Multiple Yap1p-binding Sites Mediate Induction of the Yeast Major Facilitator FLR1 Gene in Response to Drugs, Oxidants, and Alkylating Agents
2001; Elsevier BV; Volume: 276; Issue: 2 Linguagem: Inglês
10.1074/jbc.m008377200
ISSN1083-351X
AutoresDuc-Thang Nguyên, Anne‐Marie Alarco, Martine Raymond,
Tópico(s)RNA Research and Splicing
ResumoThe bZip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the FLR1 gene, encoding a multidrug transporter of the major facilitator superfamily, is a transcriptional target of Yap1p. The FLR1 promoter contains three potential Yap1p response elements (YREs) at positions −148 (YRE1), −167 (YRE2), and −364 (YRE3). To address the function of these YREs, the three sites have been individually mutated and tested in transactivation assays. Our results show that (i) each of the three YREs is functional and important for the optimal transactivation of FLR1 by Yap1p and that (ii) the three YREs are not functionally equivalent, mutation of YRE3 being the most deleterious, followed by YRE2 and YRE1. Simultaneous mutation of the three YREs abolished transactivation of the promoter by Yap1p, demonstrating that the three sites are essential for the regulation of FLR1 by Yap1p. Gel retardation assays confirmed that Yap1p differentially binds to the three YREs (YRE3 > YRE2 > YRE1). We show that the transcription of FLR1 is induced upon cell treatment with the oxidizing agents diamide, diethylmaleate, hydrogen peroxide, and tert-butyl hydroperoxide, the antimitotic drug benomyl, and the alkylating agent methylmethane sulfonate and that this induction is mediated by Yap1p through the three YREs. Finally, we show that FLR1 overexpression confers resistance to diamide, diethylmaleate, and menadione but hypersensitivity to H2O2, demonstrating that the Flr1p transporter participates in Yap1p-mediated oxidative stress response in S. cerevisiae. The bZip transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance in Saccharomyces cerevisiae. We have previously demonstrated that the FLR1 gene, encoding a multidrug transporter of the major facilitator superfamily, is a transcriptional target of Yap1p. The FLR1 promoter contains three potential Yap1p response elements (YREs) at positions −148 (YRE1), −167 (YRE2), and −364 (YRE3). To address the function of these YREs, the three sites have been individually mutated and tested in transactivation assays. Our results show that (i) each of the three YREs is functional and important for the optimal transactivation of FLR1 by Yap1p and that (ii) the three YREs are not functionally equivalent, mutation of YRE3 being the most deleterious, followed by YRE2 and YRE1. Simultaneous mutation of the three YREs abolished transactivation of the promoter by Yap1p, demonstrating that the three sites are essential for the regulation of FLR1 by Yap1p. Gel retardation assays confirmed that Yap1p differentially binds to the three YREs (YRE3 > YRE2 > YRE1). We show that the transcription of FLR1 is induced upon cell treatment with the oxidizing agents diamide, diethylmaleate, hydrogen peroxide, and tert-butyl hydroperoxide, the antimitotic drug benomyl, and the alkylating agent methylmethane sulfonate and that this induction is mediated by Yap1p through the three YREs. Finally, we show that FLR1 overexpression confers resistance to diamide, diethylmaleate, and menadione but hypersensitivity to H2O2, demonstrating that the Flr1p transporter participates in Yap1p-mediated oxidative stress response in S. cerevisiae. multidrug resistance benomyl cysteine-rich domain diamide diethylmaleate electrophoretic mobility shift assay fluconazole resistance 1 methylmethane sulfonate menadione open reading frame polymerase chain reaction tert-butyl hydroperoxide Yap1p response element synthetic dextrose wild type glyceraldehyde-3-phosphate dehydrogenase The Saccharomyces cerevisiae transcription factor Yap1p plays an important role in oxidative stress response and multidrug resistance (MDR)1 by activating target genes involved in cellular detoxification (1Bauer B.E. Wolfger H. Kuchler K. Biochim. Biophys. Acta. 1999; 1461: 217-236Crossref PubMed Scopus (228) Google Scholar, 2Toone W.M. Jones N. Curr. Opin. Genet. Dev. 1999; 9: 55-61Crossref PubMed Scopus (100) Google Scholar, 3Kolaczkowska A. Goffeau A. Drug Resist Update. 