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• W2081418938 abstract "Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of γ-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependant activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependant mechanism. Depletion of the cellular pool of glutathione is detrimental to eukaryotic cells and in Saccharomyces cerevisiae leads to sensitivity to oxidants and xenobiotics and an eventual cell cycle arrest. Here, we show that the Yap1 and Met4 transcription factors regulate the expression of γ-glutamylcysteine synthetase (GSH1), encoding the rate-limiting enzyme in glutathione biosynthesis to prevent the damaging effects of glutathione depletion. Transcriptional profiling of a gsh1 mutant indicates that glutathione depletion leads to a general activation of Yap1 target genes, but the expression of Met4-regulated genes remains unaltered. Glutathione depletion appears to result in Yap1 activation via oxidation of thioredoxins, which normally act to down-regulate the Yap1-mediated response. The requirement for Met4 in regulating GSH1 expression is lost in the absence of the centromere-binding protein Cbf1. In contrast, the Yap1-mediated effect is unaffected, indicating that Met4 acts via Cbf1 to regulate the Yap1-mediated induction of GSH1 expression in response to glutathione depletion. Furthermore, yeast cells exposed to the xenobiotic 1-chloro-2,4-dintrobenzene are rapidly depleted of glutathione, accumulate oxidized thioredoxins, and elicit the Yap1/Met4-dependent transcriptional response of GSH1. The addition of methionine, which promotes Met4 ubiquitination and inactivation, specifically represses GSH1 expression after 1-chloro-2,4-dintrobenzene exposure but does not affect Yap1 activation. These results indicate that the Yap1-dependant activation of GSH1 expression in response to glutathione depletion is regulated by the sulfur status of the cell through a specific Met4-dependant mechanism. The ability to regulate biosynthetic pathways is a fundamental aspect of adaptation to life in a changing environment. In response to a stress, cells must be able to prioritize the allocation of resources to support the increased demand for defensive mechanisms. Understanding the mechanisms that facilitate stress-induced changes is important in determining how a cell co-ordinates different areas of metabolism. An inducible stress response has been described for the biosynthesis of glutathione (GSH) in the yeast Saccharomyces cerevisiae. GSH is a tripeptide (γ-l-glutamyl-l-cysteinylglycine) that plays an important role in protecting yeast cells against damage induced by oxidative stress (1.Grant C.M. MacIver F.H. Dawes I.W. Curr. Genet. 1996; 29: 511-515Crossref PubMed Scopus (271) Google Scholar, 2.Stephan D.W.S. Jamieson D.J. FEMS Lett. 1996; 141: 207-212Crossref Google Scholar, 3.Izawa S. Inoue Y. Kimura A. FEBS Lett. 1995; 368: 73-76Crossref PubMed Scopus (224) Google Scholar). GSH counters the potentially damaging effect of reactive oxygen species through direct scavenging of free radicals and through the action of antioxidant enzymes such as the glutathione peroxidases (4.Meister A. Anderson M.E. Ann. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar). In addition GSH has other protective roles within the cell, such as the detoxification of xenobiotics and heavy metals through the formation of GSH conjugates and their subsequent export into the vacuole (5.Li Z.-S. Lu Y.-P. Zhen R.-G. Szczypka M. Thiele D.J. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (491) Google Scholar). The reversible binding of GSH to protein sulfhydryl groups can protect them from irreversible oxidative damage (6.Cotgreave I.A. Gerdes R.G. Biochem. Biophys. Res. Commun. 1998; 242: 1-9Crossref PubMed Scopus (430) Google Scholar, 7.Thomas J.A. Poland B. Honzatko R. Arch. Biochem. Biophys. 