Thioredoxin Deficiency Causes the Constitutive Activation of Yap1, an AP-1-like Transcription Factor in Saccharomyces cerevisiae
1999; Elsevier BV; Volume: 274; Issue: 40 Linguagem: Inglês
10.1074/jbc.274.40.28459
ISSN1083-351X
AutoresShingo Izawa, Keiko Maeda, Kei‐ichi Sugiyama, Jun’ichi Mano, Yoshiharu Inoue, Akira Kimura,
Tópico(s)Fungal and yeast genetics research
ResumoYap1 is a transcription factor that responds to oxidative stress in Saccharomyces cerevisiae. The activity of Yap1 is regulated at the level of its intracellular localization, and a cysteine-rich domain at the C terminus of Yap1 is involved in this regulation. We investigated the effects of redox-regulatory proteins, thioredoxin and glutaredoxin, on the regulation of Yap1, using the deficient mutants of these thiol-disulfide oxidoreductases. In the thioredoxin-deficient mutant (trx1Δ/trx2Δ), Yap1 was constitutively concentrated in the nucleus and the level of expression of the Yap1 target genes was high under normal conditions, while this was not the case for the glutaredoxin-deficient mutant (grx1Δ/grx2Δ). No distinct difference was observed in the levels of Yap1 protein between the wild type andtrx1Δ/trx2Δ. The constitutive activation of Yap1 in trxΔ/trx2Δ was observed under aerobic conditions but not under anaerobic conditions. These findings suggest that thioredoxin has negative effects on this regulation via the redox states. We also show the synthetic lethality betweenyap1Δ and trx1Δ/trx2Δ mutation, but theyap1Δ/grx1Δ/grx2Δ triple mutant was viable, suggesting a difference of the functions between thioredoxin and glutaredoxin and a genetic interaction between Yap1 and thioredoxin in vivo. Yap1 is a transcription factor that responds to oxidative stress in Saccharomyces cerevisiae. The activity of Yap1 is regulated at the level of its intracellular localization, and a cysteine-rich domain at the C terminus of Yap1 is involved in this regulation. We investigated the effects of redox-regulatory proteins, thioredoxin and glutaredoxin, on the regulation of Yap1, using the deficient mutants of these thiol-disulfide oxidoreductases. In the thioredoxin-deficient mutant (trx1Δ/trx2Δ), Yap1 was constitutively concentrated in the nucleus and the level of expression of the Yap1 target genes was high under normal conditions, while this was not the case for the glutaredoxin-deficient mutant (grx1Δ/grx2Δ). No distinct difference was observed in the levels of Yap1 protein between the wild type andtrx1Δ/trx2Δ. The constitutive activation of Yap1 in trxΔ/trx2Δ was observed under aerobic conditions but not under anaerobic conditions. These findings suggest that thioredoxin has negative effects on this regulation via the redox states. We also show the synthetic lethality betweenyap1Δ and trx1Δ/trx2Δ mutation, but theyap1Δ/grx1Δ/grx2Δ triple mutant was viable, suggesting a difference of the functions between thioredoxin and glutaredoxin and a genetic interaction between Yap1 and thioredoxin in vivo. Many aerobic organisms show adaptive responses to oxidative stress by increasing the levels of antioxidant enzymes. A portion of the adaptive response is regulated at the transcriptional level, and several transcription factors that regulate the expression of antioxidant genes have been reported. In Escherichia coli, OxyR, SoxR, and SoxS are key transcription factors for the adaptive response to oxidative stress. OxyR regulates the expression of genes encoding H2O2-inducible proteins, and SoxR and SoxS regulate the expression of genes encoding superoxide-inducible proteins (1Jamieson D.J. Storz G. Scandalios J.G. Oxidative Stress and The Molecular Biology of Antioxidant Defenses. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1997: 91-116Google Scholar). In mammals, two transcription factors, NF-κB and AP-1, have been strongly implicated in the oxidative stress response (2Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (999) Google Scholar). The activities of these transcription factors are reversibly controlled through redox states and modulated by thiol-disulfide oxidoreductases such as thioredoxin (TRX) 1The abbreviations used are:TRXthioredoxinGRXglutaredoxinGRglutathione reductaseGFPgreen fluorescent proteinCRDcysteine-rich domainNESnuclear export sequenceDCFH-DA2′,7′-dichlorofluorescin diacetatekbkilobase(s) and glutaredoxin (GRX) (2Nakamura H. Nakamura K. Yodoi J. Annu. Rev. Immunol. 