1999; 2: 403-414Crossref PubMed Scopus (106) Google Scholar). Yap1p belongs to the bZip (basic domain/leucine zipper) family of transcription factors that includes the yeast Gcn4p and the mammalian activator protein-1 proteins Fos and Jun (4Moye-Rowley W.S. Harshman K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (242) Google Scholar). It activates transcription by binding to specific DNA sequences located in the promoter of its targets (2Toone W.M. Jones N. Curr. Opin. Genet. Dev. 1999; 9: 55-61Crossref PubMed Scopus (100) Google Scholar). Yap1p targets involved in oxidative stress response include TRX2 (thioredoxin) (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar), GSH1(γ-glutamylcysteine synthetase) (6Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar), GSH2 (glutathione synthetase) (7Sugiyama K. Izawa S. Inoue Y. J. Biol. Chem. 2000; 275: 15535-15540Abstract Full Text Full Text PDF PubMed Scopus (119) Google Scholar), TRR1 (thioredoxin reductase) (8Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (238) Google Scholar),GLR1 (glutathione reductase) (9Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (212) Google Scholar), GPX2(glutathione peroxidase) (10Inoue Y. Matsuda T. Sugiyama K. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar), TSA1 (thioredoxin peroxidase) (10Inoue Y. Matsuda T. Sugiyama K. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 11Lee 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 (461) Google Scholar), and AHP1 (alkyl hydroperoxide reductase) (12Lee 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). Yap1p also regulates the transcription of genes encoding membrane-associated transporters such as YCF1, coding for an ATP binding cassette (ABC) transporter, which functions as a glutathione S-conjugate pump (13Wemmie 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), as well as ATR1 and FLR1, coding for MDR transporters of the major facilitator superfamily (14Coleman S.T. Tseng E. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 23224-23230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 15Alarco A.M. Balan I. Talibi D. Mainville N. Raymond M. J. Biol. Chem. 1997; 272: 19304-19313Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). Large-scale studies investigating Yap1p-dependent transcription have identified several additional genes that appear to be directly or indirectly regulated by Yap1p (11Lee 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 (461) Google Scholar, 16DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3686) Google Scholar, 17Dumond H. Danielou N. Pinto M. Bolotin-Fukuhara M. Mol. Microbiol. 2000; 36: 830-845Crossref PubMed Scopus (43) Google Scholar), underscoring the importance of this transcription factor in regulating stress response pathways.Yap1p was originally identified on the basis of its ability to bind to an activator protein-1 recognition element found in the SV40 enhancer (5′-TGACTAA) (4Moye-Rowley W.S. Harshman K.D. Parker C.S. Genes Dev. 1989; 3: 283-292Crossref PubMed Scopus (242) Google Scholar, 18Harshman K.D. Moye-Rowley W.S. Parker C.S. Cell. 1988; 53: 321-330Abstract Full Text PDF PubMed Scopus (174) Google Scholar). This sequence is present in the GSH1promoter and is required for Yap1p-mediated regulation of GSH1 expression (6Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar). However, it was recently demonstrated that Yap1p preferentially interacts with the sequence 5′-TTAC/GTAA (19Fernandes L. Rodrigues-Pousada C. Struhl K. Mol. Cell. Biol. 1997; 17: 6982-6993Crossref PubMed Scopus (259) Google Scholar). This sequence, which is palindromic and contains two identical TTA half-sites, has been shown to function in an orientation-independent manner (13Wemmie 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, 14Coleman S.T. Tseng E. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 23224-23230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It is present in the TRX2, YCF1, GLR1, and ATR1promoters and mediates their transcriptional activation by Yap1p (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar, 9Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (212) Google Scholar,13Wemmie 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, 14Coleman S.T. Tseng E. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 23224-23230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). It thus appears that the consensus Yap1p response element (YRE) corresponds to the sequence 5′-TT/GAC/GTAA (2Toone W.M. Jones N. Curr. Opin. Genet. Dev. 1999; 9: 55-61Crossref PubMed Scopus (100) Google Scholar).The activity of Yap1p is predominantly regulated at the level of nuclear export. Under unstressed conditions, Yap1p shuttles between the cytosol and the nucleoplasm but is mainly cytosolic. It is actively exported from the nucleus by the exportin Crm1p, which interacts with the C-terminal cysteine-rich domain (CRD) of Yap1p (20Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar, 21Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar, 22Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (203) Google Scholar, 23Kudo N. Taoka H. Toda T. Yoshida M. Horinouchi S. J. Biol. Chem. 1999; 274: 15151-15158Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar). This domain contains a leucine-rich nuclear export sequence embedded within three cysteine residues invariably conserved among homologues of the Yap1p family (20Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar, 21Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar, 22Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (203) Google Scholar). Removal of the CRD, mutation of the nuclear export sequence or of the cysteines as well as treatment of the cells with oxidative agents such as hydrogen peroxide (H2O2), diamide (DA), or diethylmaleate (DEM) all disrupt the Crm1p/ nuclear export sequence interaction, resulting in the accumulation of Yap1p in the nucleus and Yap1p-dependent transcriptional activation (20Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar, 21Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar, 22Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (203) Google Scholar, 23Kudo N. Taoka H. Toda T. Yoshida M. Horinouchi S. J. Biol. Chem. 1999; 274: 15151-15158Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 24Coleman S.T. Epping E.A. Steggerda S.M. Moye-Rowley W.S. Mol. Cell. Biol. 1999; 19: 8302-8313Crossref PubMed Scopus (128) Google Scholar). The CRD is thought to behave as a specialized export signal that is sensitive to the redox state of the cells, the oxidation status of the cysteines affecting the accessibility of the nuclear export sequence to Crm1p, thereby regulating Yap1p activity (21Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar, 22Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (203) Google Scholar).The FLR1 gene (YBR008c) was predicted to code for an integral membrane protein with 12 transmembrane domains belonging to multidrug permease subfamily I (25Nelissen B. De Wachter R. Goffeau A. FEMS Microbiol. Rev. 1997; 21: 113-134Crossref PubMed Google Scholar). We have demonstrated that FLR1 overexpression confers resistance to cycloheximide, 4-nitroquinoline N-oxide, and the azole derivative fluconazole, a drug widely used in antifungal therapy (hence FLR1, for fluconazole resistance1) (15Alarco A.M. Balan I. Talibi D. Mainville N. Raymond M. J. Biol. Chem. 1997; 272: 19304-19313Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). FLR1 overexpression has also been shown to confer resistance to different toxic compounds including cerulenin, benomyl (BN), methotrexate, and diazaborine (26Oskouian B. Saba J.D. Mol. Gen. Genet. 1999; 261: 346-353Crossref PubMed Scopus (20) Google Scholar, 27Broco N. Tenreiro S. Viegas C.A. Sa-Correia I. Yeast. 1999; 15: 1595-1608Crossref PubMed Google Scholar, 28Jungwirth H. Wendler F. Platzer B. Bergler H. Hogenauer G. Eur. J. Biochem. 2000; 267: 4809-4816Crossref PubMed Scopus (35) Google Scholar), further demonstrating its involvement in MDR. We have also demonstrated that FLR1 is a transcriptional target of Yap1p and that the FLR1 promoter contains three potential YREs, suggesting that the regulation of FLR1 expression by Yap1p could be mediated through these sequences (15Alarco A.M. Balan I. Talibi D. Mainville N. Raymond M. J. Biol. Chem. 1997; 272: 19304-19313Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In the present study, we show that the three elements are functional, although not equivalent, and important for the optimal transactivation of FLR1 by Yap1p in response to different classes of compounds, including drugs, oxidants, and alkylating agents. We also show that FLR1 overexpression confers resistance to DA, DEM, and