Yap1 and Skn7 Control Two Specialized Oxidative Stress Response Regulons in Yeast
1999; Elsevier BV; Volume: 274; Issue: 23 Linguagem: Inglês
10.1074/jbc.274.23.16040
ISSN1083-351X
AutoresJaekwon Lee, Christian Godon, Gilles Lagniel, D. L. Spector, Jérôme Garin, Jean Labarre, Michel B. Tolédano,
Tópico(s)Genomics, phytochemicals, and oxidative stress
ResumoYap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals. Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response. The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis. Yap1 controls a large oxidative stress response regulon of at least 32 proteins. Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2. Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro. The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets. Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH. Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance. Yap1 and Skn7 are two yeast transcriptional regulators that co-operate to activate thioredoxin (TRX2) and thioredoxin reductase (TRR1) in response to redox stress signals. Although they are both important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 has a negative effect upon this response. The respective roles of Yap1 and Skn7 in the induction of defense genes by H2O2 were analyzed by two-dimensional gel electrophoresis. Yap1 controls a large oxidative stress response regulon of at least 32 proteins. Fifteen of these proteins also require the presence of Skn7 for their induction by H2O2. Although about half of the Yap1 target genes do not contain a consensus Yap1 recognition motif, the control of one such gene, TSA1, involves the binding of Yap1 and Skn7 to its promoter in vitro. The co-operative control of the oxidative stress response by Yap1 and Skn7 delineates two gene subsets. Remarkably, these two gene subsets separate antioxidant scavenging enzymes from the metabolic pathways regenerating the main cellular reducing power, glutathione and NADPH. Such a specialization may explain, at least in part, the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance. Aerobic organisms have to maintain a reduced cellular redox environment in the face of the prooxidative conditions of aerobic life. The incomplete reduction of oxygen to water during respiration leads to the formation of redox-active oxygen intermediates such as the superoxide anion radical (O⨪2), hydrogen peroxide (H2O2), and the hydroxyl radical (for review see Refs. 1Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Clarendon Press, Oxford1989Google Scholar, 2Storz G. Tartaglia L.A. Farr S.B. Ames B.N. Trends Genet. 1990; 6: 363-368Abstract Full Text PDF PubMed Scopus (267) Google Scholar, 3Halliwell B. Nutr. Rev. 1994; 52: 253-265Crossref PubMed Scopus (858) Google Scholar). Redox-active oxygen intermediates are also produced during the β-oxidation of fatty acids by exposure to radiation, light, metals, and redox active drugs. Redox-active oxygen intermediates perturbate the cell redox status and when present in high levels can induce toxic damage to lipids, proteins, and DNA, eventually leading to cell death. Living organisms constantly sense and adapt to such redox perturbations. The exposure of the yeast Saccharomyces cerevisiae to low doses of H2O2 or O⨪2-generating drugs switches on within minutes a resistance to toxic doses of these oxidants (4Collinson L.P. Dawes I.W. J. Gen. Microbiol. 1992; 138: 329-335Crossref PubMed Scopus (175) Google Scholar, 5Jamieson D.J. J. Bacteriol. 1992; 174: 6678-6681Crossref PubMed Google Scholar, 6Flattery-O'Brien J. Collinson L.P. Dawes I.W. J. Gen. Microbiol. 1993; 139: 501-507Crossref PubMed Scopus (134) Google Scholar). The adaptive response to H2O2 involves a change in the expression of at least 167 proteins (7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Such a rapid and widespread genomic response suggests the existence of specific control pathways. In S. cerevisiae, the transcription factors Yap1 (8Schnell N. Krems B. Entian K.-D. Curr. Genet. 1992; 21: 269-273Crossref PubMed Scopus (163) Google Scholar, 9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar) and Skn7 (10Krems B. Charizanis C. Entian K.-D. Curr. Genet. 1996; 29: 327-334Crossref PubMed Scopus (126) Google Scholar, 11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar) have been implicated in a cellular pathway that controls the oxidative stress response. Yap1 is a bZIP DNA-binding protein of the AP-1 family (12Moye-Rowley W.S. Harshma K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (244) Google Scholar) that binds the sequence T(T/G)ACTAA termed the Yap1 response element (YRE) (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 13Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar,14Fernandes L. Rodrigues-Pousada C. Struhl K. Mol. Cell. Biol. 1997; 17: 6982-6993Crossref PubMed Scopus (261) Google Scholar). 1The abbreviations used are: YRE, Yap1 response element; t-BOOH, tert-butyl hydroperoxide; kb, kilobase; HA, hemagglutinin; bp, base pair(s); EMSA, electrophoretic mobility shift assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; GSH, glutathione. 1The abbreviations used are: YRE, Yap1 response element; t-BOOH, tert-butyl hydroperoxide; kb, kilobase; HA, hemagglutinin; bp, base pair(s); EMSA, electrophoretic mobility shift assay; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; mAb, monoclonal antibody; GSH, glutathione. Skn7 contains a receiver domain found in the family of two-component signal transduction systems of prokaryotes and a domain similar to the DNA-binding domain of heat shock factor (Hsf1) (15Brown J.L. Bussey H. Stewart R.C. EMBO J. 1994; 13: 5186-5194Crossref PubMed Scopus (131) Google Scholar, 16Morgan B.A. Bouquin N. Merril G.F. Johnston L.H. EMBO J. 1995; 14: 5679-5689Crossref PubMed Scopus (87) Google Scholar). Skn7 is also capable of specific DNA binding, but its cognate DNA sequence has not been identified precisely (11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar). Strains inactivated for either one of these regulators are hypersensitive to killing by H2O2 (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 10Krems B. Charizanis C. Entian K.-D. Curr. Genet. 1996; 29: 327-334Crossref PubMed Scopus (126) Google Scholar, 11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar, 17Krems B. Charizanis C. Entian K.-D. Curr. Genet. 1995; 27: 427-434Crossref PubMed Scopus (84) Google Scholar). This oxidative stress phenotype is related to the role of Yap1 and Skn7 in controlling the induction of several defense genes by H2O2. Yap1 controls the expression of GSH1 (γ-glutamylcysteine synthetase) (13Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar), TRX2 (thioredoxin) (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar), GLR1(glutathione reductase) (18Grant C.M. Collinson L.P. Roe J.H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (213) Google Scholar), and YCF1 (yeast cadmium factor, a glutathione S-conjugate pump)(19). More recently, Morgan showed that the induction of TRX2 and TRR1(thioredoxin reductase) by H2O2 requires a co-operation between Yap1 and Skn7 (11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar). YAP1 function can be activated by H2O2, diamide, and diethylmaleate (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 20Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1993; 242: 250-256Crossref Scopus (80) Google Scholar), and this activation is attributed to a redox stress-imposed nuclear redistribution of the protein involving the nuclear export receptor Crm1 (Xpo1) (21Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (346) Google Scholar, 22Kuge S. Toda T. Lizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar). Yap1 is