1995; 319: 1-9Crossref PubMed Scopus (363) Google Scholar). There are also requirements for GSH in methylglyoxal detoxification, as a cofactor for ribonucleotide reductase, in protein folding, and in amino acid transport (4.Meister A. Anderson M.E. Ann. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar, 8.Inoue Y. Tsujimoto Y. Kimura A. J. Biol. Chem. 1998; 273: 2977-2983Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar). The first step in the synthesis of GSH is the conjugation of glutamate and cysteine by γ-glutamylcysteine synthetase, encoded by GSH1 (9.Ohtake Y. Yabuuchi S. Yeast. 1991; 7: 953-961Crossref PubMed Scopus (112) Google Scholar, 10.Lisowsky T. Curr. Genet. 1993; 23: 408-413Crossref PubMed Scopus (30) Google Scholar). This produces γ-glutamylcysteine, to which glycine is added by glutathione synthetase, encoded by GSH2 (11.Grant C.M. MacIver F.H. Dawes I.W. Mol. Biol. Cell. 1997; 8: 1699-1707Crossref PubMed Scopus (154) Google Scholar, 12.Inoue Y. Sugiyama K.-I. Izawa S. Kimura A. Biochim. Biophys. Acta. 1998; 1395: 315-320Crossref PubMed Scopus (44) Google Scholar). Mutant strains lacking GSH1 are unable to grow in the absence of GSH, indicating that this metabolite is essential in S. cerevisiae (1.Grant C.M. MacIver F.H. Dawes I.W. Curr. Genet. 1996; 29: 511-515Crossref PubMed Scopus (271) Google Scholar, 13.Lee J.-C. Straffon M.J. Jang T.-Y. Grant C.M. Dawes I.W. FEMS Yeast Res. 2000; 1: 57-65Google Scholar, 14.Spector D. Labarre J. Toledano M.B. J. Biol. Chem. 2001; 276: 7011-7016Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The rate-limiting step in the biosynthetic pathway is Gsh1, which is feedback-inhibited at the enzyme level by GSH (4.Meister A. Anderson M.E. Ann. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5897) Google Scholar). The rate of GSH1 expression is, therefore, very important in regulating the abundance of Gsh1 and determining the rate of GSH biosynthesis. In response to oxidative stress, GSH1 expression is increased in a Yap1-dependant manner (15.Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 16.Stephen D.W.S. Jamieson D.J. Mol. Microbiol. 1997; 23: 203-210Crossref PubMed Scopus (59) Google Scholar, 17.Dormer U.H. Westwater J. Stephen D.W. Jamieson D.J. Biochim. Biophys. Acta. 2002; 1576: 23-29Crossref PubMed Scopus (27) Google Scholar). Yap1 is a redox-sensitive bZip-transcription factor that regulates the expression of many antioxidant genes (18.Lee J. Godon C. Lagniel G. Spector D. Garin J. Labarre J. Toledano M.B. J. Biol. Chem. 1999; 274: 16040-16046Abstract Full Text Full Text PDF PubMed Scopus (456) Google Scholar, 19.Carmel-Hare O. Stearman R. Gasch A.P. Botstein D. Brown P.O. Storz G. Mol. Microbiol. 2001; 39: 595-605Crossref PubMed Scopus (93) Google Scholar, 20.Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3680) Google Scholar). Upon exposure to H2O2, Yap1 accumulates in the nucleus due to the masking of a C-terminal nuclear export signal by an intramolecular disulfide bond (21.Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar, 22.Delaunay A. Isnard A.-D. Toledano M.B. EMBO J. 2000; 19: 5157-5166Crossref PubMed Scopus (414) Google Scholar). It has recently been shown that the glutathione peroxidase like-enzyme, Gpx3, is required for the H2O2-dependent formation of the intramolecular disulfide bond (23.Delaunay A. Pflieger D. Barrault M.B. Vinh J. Toledano M.B. Cell. 2002; 111: 471-481Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar). The pathway is turned off by thioredoxin, which reduces both Gpx3 and Yap1. There is a requirement for Yap1 in the response of GSH1 to oxidants such as H2O2, tert-butyl hydroperoxide, and menadione and also to other stresses such as heat shock and cadmium (16.Stephen D.W.S. Jamieson D.J. Mol. Microbiol. 1997; 23: 203-210Crossref PubMed Scopus (59) Google Scholar, 24.Takeuchi T. Miyahara K. Hirata D. Miyakawa T. FEBS Lett. 1997; 416: 339-343Crossref PubMed Scopus (22) Google Scholar, 25.Sugiyama K.