menadione (MD) but hypersensitivity to H2O2, indicating that the Flr1p transporter, in addition to its role in MDR, also modulates oxidative stress response in S. cerevisiae.DISCUSSIONIn this study, we have investigated the function of three potential YREs identified in the FLR1 promoter by computer-assisted sequence analysis with respect to their ability to mediate FLR1 regulation by Yap1p. Two independent pieces of evidence indicate that these three sequences are functional. First, mutation of any of the three YREs decreases the ability of overexpressed Yap1p to transactivate an FLR1promoter -lacZ fusion (Fig. 2), demonstrating that the three YREs are necessary to mediate optimal induction of FLR1 by Yap1p. Second, the EMSA performed with oligonucleotides overlapping each YRE demonstrates that endogenous as well as overexpressed Yap1p can bind to the three sites (Fig. 3). As simultaneous mutation of the three YREs completely abolishes the induction of the FLR1promoter by overexpressed Yap1p, we conclude that there is no other unidentified YRE in the FLR1 promoter.Our results also demonstrate that the three YREs are not functionally equivalent. Upon transactivation of the FLR1 promoter with overexpressed Yap1p, mutation of YRE3 has the most deleterious effect (90% decrease in transactivation), followed by YRE2 and YRE1 (75 and 40% decreases, respectively) (Fig. 2). Moreover, we show by EMSA that Yap1p, endogenous and overexpressed, binds more efficiently to YRE3 than to YRE2 and then to YRE1 (Figs. 3). These results suggest that the relative importance of the three YREs can potentially be attributed to differences in the binding affinity of Yap1p for each YRE. As previously mentioned, YRE3 matches the 5′-TTA(C/G)TAA sequence, whereas YRE1 and YRE2 correspond to the SV40 activator protein-1 recognition element 5′-TGACTAA shown to be less efficient than the palindromic site in mediating the in vivo activation of a reporter gene by Yap1p (19Fernandes L. Rodrigues-Pousada C. Struhl K. Mol. Cell. Biol. 1997; 17: 6982-6993Crossref PubMed Scopus (259) Google Scholar). It is also possible that other factors such as the relative position of the YRE within the FLR1promoter, the sequence context of each YRE, or the proximity of binding sites for other regulators also modulate the ability of a YRE to mediate activation of FLR1 by Yap1p. The FLR1promoter contains consensus binding sequences for different transcription factors, including a TATA box at position 35 (5′-TATAAA), a stress response element at position 135 (5′-AGGGG) known to mediate transcriptional induction in response to different stresses, including oxidative stress (44Marchler G. Schuller C. Adam G. Ruis H. EMBO J. 1993; 12: 1997-2003Crossref PubMed Scopus (411) Google Scholar), and a pleiotropic drug response element at position −439 (5′-TGCGCGGA) for binding of the two homologous transcription factors Pdr1p and Pdr3p involved in MDR (27Broco N. Tenreiro S. Viegas C.A. Sa-Correia I. Yeast. 1999; 15: 1595-1608Crossref PubMed Google Scholar). However, the functionality of these sequences has yet to be determined. Yap1p has also been shown to collaborate with the transcription factor Skn7p to regulate the expression of different target genes in response to H2O2 (8Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (238) Google Scholar, 12Lee 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). A consensus DNA binding sequence has not been precisely defined for Skn7p; it is thus not possible to predict whether this protein can bind to the FLR1 promoter. Interestingly, we also found that the relative importance of the three YREs varies with the inducer. Induction of the FLR1 promoter by DEM and BN resulted in a relative importance of the three YREs similar to that observed upon YAP1 overexpression (YRE3 > YRE2 > YRE1) (Fig. 4). However, YRE2 was found to be more important than YRE3 for the induction of FLR1 by DA, H2O2, and t-BHP (YRE2 > YRE3 > YRE1), whereas the three YREs were similarly involved in the induction by MMS (YRE3 ≅ YRE2 ≅ YRE1). The molecular basis for these differences is unclear. It is possible that different posttranslational modifications of Yap1p and/or binding to the FLR1 promoter of additional regulatory factors underlie the relative importance of the three YREs in response to specific inducers.Taken together, our results