1997; 15: 351-369Crossref PubMed Scopus (999) Google Scholar, 3Zheng M. Åslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (974) Google Scholar). OxyR is activated through the formation of a disulfide bond between Cys199 and Cys208, and deactivated by the enzymatic reduction of the bond with GRX (3Zheng M. Åslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (974) Google Scholar). The activities of NF-κB and AP-1 are also regulated by the redox modification of cysteine residues. After the dissociation of NF-κB/IκB complex, TRX augments the DNA binding and transcriptional activities of NF-κB by reducing the Cys62 residue in its DNA-binding loop (4Matthews J.R. Wakasugi N. Virelizier J.-L. Yodoi J. Hay R.T. Nucleic Acids Res. 1992; 20: 3821-3830Crossref PubMed Scopus (726) Google Scholar, 5Hayashi T. Ueno Y. Okamoto T. J. Biol. Chem. 1993; 268: 11380-11388Abstract Full Text PDF PubMed Google Scholar, 6Qin J. Clore G.M. Kennedy W.M.P. Huth J.R. Gronenborn A.M. Structure. 1995; 3: 289-297Abstract Full Text Full Text PDF PubMed Scopus (212) Google Scholar). Similarly, redox modification of AP-1 is regulated by a nuclear redox factor, Ref-1, and the Ref-1 activity is also modulated by TRX (7Walker L.J. Robson C.N. Black E. Gillespie D. Hickson I.D. Mol. Cell. Biol. 1993; 13: 5370-5376Crossref PubMed Scopus (262) Google Scholar, 8Xanthoudakis S. Miao G.G. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 23-27Crossref PubMed Scopus (319) Google Scholar). TRX can associate directly with Ref-1 in the nucleus (9Qin J. Clore G.M. Kennedy W.P. Kuszewski J. Gronenborn A.M. Structure. 1996; 4: 613-620Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 10Hirota K. Matsui M. Iwata S. Nishiyama A. Mori K. Yodoi J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3633-3638Crossref PubMed Scopus (723) Google Scholar). thioredoxin glutaredoxin glutathione reductase green fluorescent protein cysteine-rich domain nuclear export sequence 2′,7′-dichlorofluorescin diacetate kilobase(s) The contribution of TRX and GRX toward the maintenance of the intracellular environments in reduced states is comparable to glutathione (11Åslund F. Berndt K.D. Holmgren A. J. Biol. Chem. 1997; 272: 30780-30786Abstract Full Text Full Text PDF PubMed Scopus (329) Google Scholar, 12Prinz W.A. Åslund F. Holmgren A. Beckwith J. J. Biol. Chem. 1997; 272: 15661-15667Abstract Full Text Full Text PDF PubMed Scopus (529) Google Scholar). These thiol-disulfide oxidoreductases may participate in the control of redox-regulated transcription factors via the intracellular redox states (3Zheng M. Åslund F. Storz G. Science. 1998; 279: 1718-1721Crossref PubMed Scopus (974) Google Scholar, 13Demple B. Science. 1998; 279: 1655-1656Crossref PubMed Scopus (58) Google Scholar). Yap1 (yeast AP-1) is a transcription factor crucial for oxidative stress response in Saccharomyces cerevisiae, and it regulates the expression of several genes whose gene products play major roles in the oxidative stress tolerance. The null mutant of theYAP1 gene displayed hypersensitivity to oxidative stress, and the overexpression of the YAP1 gene confers stress resistance (14Hertle K. Haase E. Brendel M. Curr. Genet. 1991; 19: 429-433Crossref PubMed Scopus (59) Google Scholar, 15Hussain M. Lenard J. Gene ( Amst. ). 1991; 101: 149-152Crossref PubMed Scopus (55) Google Scholar, 16Schnell N. Entian K.-D. Eur. J. Biochem. 1991; 200: 487-493Crossref PubMed Scopus (63) Google Scholar, 17Wu 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, 18Hirata D. Yano K. Miyakawa T. Mol. Gen. Genet. 1994; 242: 250-256Crossref PubMed Scopus (80) Google Scholar). Several target genes for Yap1 have been identified, such as GSH1 encoding γ-glutamylcysteine synthetase (19Wu A.