-I. Izawa S. Inoue Y. J. Biol. Chem. 2000; 275: 15535-15540Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar). Deleting the Yap1-responsive element (YRE) 1The abbreviations used are: YREYap1-responsive elementCDNB1-chloro-2,4-dintrobenzene.1The abbreviations used are: YREYap1-responsive elementCDNB1-chloro-2,4-dintrobenzene. in the GSH1 promoter still permits H2O2-mediated induction, which may indicate that Yap1 only plays an indirect role in the response to H2O2 (17.Dormer U.H. Westwater J. Stephen D.W. Jamieson D.J. Biochim. Biophys. Acta. 2002; 1576: 23-29Crossref PubMed Scopus (27) Google Scholar). Yap1-responsive element 1-chloro-2,4-dintrobenzene. Yap1-responsive element 1-chloro-2,4-dintrobenzene. Cadmium-inducible expression of GSH1 also requires the presence of the Met4 transcription factor (26.Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellor J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Met4 is a bZip-transcriptional activator that regulates the assimilation of extracellular sulfate into the sulfur-containing amino acids methionine and cysteine (27.Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (524) Google Scholar). Met4 will, therefore, have an indirect effect on GSH biosynthesis by regulating the supply of cysteine. Met4 lacks a DNA binding domain and is tethered to DNA in the form of a heteromeric complex involving the bZip protein, Met28, and the basic helix-loop-helix protein, Cbf1 (28.Kuras L. Barbey R. Thomas D. EMBO J. 1997; 16: 2441-2451Crossref PubMed Scopus (71) Google Scholar, 29.Kuras L. Cherest H. Surdin-Kerjan Y. Thomas D. EMBO J. 1996; 15: 2519-2529Crossref PubMed Scopus (96) Google Scholar). Met4 may also be recruited to DNA in an alternative complex involving Met28 and one of the zinc finger proteins, Met31 or Met32 (30.Blaiseau P.L. Isnard A.D. Surdin-Kerjan Y. Thomas D. Mol. Cell Biol. 1997; 17: 3640-3648Crossref PubMed Scopus (98) Google Scholar, 31.Blaiseau P.L. Thomas D. EMBO J. 1998; 17: 6327-6336Crossref PubMed Scopus (86) Google Scholar). The Met4-regulated genes of the sulfate assimilation pathway have differing requirements for one or both of these complexes. The GSH1 promoter contains binding motifs for both Cbf1 and Met31/32 (26.Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellor J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The sulfate assimilation pathway is turned off upon exposure to methionine, which triggers the ubiquination of Met4 (32.Kaiser P. Flick K. Wittenberg C. Reed S.I. Cell. 2000; 102: 303-314Abstract Full Text Full Text PDF PubMed Scopus (234) Google Scholar, 33.Rouillon A. Barbey R. Patton E.E. Tyers M. Thomas D. EMBO J. 2000; 19: 282-294Crossref PubMed Scopus (140) Google Scholar). The nature of this regulation is complex because ubiquitination inhibits Met4 transcriptional activity through altered promoter recruitment or through Met4 degradation depending upon the media composition and environment of the individual promoters (34.Kuras L. Rouillon A. Lee T. Barbey R. Tyers M. Thomas D. Mol. Cell. 2002; 10: 69-80Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). In this work we investigated how GSH biosynthesis is regulated in response to depletion of the GSH pool. We have previously determined that there is Met4-dependant induction of GSH1 expression after GSH depletion (35.Wheeler G.L. Quinn K.A. Perrone G. Dawes I.W. Grant C.M. Mol. Microbiol. 2002; 46: 545-556Crossref PubMed Scopus (31) Google Scholar). Here, we identify a role for the stress-responsive transcription factor Yap1 in this response and propose a mechanism in which these two transcription factors combine to regulate GSH1 expression. In addition, we demonstrate that the regulation of GSH1 expression by this mechanism is important in defense against xenobiotics, which deplete cellular GSH concentrations. Yeast Strains, Growth Conditions, and Plasmids—The S. cerevisiae strains used in this study are derivatives of CY4 (MATa ura3–52 leu2–3 leu2–112 trp1–1 his3–11 can1–100) (1.Grant C.M. MacIver F.H. Dawes I.W. Curr. Genet. 