also important in cadmium tolerance because deletion of its gene results in a cadmium-hypersensitive phenotype (19Wemmie J.A. Szczypka M.S. Thiele D.J. Moye-Rowley W.S. J. Biol. Chem. 1994; 269: 32592-32597Abstract Full Text PDF PubMed Google Scholar, 20Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1993; 242: 250-256Crossref Scopus (80) Google Scholar, 23Wu A. Wemmie J.A. Edgington N.P. Goebl M. Guevara J.L. Moye-Rowley W.S. J. Biol. Chem. 1993; 268: 18850-18858Abstract Full Text PDF PubMed Google Scholar). This function is attributed to the control by Yap1 of GSH1 (13Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar) and of YCF1 (19Wemmie J.A. Szczypka M.S. Thiele D.J. Moye-Rowley W.S. J. Biol. Chem. 1994; 269: 32592-32597Abstract Full Text PDF PubMed Google Scholar). In addition to its involvement in the oxidative stress response, Skn7 is implicated in the control of cell wall biosynthesis, cell cycle, and the osmotic stress response. Overexpression of SKN7 can suppress the cell wall assembly mutation kre9 (24Brown L.L. Bussey H. J. Bacteriol. 1993; 175: 6908-6915Crossref PubMed Google Scholar) and the growth defect associated with apkc1 null deletion (15Brown J.L. Bussey H. Stewart R.C. EMBO J. 1994; 13: 5186-5194Crossref PubMed Scopus (131) Google Scholar). Overexpression of SKN7also suppresses the lethality associated with loss of the G1 transcription factors SBF and MBF (16Morgan B.A. Bouquin N. Merril G.F. Johnston L.H. EMBO J. 1995; 14: 5679-5689Crossref PubMed Scopus (87) Google Scholar). Skn7 is modulated by the Sln1-Ypd1 osmosensor and contributes to regulation of the HOG osmo-stress pathway (25Ketela T. Brown J.L. Stewart R.C. Bussey H. Mol. Gen. Genet. 1998; 259: 372-378Crossref PubMed Scopus (66) Google Scholar). The involvement of Skn7 into such diverse pathways raises the question of the possible connection between these pathways. We sought to further analyze the co-operative functions of Yap1 and Skn7 in the control of the oxidative and cadmium stress responses and found that these two regulators do not always act together in these stress responses. Although both Yap1 and Skn7 are important for resistance to H2O2, only Yap1 is important for cadmium resistance, whereas Skn7 is not only dispensable but appears to negatively affect this response. The role of Yap1 and Skn7 in the induction of defense genes by H2O2 was further analyzed by two-dimensional gel electrophoresis. The data presented here identify, within a large Yap1 stress response regulon, a gene subset that also requires Skn7 for its control. This partition of the Yap1 regulon correlates with two distinct classes of defense genes. Such a specialization within the oxidative stress response may explain the dissociated function of Yap1 and Skn7 in H2O2 and cadmium resistance. All studies were performed with the wild type strain YPH98 (26Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) (MATa ura3-52 lys2-801amber ade2-101ochre trp1-Δ1leu2-Δ1) and its isogenic derivatives yap1Δ-1 (yap1::LEU2), skn7Δ-1 (skn7::TRP1), yap1Δ-1, andskn7Δ-1 (yap1::LEU2, skn7::TRP1). The composition of synthetic complete, rich broth, and glucose selective media are described elsewhere (27Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1990Google Scholar). Strains were transformed by electroporation as described (28Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 182-185Crossref PubMed Scopus (673) Google Scholar). H2O2, t-BOOH, and cadmium sulfate were purchased from Sigma. The 12CA5 anti-HA and the 9E10 anti-Myc monoclonal antibody were purchased from Roche Molecular Biochemicals. Standard protocols and buffers were used (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Gene disruptions were performed by the one-step gene disruption technique (28Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 182-185Crossref PubMed Scopus (673) Google Scholar). pSKN7 is a YEp351 plasmid carrying a 3.5-kb genomic fragment containing the entireSKN7 gene (24Brown L.L. Bussey H. J. Bacteriol. 