 °C before the final precipitation. Haploid yeast strain YOY14-4Ba carrying theCYS4 gene on a URA3-containing plasmid (pYO38) was mated with haploid strain SF838-5Aαcys4Δ::LEU2. Diploids were selected on supplemented minimal medium lacking both uracil and leucine and named YOY12/pYO38. YOY12/pYO38 cells growing in YEPD, pH 5.0, plates were patched on sporulation medium and incubated at 30 °C for 5–6 days. Tetrads were dissected on YEPD, pH 5.0, plates and incubated at 30 °C for 30–48 h to allow the spores to germinate. The CYS4locus was disrupted in a vma1Δ::URA3 strain (SF838-5Aa vma1Δ::URA3) as described above. The resulting vma1Δ cys4Δ strain was crossed to yeast strain PNY1 (33Liu Q. Leng X.-H. Newman P.R. Vasilyeva E. Kane P.M. Forgac M. J. Biol. Chem. 1997; 272: 11750-11756Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) to obtain a heterozygous diploid yeast strain (YOY13) with only one functional copy of the CYS4 gene and no wild-type allele of the VMA1 gene (the only functional copy is the C261V mutant allele, designated here as vma1–51, integrated at the URA3 locus). YOY13 was sporulated and tetrads were dissected on YEPD, pH 5.0, plates. cys4 spores, identified by their cysteine auxotrophy, were selected. Whole cell lysates were prepared from selected (cys4) spores and analyzed by Western blotting using monoclonal antibody 7D5 directed against the 69-kDaVMA1 gene product. This antibody is able to detect the product of the vma1–51 mutant allele of VMA1(33Liu Q. Leng X.-H. Newman P.R. Vasilyeva E. Kane P.M. Forgac M. J. Biol. Chem. 1997; 272: 11750-11756Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Spores in which the vma1–51 mutation had co-segregated with the cys4-Δ1 mutation were selected. One such spore, YOY13-2Ca, was used in the experiments described here. Vacuolar accumulation of quinacrine was assessed as described by Roberts et al. (34Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1990; 194: 644-661Crossref Scopus (287) Google Scholar). Once stained, cells were visualized within 10 min using a Zeiss Axioskop Routine immunofluorescence microscope. Cells were viewed under Nomarski optics to observe normal cell morphology and under a fluorescein isothiocyanate filter with a 100 × objective to observe vacuolar staining. Solubilization of vesicles and purification of the vacuolar H+-ATPase were performed basically as described (3Kane P.M. Stevens T.H. J. Bioenerg. Biomembr. 1992; 24: 383-393Crossref PubMed Scopus (56) Google Scholar, 51Uchida E. Ohsumi Y. Anraku Y. J. Biol. Chem. 1985; 260: 1090-1095Abstract Full Text PDF PubMed Google Scholar) with the following modifications. 0.5–1 mg of solubilized vesicles were layered on a 12 ml of 20–50% (w/v) glycerol gradient and centrifuged at 200,000 × g for 8 h in a Beckman Ti-75 rotor. Sixteen 700-μl fractions were collected and analyzed for ATPase activity to identify fractions containing peak ATPase activity. Fractions were diluted 1:1 with water and protein precipitated by addition of an equal volume of 20% trichloroacetic acid. Precipitated proteins were solubilized in 50 μl of cracking buffer (50 mm Tris-HCl, pH 6.8, 1 mm EDTA, 8 m urea, 5% SDS, 5% β-mercaptoethanol), separated on a 10% SDS-polyacrylamide gel, and detected by silver staining or Western blotting. To determine the specific activity of the purified V-ATPase, protein was precipitated as described above and resuspended in cracking buffer lacking β-mercaptoethanol. Protein concentration was then determined using Bio-Rad DC Protein Assay kit. Whole cell lysates and solubilized vacuolar membrane vesicles were prepared, and SDS-polyacrylamide gel electrophoresis was performed as described previously (35Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar). Immunoblots were probed with monoclonal antibodies 10D7, 7D5, 13D11, and 7A2, and polyclonal anti-27-kDa subunit antisera against the 100-, 69-, 60-, 42, and 27-kDa subunits, respectively, of the yeast V-ATPase (35Kane P.M. Kuehn M.C. Howald-Stevenson I. Stevens T.H. J. Biol. Chem. 1992; 267: 447-454Abstract Full Text PDF PubMed Google Scholar, 36Ho M.N. Hill K.J. Lindorfer M.A. Stevens H. J. Biol. Chem. 1993; 268: 221-227Abstract Full Text PDF PubMed Google Scholar). Bound antibodies were detected using alkaline phosphatase-conjugated secondary antibodies. Plasmid DNA for sequencing was purified using the QIAprep-spin Plasmid Kit from QIAGEN. Sequencing was done by the dideoxy chain termination method using SequenaseRsequencing kit with Sequenase version 2.0 (U. S. Biochemical Corp.) and 35S-dATP. Indirect immunofluorescence microscopy and preparation of vacuolar membrane vesicles were performed as described by Roberts et al. (34Roberts C.J. Raymond C.K. Yamashiro C.T. Stevens T.H. Methods Enzymol. 