1996; 29: 511-515Crossref PubMed Scopus (271) Google Scholar). Strain CY197, which is deleted for GSH1, has been described previously (13.Lee J.-C. Straffon M.J. Jang T.-Y. Grant C.M. Dawes I.W. FEMS Yeast Res. 2000; 1: 57-65Google Scholar) as have the yap1 mutant (36.Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Molec. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (210) Google Scholar) and the met4 and gsh1 met4 mutants (35.Wheeler G.L. Quinn K.A. Perrone G. Dawes I.W. Grant C.M. Mol. Microbiol. 2002; 46: 545-556Crossref PubMed Scopus (31) Google Scholar). Strain CY813, which is deleted for CBF1, was made by back-crossing CY4 with EUROSCARF strain Y16858 (MATα his3D1 leu2D0 lys2D0 ura3D0 cbf1::kanMX4). These strains were used to construct the gsh1 yap1, gsh1 cbf1, met4 cbf1, yap1 cbf1, gsh1 met4 cbf1, and gsh1 yap1 cbf1 mutants using standard yeast genetic methods. Strains were grown in rich YEPD medium (2% w/v glucose, 2% w/v Bacto-peptone, 1% w/v yeast extract) or minimal SD media (0.17% w/v yeast nitrogen base without amino acids, 5% w/v ammonium sulfate, 2% w/v glucose) supplemented with appropriate amino acids and bases (2 mm leucine, 0.3 mm histidine, 0.4 mm tryptophan, 1 mm lysine, 0.15 mm adenine, and 0.2 mm uracil). Media were solidified by the addition of 2% (w/v) agar. Where appropriate, media were further supplemented by a non-repressive concentration of methionine (0.05 mm) to overcome the methionine auxotrophy of cbf1 and/or met4 mutants. Sensitivity to 1-chloro-2,4-dintrobenzene (CDNB) was determined by growing cells to stationary phase (48 h growth) and spotting onto agar plates. The plasmids used in this study containing the GSH1::lacZ (pyDJ73 and pyDJ76 (26.Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellor J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar)) and YRE::lacZ (15.Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar) reporter constructs have been described previously. Determination of Glutathione and Thioredoxin Redox States—Concentrations of free (GSH and GSSG) and protein-bound (GSSP) glutathione were determined as described previously (37.Grant C.M. Perrone G. Dawes I.W. Biochem. Biophys. Res. Commun. 1998; 253: 893-898Crossref PubMed Scopus (155) Google Scholar). The redox state of thioredoxins was measured by covalent modification with the thiolreactive probe 4-acetamido-4′maleimidyldystilbene-2,2′-disulfonic acid (Molecular Probes) as described previously (38.Frand A.R. Kaiser C.A. Mol. Cell. 1999; 4: 469-477Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar). Western Blot Analysis—Protein extracts were subjected to electrophoresis on 18% SDS-PAGE minigels and electroblotted onto a poly(vinylidene difluoride) membrane (Amersham Biosciences). Blots were incubated in anti-thioredoxin antibody (1:1000 dilution) as described previously (39.Trotter E.W. Grant C.M. EMBO Rep. 2003; 4: 184-189Crossref PubMed Scopus (108) Google Scholar). Bound antibody was visualized by chemiluminescence (ECL, Amersham Biosciences) after incubation of the blot in donkey anti-rabbit immunoglobulin-horseradish peroxidase conjugate (Santa Cruz, CA). β-Galactosidase Assays—For the determination of β-galactosidase activity, transformants were assayed essentially as described previously (40.Rose M. Botstein D. Methods Enzymol. 1983; 101: 167-180Crossref PubMed Scopus (271) Google Scholar). Activity is expressed as nmol of o-nitrophenyl-β-d-galacto-pyranoside hydrolyzed/min/μg of total protein (units). All β-galactosidase experiments were repeated at least twice, and a representative plot is shown. Values shown are the means of at least two independent determinations. Error bars denote S.E. Northern Blot Analysis—Yeast cells were grown to mid-exponential phase (A600 = 0.6–0.7) in minimal media in the presence of 1 mm methionine or 1 mm GSH where appropriate. Total RNA was prepared by Trizol extraction according to the manufacturer's specifications (Invitrogen). RNA (50 μg) was electrophoresed on 