-L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar), GLR1 encoding glutathione reductase (20Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (213) Google Scholar),TRX2 encoding TRX (21Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar), and TRR1 encoding thioredoxin reductase (22Morgan 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). These gene products are involved in the adaptive response to oxidative stress. Yap1 was originally identified as a functional homologue of mammalian AP-1 on the basis of its ability to bind to an AP-1 recognition element (23Moye-Rowley W.S. Harshman K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (245) Google Scholar). Yap1 binds to the Yap1 recognition element (YRE: 5′-TTAGT(C/A)A-3′) in the promoter region of target genes (24Wemmie 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). The N terminus of Yap1 contains a bZip domain, which is conserved among the AP-1 family, including mammalian Jun, Fos, and S. cerevisiaeGcn4 (23Moye-Rowley W.S. Harshman K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (245) Google Scholar). A cysteine-rich domain (CRD) at the C terminus of Yap1, containing three Cys-Ser-Glu sequence motifs, plays an important role in the control of Yap1, especially for the intracellular localization of Yap1 (25Alarco A.-M. Balan I. Talibi D. Mainville N. Raymond M. J. Biol. Chem. 1997; 272: 19304-19313Abstract Full Text Full Text PDF PubMed Scopus (168) Google Scholar, 26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar). In the response to oxidative stress, the localization of Yap1 changes dramatically, while the increase in the DNA binding activity is modest and the levels of Yap1 do not increase (26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar, 27Takeuchi T. Miyahara K. Hirata D. Miyakawa T. FEBS Lett. 1997; 416: 339-343Crossref PubMed Scopus (22) Google Scholar, 28Wemmie J.A. Steggerda S.M. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 7908-7914Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Yap1 exists both in the cytoplasm and the nucleus under non-stressed conditions, while it is concentrated in the nucleus under oxidative conditions (26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar). Recently, it has been reported that the localization of Yap1 is controlled by Crm1-mediated nuclear export and that Yap1 has an nuclear export sequence (NES) embedded within the CRD (29Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar, 30Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (205) Google Scholar). Mutational analysis suggested that cysteine residues within the CRD serve as redox sensors, which regulate the availability of the NES (30Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (205) Google Scholar). Therefore, removal of the CRD causes constitutive accumulation of Yap1 in the nucleus, which results in the increase of transcription of the Yap1 target genes (26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar). Additionally, the CRD is required for Yap1 to discriminate the stresses elicited by H2O2, diamide, and CdCl2 (27Takeuchi T. Miyahara K. Hirata D. Miyakawa T. FEBS Lett. 1997; 416: 339-343Crossref PubMed Scopus (22) Google Scholar, 28Wemmie J.A. Steggerda S.M. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 7908-7914Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). The importance of the CRD raised the possibility that TRX and/or GRX modulate the activity of Yap1 by the redox modification of cysteine residues, like OxyR, NF-κB, and AP-1. To examine whether TRX and/or GRX attend the redox regulation of Yap1, we analyzed the phenotypes of the TRX- and GRX-deficient mutants with respect to the Yap1-mediated gene expression and the localization of Yap1. Here we show that TRX deficiency (trx1Δ/trx2Δ) caused the constitutive activation of Yap1 under aerobic conditions but not under anaerobic conditions, and that GRX deficiency (grx1Δ/grx2Δ) did not affect the Yap1 activity. We also provide the evidence that theyap1Δ/trx1Δ/trx2Δ triple mutant shows the synthetic lethality while theyap1Δ/grx1Δ/grx2Δ triple mutant was viable. S. cerevisiae YPH250 and YPH252 were obtained from the Yeast Genetic Stock Center, University of California, Berkeley, CA. Cells were cultured in 50 ml of SD minimal medium (2% glucose, 0.67% yeast nitrogen base without amino acids, pH 5.5) with appropriate amino acids and bases at 30 °C with reciprocal shaking in 200-ml Erlenmeyer flasks. For anaerobic cultivation, the medium was flushed with nitrogen gas, sealed up, and incubated without shaking at 30 °C. Exponentially growing cells were harvested at A610 = 0.5. Theyap1Δ mutants were constructed by using pSM27 as described by Wu et al. (17Wu 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). pSM27 was digested with EcoRI and transformed to YPH250 (MAT a trp-Δ1 his3-Δ200 lys2–801 leu2-Δ1 ade2–101 ura3–52) and YPH252 (MATα trp-Δ1 his3-Δ200 lys2–801 leu2-Δ1 ade2–101 ura3–52) to yield theyap1Δ and YA-1α, respectively. All mutants used in this study were derived from YPH250 except for YA-1α and YT-1α (see below). S. cerevisiae has two cytosolic TRX genes, TRX1and TRX2 (31Gan Z.-R. J. Biol. Chem. 1991; 266: 1692-1696Abstract Full Text PDF PubMed Google Scholar); one mitochondrial TRX gene, TRX3(32Pedrajas J.R. Kosmidou E. Miranda-Vizuete A. Gustafsson J.-Å. Wright A.P.H. Spyrou G. J. Biol. Chem. 1999; 274: 6366-6373Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar); and two GRX genes, GRX1 and GRX2(TTR1) (33Gan Z.-R. Biochem. Biophys. Res. Commun. 1992; 187: 949-955Crossref PubMed Scopus (20) Google Scholar, 34Luikenhuis S. Perrone G. Dawes I.W. Grant C.M. Mol. Biol. Cell. 1998; 9: 1081-1091Crossref PubMed Scopus (198) Google Scholar). To disrupt the TRX1 gene, theTRX1 gene was amplified using the following oligonucleotide primers: 5′-GATCAGAATGATTGAAATCA-3′ and 5′-GACGAGCTATAGGATGATGA-3′. The amplicon (2.0 kb) was treated by Klenow fragment, and then cloned, respectively, to the HincII site of pUC19 (pTR-1) and to the BamHI site of YEp13, which was also treated by Klenow fragment (YEpTRX1). The URA3 gene (1.2 kb) isolated from YEp24 was treated with Klenow fragment and inserted between the MunI/MunI sites internal to the TRX1 gene in pTR-1, which was also treated by Klenow fragment, to yield pTRD1. pTRD1 was digested with ApaLI andDraI, and the trx1Δ::URA3fragment was transformed to YPH250 and YA-1α to yield thetrx1Δ and YT-1α, respectively. Disruption of theTRX2 gene was done using thetrx2::HIS3 disruption plasmid, kindly provided by Drs. S. Kuge and N. Jones (21Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar). Thetrx2::HIS3 disruption plasmid was digested with SphI, and thetrx2Δ::HIS3 fragment was transformed to YPH250 and the trx1Δ mutant to yield thetrx2Δ mutant and the trx1Δ/trx2Δ mutant, respectively. Additionally, thetrx2::LEU2 disruption plasmid was constructed. The TRX2 gene was amplified by using the following oligonucleotide primers: 5′-GATCAGCATAACTTGAGTGC-3′ and 5′-GATCGCATGGAACGCCAAGC-3′. The amplicon (0.8 kb) was treated with Klenow fragment, and then cloned to the HincII site of pUC19 (pTR-2), and to the BamHI site of YEp13 (YEpTRX2), respectively. The LEU2 gene (2.2 kb) isolated from YEp13 was treated with Klenow fragment and inserted between theHincII/EcoO65I sites internal to theTRX2 gene in pTR-2, which was also treated with Klenow fragment, to yield pTRD2. pTRD2 was digested with PstI andSmaI, and the trx2Δ::LEU2fragment was transformed to the yap1Δ mutant to yield YT-2a. Mitochondrial thioredoxin gene, TRX3 (YCR083w), was also amplified using the following oligonucleotide primers: 5′-GGCGGAGAATAGGGATCCACTGCGA-3′ and 5′-GTCTCCGCTGGATCCAGAATATAAC-3′. The amplicon (1.4 kb) was digested with BamHI and cloned to the BamHI site of YEp13 (YEpTRX3). To disrupt the GRX genes, the GRX1 gene was amplified using the following primers: 5′-CATCCTTAGAAAGGATCCCACATTG-3′ and 5′-CGAGACGTACGGGATCCTAAAGTGG-3′. The amplicon (1.1 kb) was digested with BamHI and cloned to the BamHI site of YEp13 (YEpGRX1), and also to the BamHI site of pUC19 (pGR-1). A 1.2-kb BglII-ClaI fragment containing theTRP1 gene from pRS414 was inserted