1990; 194: 644-661Crossref Scopus (287) Google Scholar). Vacuolar ATPase activity was measured in a coupled enzyme assay as described previously (37Lotscher H.-R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4128-4134Crossref PubMed Scopus (40) Google Scholar). The effects of various oxidizing and reducing agents on V-ATPase activity were tested by preincubating 5-μg vesicles with the indicated agent in 100 μl of buffer (15 mm MES-Tris, pH 7.0, 4.8% glycerol). At the end of the incubation period, the whole reaction mixture was assayed for ATPase activity as described (37Lotscher H.-R. deJong C. Capaldi R.A. Biochemistry. 1984; 23: 4128-4134Crossref PubMed Scopus (40) Google Scholar). Equivalent concentrations of the oxidizing and reducing agents were shown not to inhibit the enzymes of the coupled ATPase assay system. Purification of the V-ATPase was carried out as described. 2Y. E. Oluwatosin and P. M. Kane, submitted for publication. Protein was determined by the method of Lowry (38Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) and silver staining was performed as described (39Wray W. Boulikas T. Wray V.P. Hancock R. Anal. Biochem. 1981; 118: 197-203Crossref PubMed Scopus (2502) Google Scholar). The MEY14 strain, carrying the vma41-1 mutation was obtained in a genetic screen designed to identify genes required for vacuolar membrane ATPase activity in yeast.2 The screen was based on the set of growth phenotypes characteristic of mutants with loss of vacuolar membrane V-ATPase activity (vma mutants). The Vma− growth phenotypes include inability to grow in medium buffered to pH 7 or above, medium containing 100 mmCaCl2, or medium containing a non-fermentable carbon source. After backcrossing to the original MEY14 strain to remove background mutations, one Vma− spore, YOY14-4Ba, was selected for further analysis. In addition to Vma− phenotypes, both the MEY14 mutant and the YOY14-4Ba strain are unable to grow on minimal medium, suggesting the cosegregation of the Vma− phenotype and an undetermined nutritional auxotrophy. The VMA41 gene was cloned by complementation of the pH-dependent growth phenotype of the YOY14-4Ba strain. The YOY14-4Bamutant strain was transformed with a yeast DNA library on a single copy (CEN) plasmid (YCp50). Transformants were selected on SD-ura, pH 7.5, medium and 15 independent Ura+Vma+transformants were obtained. Plasmids were recovered from transformants and re-checked for complementation. All 15 plasmids were able to restore growth at pH 7.5 to the YOY14-4Ba strain. Restriction endonuclease analyses revealed that all 15 contain the same 11-kb yeast DNA insert (Fig. 1). Various subclones of plasmid pMEY14-1 were generated in the yeast shuttle vector pRS316. Analyses of these sublones indicated that a 2-kbXbaI-SphI fragment is sufficient for complementation (Fig. 1). A 350-base pair region internal to this fragment was sequenced and used to search for homology to any sequences in the GenEMBL data base. This analysis revealed that the sequenced region (indicated by the arrows in Fig. 1) lies within the reported nucleotide sequence of the yeast NHS5 gene for β-thionase, also known as the STR4 or CYS4 gene for cystathionine-β-synthase (40Cherest H. Thomas D. Surdin-Kerjan Y. J. Bacteriol. 1993; 175: 5366-5374Crossref PubMed Google Scholar, 41Kruger W.D. Cox D.R. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 6614-6618Crossref PubMed Scopus (69) Google Scholar). For clarity, theCYS4 nomenclature is used throughout this report. TheCYS4 gene product is essential for cysteine metabolism; as a result, cys4 mutants exhibit a cysteine-dependent growth phenotype (40Cherest H. Thomas D. Surdin-Kerjan Y. J. Bacteriol. 