1993; 175: 6908-6915Crossref PubMed Google Scholar). pSKN7-HA is a tagged version ofSKN7 in which the HA epitope was introduced at theSKN7 PstI site 28 codons downstream of the ATG (15Brown J.L. Bussey H. Stewart R.C. EMBO J. 1994; 13: 5186-5194Crossref PubMed Scopus (131) Google Scholar). Theskn7::TRP1 construct used to createskn7Δ-1 lacks an internal 1.5-kbPvuII-HincII fragment of the wild typeSKN7 gene, which has been replaced by the TRP1gene (24Brown L.L. Bussey H. J. Bacteriol. 1993; 175: 6908-6915Crossref PubMed Google Scholar). The yap1::LEU2 construct used to createyap1Δ-1 was prepared by removing the YAP1coding sequence from the BamHI site (+186) to theKpnI site (+1650) relative to the ATG and replacing it with the LEU2 gene. pYAP1 was constructed by subcloning a 2.5-kb EcoRI DNA fragment carrying the entireYAP1 gene (12Moye-Rowley W.S. Harshma K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (244) Google Scholar) into pRS426. pMF6(X-H) is a PUC18 plasmid lacking the AccI polylinker site and carrying the same 2.5-kb EcoRI YAP1 fragment. To generate pYAP1-9Myc, a polymerase chain reaction-amplified 390-bp sequence encoding 9 Myc epitopes was first introduced in vector pMF6(X-H) into the YAP1 AccI site located two codons downstream of the ATG; The YAP1-9Myc fusion was then subcloned at the EcoRI site of pRS426 to generate pYAP1-9Myc. The functionality of the YAP1-9Myc construct was evaluated by its ability to rescue the H2O2-hypersensitive phenotype ofyap1Δ-1. The TSA1-lacZ gene fusions were constructed as follows: a 4.5-kb DNA fragment spanning the entirelacZ coding sequence from the BamHI site, two codons downstream of the ATG to a KpnI site approximately 1 kb from the stop codon, was subcloned into pRS424 to generate pMT1. ANaeII to KpnI 328-bp plasmid fragment containing the α peptide lacZ sequence was then removed from pMT1 to generate pMT11. TSA1 promoter fragments corresponding to −1000, −837, −403, −243, and −204 to +1 relative to the ATG were amplified by polymerase chain reaction from genomic DNA and subcloned between the XhoI and BamHI sites of pMT11. Patch assays were performed as follows: 10-μl aliquots containing approximately 2 × 103cells of an overnight culture were spotted on rich broth or synthetic complete solid plates containing H2O2, t-BOOH, or cadmium sulfate at the indicated concentration. Plates were monitored after 3–6 days incubation at 30 °C. Yeast cells from overnight culture were diluted to an A 600 of 0.01 in synthetic complete medium and incubated with shaking at 30 °C until they reached an A 600 of 0.3. The cells were then aliquoted and incubated in the absence or in the presence of H2O2 (0.2 mm) for 20 min. Total RNA was prepared by the hot phenol method (30Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1152) Google Scholar). For each condition tested, 20-μg RNA samples were loaded per lane on an agarose gel containing formaldehyde, separated by electrophoresis, transferred to a nylon membrane (Bio-Rad), and hybridized with the indicated random primed (Roche Molecular Biochemicals) 32P-labeled DNA probe. Hybridization of each blot with a small nuclear RNAU3 (SNR17A) specific 32P-labeled DNA probe served as a RNA loading control. Pre-hybridization, hybridization, and washes were carried out as described (29Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Hybridized membranes were exposed for autoradiography. Yeast crude extracts used in EMSAs were prepared as follows. Cells were grown to an A 600 of 0.3 and were left untreated or were treated with H2O2(0.6 mm) for 5 min and harvested. Extracts were prepared by glass bead disruption as described previously (7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar), except