1.2% agarose-formaldehyde gels, blotted overnight onto a Nylon+ membrane, and hybridized with 32P-labeled DNA specific probes. DNA probes were synthesized by PCR using the following oligonucleotides: MET3 (MET3–1, 5′-GGGTCTCTCTCTGTCGTAACAGTTG-3′; MET3–2, 5′-TTGAGATGGGAGCATTTTATGACGA-3′); MET16 (MET16–1, 5′-CAAAGGTATCAACCCATAGCAACTC-3′; MET16–2, 5′-CGTACAGCGCGA ATTCTCCGCCAGC-3′); MET25 (MET25–1, 5′-CAATTCTATTACCCCCATCCATACA-3′; MET25–2, 5′-TAATTTTACAAC CATTACGCACAC-3′). Probes were labeled with the Megaprime kit (Amersham Biosciences). ACT1 was used as a control for RNA loading. Microarray Hybridizations and Data Analysis—Yeast cells were grown in triplicate to mid-exponential phase in minimal SD media. Growth conditions for the gsh1 mutant were predetermined to allow maximal GSH depletion in mid-exponential phase without a decrease in growth rate. Total depletion of the GSH pool in the gsh1 mutant leads to a cell cycle arrest. To examine the transcriptome in GSH-depleted cells that were still growing normally, cultures of the gsh1 mutant were inoculated with the lowest volume of a GSH-containing stationary phase culture that still allowed a normal growth rate to an A600 of 0.5–0.6. Preparation of RNA, probes, and hybridization to whole yeast genome microarrays (YG-S98, Affymetrix) was performed as described on the Consortium for Functional Genomics of Microbial Eukaryotes (COGEME) web site (www.cogeme.man.ac.uk). Data acquisition was performed using Affymetrix Microarray Suite Version 5.0 software and analyzed using dChip v1.1 software (41.Li C. Wong W.H. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 31-36Crossref PubMed Scopus (2691) Google Scholar). The mean expression values from three independently grown yeast cultures were used for comparative analysis. Gene expression was deemed to be significantly different between strains if it fulfilled the following criteria; 1) the fold change of gene expression was greater than 2 at the 90% confidence level, 2) the mean expression values are significantly different using an unpaired t test (p < 0.05), 3) the mean expression values differ by greater than 50, 4) the gene is called present in greater than 60% of the arrays. For the comparison to other microarray data sets, data from the YAP1 overexpression and trr1 mutant analyses were downloaded from Stanford Genomic Resources web site (genome-www.stanford.edu/yeast_stress and genome-www.stanford.edu/trr1, respectively) (19.Carmel-Hare O. Stearman R. Gasch A.P. Botstein D. Brown P.O. Storz G. Mol. Microbiol. 2001; 39: 595-605Crossref PubMed Scopus (93) Google Scholar, 20.Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3680) Google Scholar). Genes exhibiting a log2 expression ratio higher than 1.0 were compared with the data set obtained in the gsh1 mutant. The identification of prospective regulatory sequences within promoters was performed by RSAT (Regulatory Sequence Analysis Tools, rsat.ulb.ac.be/rsat). The Regulation of the Sulfate Assimilation Pathway Is Not Affected by Cellular GSH Concentrations—The regulation of metabolic pathways is essential for cell adaptation to differing environmental conditions. In this paper we have investigated the mechanisms regulating the biosynthesis of the key antioxidant, GSH. Our previous work indicated that expression of GSH1 is strongly induced in response to GSH depletion and that this response is dependent on the presence of the Met4 transcription factor (35.Wheeler G.L. Quinn K.A. Perrone G. Dawes I.W. Grant C.M. Mol. Microbiol. 2002; 46: 545-556Crossref PubMed Scopus (31) Google Scholar). To understand the mechanisms regulating GSH1 expression, we were particularly interested in the transcriptional responses of other Met4-dependent genes in response to GSH depletion. We, therefore, analyzed the global transcriptional response to GSH depletion in a gsh1 mutant. Microarray analysis revealed that there is a significant effect on the transcriptome, with 151 open reading frames significantly up-regulated and 38 down-regulated. Here we will only refer to the findings that are relevant to the regulation of GSH1 expression, since a detailed analysis of these data is beyond the scope of this paper and will be described elsewhere. 