betweenBglII/ClaI sites internal to the GRX1gene in pGR-1 (pGRD1). pGRD1 was digested with SmaI andSalI, and the grx1Δ::TRP1fragment was transformed to YPH250 to yield the grx1Δ mutant. The GRX2 gene was also amplified using the following primers: 5′-GGGTCATTGCCGTGGATCCTACAAAAC-3′ and 5′-TACACGTGGATCCTGATGCTGAAGT-3′. The amplicon (1.2 kb) was digested with BamHI and cloned to the BamHI site of YEp13 (YEpGRX2), and also to the BamHI site of pUC19 (pGR-2). The URA3 gene (1.2 kb) isolated from YEp24 was treated with Klenow fragment and inserted between theBsaAI/BsaAI sites internal to the GRX2gene (pGRD2). pGRD2 was digested with SmaI andSacI, and the grx2Δ::URA3fragment was transformed to YPH250 and the grx1Δ to yield the grx2Δ mutant and thegrx1Δ/grx2Δ mutant, respectively. Disruption of each gene was verified by polymerase chain reaction. Thetrx1Δ/trx2Δ mutant showed the methionine auxotrophy, the increase of cell size, and elongation of generation time as reported by Muller (35Muller E.G.D. J. Biol. Chem. 1991; 266: 9194-9202Abstract Full Text PDF PubMed Google Scholar). TheGSH1 promoter fragment containing the region from −800 to +33 (+1 representing the start of translation) was generated by polymerase chain reaction using the primers: 5′-TAATCTTATGAATCCCGGGGATTTTATCGG-3′ and 5′-CTAGACTCAAACCCGGGCAAAGGCGTGCCC-3′. The amplicon was digested with SmaI and cloned into the SmaI site of pMC1871 containing the coding region of lacZ (Amersham Pharmacia Biotech), to yield pMC-GSH1lacZ. As a result of this construction, the first 11 amino acid residues of Gsh1 were fused to β-galactosidase whose first 8 amino acids were deleted. TheGSH1-lacZ in pMC-GSH1lacZ was isolated by digestion withSalI and cloned into the SalI site of pRS414 (pRS-GSH1lacZ). A Yap1-dependent lacZ reporter gene containing three SV40 AP-1 sites and the TATA element of theCYC1 promoter was kindly gifted by Dr. Kuge (21Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar, 29Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar). β-Galactosidase activity was measured as described by Miller (36Miller J.H. Experiments in Molecular Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1972: 352-356Google Scholar). One unit of the activity was defined as the amount of enzyme increasing A420 per hour at 30 °C. Protein was determined by the method of Lowry et al.(37Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Northern hybridization was performed using 25 μg of total cellular RNA isolated from yeast cells by the method of Schmitt et al. (38Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1991; 18: 3091-3092Crossref Scopus (1152) Google Scholar). The probes were generated by random primed labeling of the 0.4-kbEcoRV-BamHI fragment of GSH1 gene, 0.7-kb PvuII-HincII fragment of TRR1gene, and 1.1-kb EcoRI-PstI fragment ofACT1 gene, respectively, with [α-32P]dCTP using a kit (Random Primer DNA labeling kit version 2, Takara). pRS cup1 cp-GFP-YAP1 was kindly provided by Dr. Kuge (26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google Scholar). To visualize DNA, the cells were stained with 1 μg/ml 4′,6′-diamidino-2-phenylindole dihydrochloride. ANdeI/BamHI fragment encoding the whole coding region of the TRX2 gene was inserted into theNdeI/BamHI site of pET15-b (Novagen) to construct pET-TRX2. The resulting protein contained six histidine tag residues fused in-frame with the TRX2 coding region. pET-TRX2 was transferred into E. coli strain BL21(DE3). Histidine-tagged Trx2 was purified using histidine affinity column chromatography (His-trap, Amersham Pharmacia Biotech), followed by gel filtration chromatography (Superdex 75, Amersham Pharmacia Biotech). Purified fusion protein was then used for the production of anti-yeast TRX antibody using New Zealand White rabbits. Immunization and purification of anti-Trx antibody were accomplished by Sawady Technology Co., Ltd., Tokyo, Japan. This anti-TRX antibody was specific to yeast TRX and was able to detect both Trx1 and Trx2. Immunofluorescence microscopic observation of yeast was performed by the methods of Rose et al. (39Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). Anti-rabbit IgG (H+L)-fluorescein isothiocyanate (FI-1000, Vector Laboratory Inc.) was used as the secondary antibody. Cell extracts were prepared according to Wemmie et al. (28Wemmie J.A. Steggerda S.M. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 7908-7914Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and 170 μg of total protein of each sample was run on 10% SDS-polyacrylamide gels. Proteins were electrically transferred to polyvinylidene difluoride membrane (Immobilon, Millipore). Anti-Yap1 antiserum raised in rabbit was kindly gifted by Dr. Moye-Rowley. Horseradish peroxidase-conjugated secondary antibody (Jackson Immunoresearch Laboratory, Inc.) and diaminobenzidine were used to visualize immunoreactive protein. The activity of glutathione reductase, total glutathione, and the susceptibility and adaptation to H2O2 were determined as described previously (40Izawa S. Inoue Y. Kimura A. FEBS Lett. 1995; 368: 73-76Crossref PubMed Scopus (225) Google Scholar, 41Izawa S. Inoue Y. Kimura A. Biochem. J. 1996; 320: 61-67Crossref PubMed Scopus (203) Google Scholar). Intracellular oxidation level of yeast was measured using the oxidant-sensitive probe 2′,7′-dichlorofluorescin diacetate (DCFH-DA) purchased from Molecular Probes (42Davidson J.F. Whyte B. Bissinger P.H. Schiestl R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 5116-5121Crossref PubMed Scopus (372) Google Scholar). Cells growing exponentially in SD medium were collected and resuspended in fresh SD medium containing 0.1 mmDCFH-DA and incubated at 30 °C for 20 min. After the incubation, cells were washed, resuspended in distilled water, and disrupted by vortexing with glass beads. Cell extracts (70 μl) were mixed in 500 μl of distilled water, and fluorescence was measured with λex = 490 nm and λem = 524 nm using a Hitachi F-3000 spectrofluorometer. Mating, sporulation, dissection, and tetrad analysis of yeast were done as described by Roseet al. (39Rose M.D. Winston F. Hieter P. Methods in Yeast Genetics: A Laboratory Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1990Google Scholar). In order to see the effect of TRX and GRX on the activity of Yap1, we investigated the levels of cellular glutathione and glutathione reductase (GR) activity in the TRX- and GRX-deficient mutants. The GSH1 and GLR1 genes, which are target genes for Yap1 (19Wu A.-L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 20Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (213) Google Scholar), encode γ-glutamylcysteine synthetase (Gsh1) and GR, respectively (43Ohtake Y. Yabuuchi S. Yeast. 1991; 7: 6953-6961Crossref Scopus (112) Google Scholar, 44Collinson L.P. Dawes I. Gene ( Amst. ). 1995; 156: 123-127Crossref PubMed Scopus (67) Google Scholar). The Gsh1 catalyzes the first and rate-limiting step of glutathione synthesis (45Meister A. Anderson M.E. Annu. Rev. Biochem. 1983; 52: 711-760Crossref PubMed Scopus (5972) Google Scholar). Therefore, it is conceivable that the levels of intracellular glutathione and GR activity reflect the activity of Yap1. Table I shows the levels of total glutathione and GR activity in various mutants. As reported previously, the yap1Δ mutant showed lower levels of total glutathione and GR activity than the wild type (19Wu A.-L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 20Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (213) Google Scholar). Total glutathione and GR activity in the wild type were increased by the treatment with 0.2 mm H2O2, but not in theyap1Δ mutant (46Izawa S. Maeda K. Miki T. Mano J. Inoue Y. Kimura A. Biochem. J. 1998; 330: 811-817Crossref PubMed Scopus (109) Google Scholar). The levels of glutathione and GR activity in trx1Δ/trx2Δ were constitutively high under non-stressed conditions, and the values were higher than those in the wild type which was treated with H2O2. This indicates that the loss of both Trx1 and Trx2 affects intracellular glutathione metabolism. GRX deficiency (grx1Δ/grx2Δ) and single-gene mutation of TRX or GRX genes did