for the use of a modified breakage buffer containing 200 mm Tris-HCl, pH 8.0, 10 mm MgCl2, 10% glycerol, complete mixture inhibitor (Roche Molecular Biochemicals). The DNA binding reactions were carried out in 1× TC buffer (25 mmTris-HCl, pH 7.5, 50 mm NaCl, 2 mm EDTA, pH 8, 5 mm MgCl2, 0.1% (v/v) CHAPS, 10% (v/v) glycerol) with 20 μg of crude yeast extracts, 5–15 fmol [32P]ATP-labeled probe, 1 μg of poly(dI-dC) (Amersham Pharmacia Biotech) in a total volume of 20 μl. The binding reaction was incubated for 5 min at room temperature and subjected to electrophoresis in a 6% polyacrylamide gel (acrylamide/N,N′-methylenebisacrylamide weight ratio, 27.5:1) in 45 mm Tris/45 mm boric acid/1 mm EDTA for 1 h at 200 V. Measurement of the H2O2 response was carried out as described previously (7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). Basically, mid-log cells (A 600 = 0.3) were exposed or not to H2O2 (0.2 mm) for 15 min, pulse labeled with [35S]methionine for another 15 min, and harvested. An equal aliquot of 3H-labeled cells was mixed to the35S-labeled cells and served as an internal protein concentration standard for each two-dimensional gel spot. Cell mixtures were extracted and subjected to comparative two-dimensional gel analysis. Accordingly, the previously identified 71 proteins of the H2O2 stimulon were analyzed in wild type and isogenic yap1Δ-1, and skn7Δ-1. Uninduced and H2O2-induced synthesis rate indexes (ratio of individual [35S]/[3H] spot ratios to the Act1p [35S]/[3H] spot ratio) were calculated in each strain for the 71 H2O2-stimulated proteins. Specific DNA sequences were searched within 1 kilobase from the initiation codon of identified genes with the Saccharomyces Genome Data Base package. Searches were done with the following query sequences: T(T/G)ACTAA, which corresponds to the known Yap1 recognition sequences (YRE) (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 13Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 14Fernandes L. Rodrigues-Pousada C. Struhl K. Mol. Cell. Biol. 1997; 17: 6982-6993Crossref PubMed Scopus (261) Google Scholar), and CAGCAGCCGAAAAGA, which correspond to a 23-bp TRX2 promoter sequence capable of binding Skn7in vitro (11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar). Although strains carrying deletions ofYAP1 (yap1Δ-1)or SKN7(skn7Δ-1) are both hypersensitive to killing by H2O2 (9Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 10Krems B. Charizanis C. Entian K.-D. Curr. Genet. 1996; 29: 327-334Crossref PubMed Scopus (126) Google Scholar, 11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar), skn7Δ-1 can tolerate higher concentrations of H2O2 (Fig.1 A). The same phenotypic profile is seen in the tolerance of t-BOOH (Fig. 1 B). Another functional distinction is the ability of YAP1overexpression to partially rescue the skn7Δ-1 peroxide-hypersensitive phenotype, whereas SKN7overexpression has no effect in yap1Δ-1 (Fig.1 B). Analysis of the cadmium tolerance further demonstrates the distinctive roles of Yap1 and Skn7 in controlling stress responses (Fig. 1 C). Whereas yap1Δ-1 is hypersensitive to cadmium (19Wemmie J.A. Szczypka M.S. Thiele D.J. Moye-Rowley W.S. J. Biol. Chem. 1994; 269: 32592-32597Abstract Full Text PDF PubMed Google Scholar, 20Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1993; 242: 250-256Crossref Scopus (80) Google Scholar, 23Wu A. Wemmie J.A. Edgington N.P. Goebl M. Guevara J.L. Moye-Rowley W.S. J. Biol. Chem. 1993; 268: 18850-18858Abstract Full Text PDF PubMed Google Scholar), skn7Δ-1 is significantly more resistant than the wild type strain to this toxic metal. The double delete yap1Δ-1,skn7Δ-1 is hypersensitive to cadmium, suggesting that the skn7Δ-1 cadmium hyperresistance phenotype is dependent upon YAP1. Therefore, Yap1 and Skn7 have distinctive roles in peroxide stress tolerance and