1993; 175: 5366-5374Crossref PubMed Google Scholar). A LEU2-disrupted copy (plasmid pYO50) of the CYS4 open reading frame was constructed as shown in Fig. 1. Haploid yeast strain SF838-5Aα was transformed with ApaI/SacII-digested pYO50 and stable Leu+ transformants selected. DNA was extracted from three independent transformants and the parental wild type strain and analyzed by polymerase chain reaction to confirm the disruption of theCYS4 locus in the transformants. The results (not shown) indicate that the 968-base pair fragment, expected from the wild typeCYS4 locus, was replaced by a larger, 2.3-kb fragment in the transformants, indicating that the CYS4 gene has been disrupted in these cells. The growth phenotypes of cys4Δcells are indistinguishable from those of the original YOY14-4Ba mutant. In addition to the characteristic Vma− growth phenotypes, cys4Δ cells are unable to grow on minimal medium without externally supplied cysteine. These results suggest that a single mutation is responsible for both the Vma− phenotypes and the cysteine auxotrophy of the mutant cells. To determine if the Vma− growth phenotype of thevma41 strain is directly related to its cysteine auxotrophy, YOY14-4Ba and cys4Δ cells were supplied with cysteine or reduced glutathione. Under these conditions, the mutant cells were able to grow on minimal medium. Interestingly, externally supplied cysteine was able to partially complement the growth phenotype of YOY14 strain on YEPD medium buffered to pH 7.5 (Fig.2), with glutathione giving better complementation than cysteine. Conversely, depletion of intracellular glutathione in wild type cells by addition of 50 μm1-chloro-2,4-dinitrobenzene (57Li Z.-S. Szczypka M. Lu Y.-P. Thiele D.J. Rea P.A. J. Biol. Chem. 1996; 271: 6509-6517Abstract Full Text Full Text PDF PubMed Scopus (382) Google Scholar) prevented growth of the cells on YEPD medium buffered to pH 7.5 (data not shown) under conditions where growth of cells on YEPD buffered to pH 5.0 continued. These results indicate that glutathione deficiency results in a pH-dependent (Vma−) growth phenotype in yeast. To confirm that vma41–1 is indeed an allele ofCYS4, diploid strain YOY12/pYO38, obtained from a cross between cys4-Δ1 and YOY14-4Ba/pYO38 was sporulated and the resulting tetrads dissected. We found that YOY12 cured of the plasmid pYO38 was unable to sporulate, even after 2 weeks in sporulation medium. This is consistent with previous results indicating that glutathione auxotrophic mutants ofSchizosaccharomyces pombe are defective in sporulation (42Chaudhuri B. Ingavale S. Bachhawat A.K. Genetics. 1997; 145: 75-83Crossref PubMed Google Scholar). Tetrad analysis indicated a 4:0 segregation of the Vma−phenotype in YOY12 spores since all Ura− spores were Vma− and all Ura+ spores were also Vma+. Moreover, when Ura+ spores lost the plasmid pYO38, they became Vma−. Also, YOY12 diploids lacking plasmid pYO38 exhibit a pH-dependent growth phenotype. These results confirm that vma41-1 is allelic toCYS4. All knownvma mutants are unable to accumulate the fluorescent weak base, quinacrine, in their vacuoles as a result of loss of vacuolar acidification (25Yamashiro C.T. Kane P.M. Wolczyk D.F. Preston R.A. Stevens T.H. Mol. Cell. Biol. 1990; 10: 3737-3749Crossref PubMed Scopus (146) Google Scholar). Quinacrine vital staining was used to assess vacuolar acidification in YOY14-4Ba (vma41-1) andcys4Δ mutant cells. Our results (Fig.3) show that mutants lacking a functionalCYS4 gene are unable to accumulate quinacrine in their vacuoles, indicating loss of vacuolar acidification in these mutants. Since GSH was able to complement the pH-dependent growth phenotype of cys4 mutants, it may also be able to restore quinacrine accumulation in the vacuole. Fig. 3 shows partial restoration of vacuolar acidification by GSH as indicated by partial vacuolar staining with quinacrine in the presence of GSH. To begin to understand the basis of the Vma− growth and vacuolar acidification defects of cys4 mutants, we examined the steady-state levels of several V-ATPase subunits and one specific assembly factor in these cells. Whole cell protein extracts prepared from YOY14-4Ba and cys4Δ cells were analyzed by Western blotting. The results show that the 69-, 60-, 54-, 42-, and 27-kDa peripheral subunits of the V-ATPase and the 25-kDa (Vma12p) V-ATPase assembly factor are present in cys4 mutant cells at normal levels compared with wild type cells (Fig.4 A). Addition of GSH to the growth medium does not increase the steady state levels of the V-ATPase subunits in cys4-Δ1 cells (Fig. 4 A). These results indicate that, even in the absence of additional extracellular glutathione, there is sufficient cysteine in the mutants during growth in rich medium to support normal levels of subunit biosynthesis. Previous results (43Doherty R.D. Kane P.M. J. Biol. Chem. 1993; 268: 16845-16851Abstract Full Text PDF PubMed Google Scholar) have shown that the presence of V-ATPase subunits at normal levels does not always imply assembly of the enzyme. Therefore,
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