not affect total glutathione levels and GR activity (data for single-gene mutants are not shown), and our results are consistent with the previous reports (34Luikenhuis S. Perrone G. Dawes I.W. Grant C.M. Mol. Biol. Cell. 1998; 9: 1081-1091Crossref PubMed Scopus (198) Google Scholar, 47Muller E.G.D. Mol. Biol. Cell. 1996; 7: 1805-1813Crossref PubMed Scopus (165) Google Scholar). The results in Table Iimply that Yap1 is constitutively activated intrx1Δ/trx2Δ even though the cells are not exposed to oxidative stress.Table ICellular total glutathione contents and GR activity in various mutantsStrainH2O2GlutathioneGR activityμmol/g cellmilliunits/mgWild type−1.81 ± 0.1274.2 ± 5.8+2.08 ± 0.1197.3 ± 6.4yap1Δ−0.85 ± 0.0946.5 ± 4.2+0.87 ± 0.0951.0 ± 5.0grx1Δ/grx2Δ−1.76 ± 0.1276.0 ± 5.4+1.90 ± 0.1295.4 ± 3.8trx1Δ/trx2Δ−2.78 ± 0.22117.0 ± 8.4+3.14 ± 0.20143.0 ± 8.8Cells growing exponentially were treated with or without 0.2 mm H2O2 for 60 min. Data are means ± S.D. from three independent experiments. One unit of GR was defined as the amount of enzyme reducing 1.0 μmol of GSSG/min at 25 °C. Open table in a new tab Cells growing exponentially were treated with or without 0.2 mm H2O2 for 60 min. Data are means ± S.D. from three independent experiments. One unit of GR was defined as the amount of enzyme reducing 1.0 μmol of GSSG/min at 25 °C. To confirm the constitutive activation of Yap1 in the TRX-deficient mutant, we next assessed the expression of the lacZ reporter gene under the control of the GSH1 promoter. A fusion gene was constructed containing the lacZ coding region fused to the GSH1 promoter sequence which contains the Yap1 binding site (5′-TTAGTCA-3′) (19Wu A.-L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar). As shown in Fig.1 A, β-galactosidase activity was increased with H2O2 treatment in the wild type, but not in the yap1Δ, as reported previously (48Stephan D.W.S. Rivers S.T. Jamieson D.J. Mol. Microbiol. 1995; 16: 415-423Crossref PubMed Scopus (179) Google Scholar). The β-galactosidase activities in single mutants (trx1Δ or trx2Δ) under non-stressed conditions were the same to that in the wild type. On the contrary, the basal level of β-galactosidase activity in trx1Δ/trx2Δ was significantly higher than that of the wild type, and the value was almost the same as that of the wild type treated with 0.2 mm H2O2 (the wild type with H2O2, 88.8 ± 1.3;trx1Δ/trx2Δ without H2O2, 95.1 ± 1.2 units/mg). The β-galactosidase activity in trx1Δ/trx2Δ was scarcely increased by the treatment with H2O2, suggesting that Yap1 activity was constitutively high and presumably saturated in the trx1Δ/trx2Δ mutant under non-stressed conditions. We reconfirmed the increased expression of the GSH1 gene intrx1Δ/trx2Δ by Northern blotting analysis (Fig. 1 B). Basal level of the GSH1 gene expression in trx1Δ/trx2Δ was almost the same as that of the H2O2-treated wild type. Expression of the GSH1 gene intrx1Δ/trx2Δ did not increase with 0.2 mm H2O2 stress. These results were in good agreement with those obtained by using the GSH1-lacZreporter gene assay. Expression of the TRR1 gene, another target gene for Yap1 (22Morgan 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), was also constitutively high intrx1Δ/trx2Δ, but not ingrx1Δ/grx2Δ (Fig. 1 C). In yeast and other eukaryotic cells, expression of most genes is controlled by multiple transcription factors acting co-operatively (49Robertson L.M. Kerppola T.K. Vandrell M. Luk D. Smeyne R.J. Bocchiaro C. Morgan J.I. Curran T. Neuron. 1995; 14: 241-252Abstract Full Text PDF PubMed Scopus (272) Google Scholar). To see whether other factor(s) that may bind to theGSH1 promoter region are involved in increased expression of the GSH1-lacZ reporter gene in thetrx1Δ/trx2Δ mutant, we also examined the expression of a lacZ reporter gene driven by three SV40 AP-1 sites and the TATA element of the CYC1 promoter (21Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (387) Google Scholar, 26Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (347) Google S
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