opposite effects upon cadmium tolerance, with Yap1 acting positively and Skn7 acting negatively. A negative role for Skn7 is also suggested by the decreased cadmium tolerance observed upon overexpression ofSKN7 (not shown). These results prompted us to evaluate the role of Yap1 and Skn7 in the control of known H2O2-inducible target genes. Yap1 and Skn7 co-operate to activate TRX2 and TRR1 in response to H2O2 (11Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (239) Google Scholar). We thus evaluated by Northern blot the respective roles of Yap1 and Skn7 in the control of three other known Yap1 targets, SSA1 (31Stephen D.W.S. Rivers S.L. Jamieson D.J. Mol. Microbiol. 1995; 16: 415-423Crossref PubMed Scopus (177) Google Scholar), GSH1 (13Wu A. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar), and GLR1 (18Grant C.M. Collinson L.P. Roe J.H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (213) Google Scholar), and of four other H2O2-inducible genes, TSA1 (TSA or peroxiredoxin), AHP1 (an alkyl hydroperoxide reductase) (32Lee J. Spector D. Godon C. Labarre J. Toledano M.B. J. Biol. Chem. 1999; 274: 4537-4544Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar), CCP1 (cytochrome c peroxidase), andHSP82 (7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar) (Fig. 2). The genes analyzed were all potently induced by H2O2 in wild type cells, and this induction was abolished inyap1Δ-1 (Fig. 2, A and B). Inskn7Δ-1 cells, the induction by H2O2 was also abolished for TSA1,CCP1, TRR1, HSP82, and SSA1and significantly diminished for TRX2 and AHP1(Fig. 2 A). In contrast, induction of GLR1 andGSH1 by H2O2 was actually stronger in skn7Δ-1 than in wild type cells (Fig. 2B). However, inyap1Δ-1,skn7Δ-1 double null cells, the induction of GSH1 by H2O2 was totally abolished, demonstrating that the H2O2-superinduced levels seen inskn7Δ-1 cells are dependent upon YAP1. Therefore, Yap1 and Skn7 co-operate in the control of several H2O2 target genes but have opposite effects in the control of other H2O2 target genes, with Yap1 acting positively and Skn7 acting negatively. These results may explain, at least in part, the opposite functions of Yap1 and Skn7 in cadmium tolerance. To further dissect the intricate functions of Yap1 and Skn7 in the control of the oxidative and metal stress responses, we searched for other target genes by comparative two-dimensional gel electrophoresis of total soluble yeast proteins. We recently identified with this method 71 proteins whose synthesis rate is significantly increased minutes after exposure to H2O2 (7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar). We sought to identify among these proteins those whose induction by H2O2 would be lost or significantly diminished in yap1Δ-1. Exponentially growing wild type andyap1Δ-1 cells were pulse-labeled after exposure to H2O2 for 15 min and then subjected to two-dimensional gel electrophoresis (Fig.3, A–C ). Uninduced and H2O2-induced synthesis rate indexes of the 71 H2O2 targets were calculated inyap1Δ-1 and divided by those of the wild type strain (yap1Δ-1/WT). 31 proteins with ayap1Δ-1/WT-induced synthesis rate index ratio equal to or below the value of 0.6 were considered as dependent upon Yap1 for their induction by H2O2. Their synthesis rate indexes are represented in Fig. 4. These proteins were sorted into functional classes (TableI). The Yap1 regulon includes most of the oxidant scavenging enzymes. These are, in addition to those mentioned above, cytosolic catalase (Ctt1p), copper/zinc and manganese superoxide dismutases (Sod1p and Sod2p), YDR453Cp, and YOL151Wp. YDR453Wp is an AhpC/TSA family member, and YOL151Wp is similar to plant NADPH isoflavonoid reductases shown to rescue the diamide hypersensitivity phenotype of a yap1 null strain (33Babiychuck E. Kushnir S. Belles-Boix E. Van Montagu M. Inzé D. J. Biol. Chem. 1995; 270: 