2G. L. Wheeler and C. M. Grant, manuscript in preparation. The microarray analysis of the gsh1 mutant indicates that lower cellular GSH concentrations do not lead to a general induction of the sulfate assimilation pathway. There is no significant effect on the expression of genes involved in sulfur metabolism other than a 3.3-fold increase in STR3 transcripts. The STR3 gene encodes cystathionine β-lyase, which catalyzes the conversion of cysteine to cystathionine and would, thus, prevent a toxic accumulation of cysteine in the gsh1 mutant (42.Hansen J. Johannesen P.F. Mol. Gen. Genet. 2000; 263: 535-542Crossref PubMed Scopus (97) Google Scholar). To confirm the effect of GSH depletion on the regulation of the sulfate assimilation pathway, we analyzed the transcriptional response of MET3, MET16, and MET25 using Northern blot analysis (Fig. 1A). These genes have previously been shown to be controlled by the Met4 transcriptional activator protein (27.Thomas D. Surdin-Kerjan Y. Microbiol. Mol. Biol. Rev. 1997; 61: 503-532Crossref PubMed Scopus (524) Google Scholar). However, none of these genes was induced by GSH depletion in the gsh1 mutant nor were they repressed by the exogenous addition of 1 mm GSH. This is in strong contrast with the induction of GSH1 expression observed after GSH depletion (Fig. 1B). The addition of 1 mm methionine severely repressed the expression of all three sulfate assimilation genes and also repressed the GSH1 induction observed in the gsh1 mutant. Thus, although the expression of the GSH1 gene is regulated in response to GSH availability, the expression of other known Met4 target genes is unaffected under these conditions. This indicates that Met4 acts to specifically up-regulate GSH biosynthesis in response to GSH depletion. We, therefore, examined the microarray data generated from the gsh1 mutant for evidence that other transcription factors may contribute to this specificity. GSH Depletion Activates Yap1-regulated Genes—Examination of the microarray data revealed that a number of genes that are regulated by the Yap1 transcriptional activator protein are up-regulated in the gsh1 mutant (Table I). For example, mutant strains of yeast lacking thioredoxin reductase (trr1) exhibit a constitutive Yap1 response, indicating that the thioredoxin system is integral for the redox regulation of this transcription factor (19.Carmel-Hare O. Stearman R. Gasch A.P. Botstein D. Brown P.O. Storz G. Mol. Microbiol. 2001; 39: 595-605Crossref PubMed Scopus (93) Google Scholar). Comparison of the whole-genome expression data identified 16 genes in the gsh1 mutant from a set of 35 genes in the trr1 mutant that are strongly induced (greater than 2-fold) in both mutants. The group of genes up-regulated in a gsh1 mutant also contains 25 genes that have previously been shown to be up-regulated (from a set of 118 genes) after the overexpression of plasmid-borne YAP1 in wild-type cells (20.Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3680) Google Scholar). Furthermore, analysis of the promoter regions of the genes up-regulated in the gsh1 mutant indicates that a significant proportion (43 of 151) contain a putative Yap1 binding site (TTAC/GTAA or TGACTAA).Table IComparison of gene expression profiles in a wild-type and gsh1 mutant identifies many genes that are regulated by Yap1Gene nameAccession no.FunctionFold change2-μm YAP1aElevated expression in cells containing YAP1 on a 2 μm plasmid (20).trr1bElevated expression in a trr1 mutant (19).YREcContain a putative YRE in their promoter, either the TTAG/CTAA motifidentified in TRX2 (21) or the TTAGTCA motif from GSH1 (15).ARN1YHL040CSiderophore transporter27.52+HSP26YBR072WHeat shock protein 2625.42+YOL153C17.62+YGR043C14.05+AAD6YFL056CPutative