26224-26231Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). The Yap1 regulon also includes several carbohydrate metabolism enzymes, a few heat shock proteins and proteases, amino acid metabolism enzymes, and other unclassified or unknown proteins.Figure 4Basal and H2O2-stimulated synthesis rate indexes of the proteins of the Yap1-controlled regulon. Histogram representation of uninduced (black bars) and H2O2-induced (white bars) synthesis rate indexes calculated in wild type (bars 1),yap1Δ-1 (bars 2), and skn7Δ-1 (bars 3) cells as described under “Materials and Methods.” For each protein spot, values were normalized to the wild type uninduced level that was arbitrarily given the value of 1. The names of proteins are indicated above the histograms.View Large Image Figure ViewerDownload (PPT)Table IIdentification of proteins dependent upon Yap1, Skn7, or both regulators for their induction by H2O2Gene nameH2O2 stimulation indexaStimulation indexes were reported in Ref. 7.yap1Δ-1/WTskn7Δ-1/WTYRE coordinatesProtein functionYap1 and Skn7-dependent proteinsAntioxidant defensesCCP16<0.10.07nonecytochrome c peroxidaseCTT114.70.300.49nonecatalase TSOD14.30.240.77143copper/zinc superoxide dismutaseSOD25.90.10<0.1266, 862manganese superoxide dismutaeTRR113.2<0.10.17186thioredoxin reductae/NADPH-dependentTRX211.5 15<0.1<0.10292, 467, 583similarity to Tsa1pAHP13.10.270.53494Alkyl hydroperoxide reductaseHeat shock proteinsSSA12.70.510.28noneheat shock proteinHSP82NDbND, not determined. These proteins were only analyzed by Northern blot.NDND767heat shock proteinHSP783.90.310.13nonemitochondrial proteaseAmino acid metabolismLYS201.80.410.17546, 688probable homocitrate synthaseYAP1-dependent proteinsAntioxidant defensesGLR12.10.211.0191glutathione reductaseGSH1NDNDND383γ-glutamyl cysteine synthaseCYS33.20.311.30235cystathionine γ lyaseYOL151W5.90.181.18nonesimilarity to plant dihydroflavonol-4-reductasesCarbohydrate Metabolism Enzymespentose phosphate pathwayTAL14.10.280.89nonetransaldolaseZWF120.440.80293glucose-6-phoshate dehydrogenaseGlycerol metabolismDAK11.90.620.7749similarity to dihydroxyacetone kinaseTrehalose synthesisPGM24.40.631.70nonephosphoglucomutaseTPS13.80.311.22nonetrehalose-6-phosphate synthaseProteasesCIM51.70.520.90noneproteasome subunitMPR13.00.421.0979519 S proteasome subunit (deubiquitinase)UBA11.60.391.01noneubiquitin-activating enzymeNot classifiedOYE32.00.101.25158, 205NADPH dehydrogenaseCDC481.60.480.98835ATPase familyPDI2.00.181.03noneProtein-disulfide isomeraseUnknown functionYMR318C2.10.211.43nonesimilarity to alcohol dehydrogenaseYNL134C2.60.211.48182, 289, 758weak similarity to alcohol dehydrogenaseYNL274C2.10.491.01nonesimilarity to α-ketoisocaproate reductaseYDR032C2.50.480.91259similarity to flavodoxinSkn7-dependent proteinsUnknown functionYBR025C2.40.910.33826similarity to the GTP-binding YCHF protein familyDNM12.11.050.36915dynamin-related proteina Stimulation indexes were reported in Ref. 7Godon C. Lagniel G. Lee J. Buhler J.-M. Kieffer S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. 1998; 273: 22480-22489Abstract Full Text Full Text PDF PubMed Scopus (498) Google Scholar.b ND, not determined. These proteins were only analyzed by Northern blot. Open table in a new tab Skn7 target genes were similarly identified by comparative two-dimensional gel electrophoresis (Fig. 3, A–C ). Thirteen proteins with a skn7Δ-1 to wild type (skn7Δ-1/WT)-induced synthesis rate index ratio equal to or below 0.6 were considered as dependent upon Skn7 for their induction by H2O2 (Table I). Their synthesis rates indexes are represented in Fig. 4. Most of these proteins were also identified as Yap1 target genes. However, two of them were not identified as Yap1 targets. Conversely, several Yap1 target genes were still normally induced or even superinduced in the skn7 null strain (Fig.
Referência(s)