aryl-alcohol dehydrogenase12.05+++OYE3YPL171CNAD(P)H dehydrogenase11.99++GTT2YLL060CGlutathione transferase10.84+++ECM4YKR076WExtracellular mutant9.91+++DCS2YOR173W7.11+GPM2YDL021WPhosphoglycerate mutase6.62+MRS4YKR052CMitochondrial carrier protein6.5+YLL055W6.42+HXK1YFR053CHexokinase I6.4+RTN2YDL204WMember of the RTNLA subfamily6.28+YBR116C6.28+YBR116C6.28+AAD16YFL057CPutative aryl-alcohol dehydrogenase5.89+YKL071W5.77+++NMA2YGR010WNicotinamide adenylyltransferase5.68+YML131W5.52+++YHR087W5.43+LAP4YKL103CVacuolar aminopeptidase5.2+TMT1YER175CTrans-aconitate methyltransferase 14.94+ALD4YOR374WAldehyde dehydrogenase4.55+YOL048C4.53+YIL167W4.47+YMR090W4.43++YOL164W4.06+YLR108C3.98++ATR1YML116WAminotriazole resistance3.9++ISU2YOR226CIron-sulfur cluster assembly3.84+++FRE1YLR214WFerric reductase3.57++YLR046C3.47+YDL146W3.46+AAD4YDL243CPutative aryl-alcohol dehydrogenase3.43++VHT1YGR065CVitamin H transporter3.3+GPH1YPR160WGlycogen phosphorylase3.23+GRE2YOL151WInduced by osmotic stress3.12++COT1YOR316CInvolved in cobalt accumulation3.07+DIT1YDR403WFirst enzyme in dityrosine synthesis3.07+YDL124W3.05++YLR460C2.97+++SDL1YIL168Wl-Serine dehydratase2.89++TSL1YML100WTrehalose-6-phosphate synthase2.85+YCR102C2.76++HAL1YPR005CInvolved in halotolerance2.74+YNL134C2.71+++GCY1YOR120WGalactose-induced transcript2.7+MRL1YPR079WMannose 6-phosphate receptor-like2.64+YGR154C2.62+YHR199C2.6+YDR533C2.59+AMS1YGL156WVacuolar α-mannosidase2.54+YDL199C2.52+CVT19YOL082WRequired for protein-vacuolar largeting2.49+FLR1YBR008CMajor facilitator transporter2.41++PRX1YBL064CMitochondrial thioredoxin peroxidase2.26+a Elevated expression in cells containing YAP1 on a 2 μm plasmid (20.Gasch A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3680) Google Scholar).b Elevated expression in a trr1 mutant (19.Carmel-Hare O. Stearman R. Gasch A.P. Botstein D. Brown P.O. Storz G. Mol. Microbiol. 2001; 39: 595-605Crossref PubMed Scopus (93) Google Scholar).c Contain a putative YRE in their promoter, either the TTAG/CTAA motifidentified in TRX2 (21.Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar) or the TTAGTCA motif from GSH1 (15.Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar). Open table in a new tab To determine whether Yap1 oxidation in response to GSH depletion was due to an altered redox status, we performed microarray analysis on the glr1 mutant. Mutant strains lacking GLR1, encoding glutathione reductase, are unable to recycle oxidized GSSG back to GSH, and consequently, the redox status of the GSH pool is much more oxidized (36.Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Molec. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (210) Google Scholar). However, oxidation of the GSH pool does not result in Yap1 activation because no target genes were up-regulated in the glr1 mutant (data not shown). Given the elevated expression of Yap1 targets genes in the gsh1 mutant, we confirmed that Yap1 is activated in a gsh1 mutant using a YRE::lacZ reporter construct. This construct contains three YRE from the promoter of GSH1 fused upstream of the β-galactosidase reporter gene (15.Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar). The gsh1 mutant strain exhibited a 5-fold increase in YRE::lacZ expression compared with the wild-type control (data not shown). These data indicate that GSH depletion leads to an activation of the Yap1 transcription factor, which has previously been shown to regulate GSH1 expression in response to oxidative stress (43.Stephen D.W.S. Rivers S.L. Jamieson D.J. Mol." @default.
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• W2081418938 title "Coupling of the Transcriptional Regulation of Glutathione Biosynthesis to the Availability of Glutathione and Methionine via the Met4 and Yap1 Transcription Factors" @default.
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• W2081418938 doi "https://doi.org/10.1074/jbc.m310156200" @default.
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