demonstrate that (i) the FLR1promoter contains three functional YREs, (ii) the two types of YRE sequences (5′-TTA(C/G)TAA and 5′-TGACTAA) coexist and function within the same promoter, and (iii) YREs differentially mediate transcriptional induction by Yap1p in response to various inducers. Unlike other Yap1p target genes described so far, the FLR1promoter is the only one found to contain three YREs. The ATR1, YCF1, GSH1, and GLR1promoters contain a single YRE (6Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 9Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (212) Google Scholar, 13Wemmie 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, 14Coleman S.T. Tseng E. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 23224-23230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The TRX2promoter contains two YREs, but it is not known whether both YREs are functional and, if so, whether they are equally important for the transactivation of TRX2 by Yap1p (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar). Interestingly, DNA microarray studies have identified an ORF (YKL071w) regulated by YAP1 and whose promoter is predicted to contain five YREs clustered within 60 base pairs (16DeRisi J.L. Iyer V.R. Brown P.O. Science. 1997; 278: 680-686Crossref PubMed Scopus (3686) Google Scholar). It will be interesting to see whether these five YREs are functional and equivalent for the regulation of YKL071w by Yap1p.We show that FLR1 transcription is induced by Yap1p upon treatment of the cells with different oxidizing agents. FLR1transcription was induced by DA, DEM, H2O2, and t-BHP but not by MD, indicating that the induction of FLR1 is specific for certain oxidants. This induction pattern is similar to that reported for TRX2, which is also induced by DA, H2O2, and t-BHP but not by MD (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar, 8Morgan B.A. Banks G.R. Toone W.M. Raitt D. Kuge S. Johnston L.H. EMBO J. 1997; 16: 1035-1044Crossref PubMed Scopus (238) Google Scholar, 45Stephen D.W. Rivers S.L. Jamieson D.J. Mol. Microbiol. 1995; 16: 415-423Crossref PubMed Scopus (176) Google Scholar), suggesting that the two genes are regulated by similar mechanisms. However, the demonstration that GSH1, conversely to FLR1 and TRX2, can be induced by MD (46Stephen D.W. Jamieson D.J. Mol. Microbiol. 1997; 23: 203-210Crossref PubMed Scopus (59) Google Scholar) indicates that Yap1p targets are differentially regulated in response to specific oxidants. Again, it is possible that additional transcription factors bind to Yap1p-regulated promoters to confer oxidant-specific induction. Taken together, these results demonstrate that FLR1 belongs to the network of genes controlled by Yap1p in response to oxidative stress and that this network does not only consist of enzymes and molecules with antioxidant properties but also of transporters.It has been recently reported that yeast exposure to MMS results in increased levels of FLR1 transcripts (42Jelinsky S.A. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1486-1491Crossref PubMed Scopus (379) Google Scholar). We show here that this increase occurs at the transcriptional level, is mediated by Yap1p, and requires the three YREs (Fig. 4). The mechanisms by which Yap1p induces transcription in response to MMS are still unknown but appear to somehow differ from those involved in oxidative stress response. Transcriptional activation by Yap1p in response to oxidants has been shown to involve relocalization of Yap1p to the nucleus without affecting the levels of YAP1 expression (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar, 20Kuge S. Jones N. Nomoto A. EMBO J. 1997; 16: 1710-1720Crossref PubMed Scopus (344) Google Scholar). Unlike oxidants, however, MMS exposure increases by ∼6-fold the levels of YAP1 transcripts (42Jelinsky S.A. Samson L.D. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1486-1491Crossref PubMed Scopus (379) Google Scholar). In addition, we find that YRE1, which plays virtually no role in the activation of FLR1 by oxidants, is important for the induction of FLR1 by MMS (Fig. 4), uncovering further differences in the mechanism of induction by Yap1p in response to oxidants and alkylating agents. Whether MMS also affects the nuclear localization of Yap1p is under investigation.To evaluate the biological consequences of FLR1 induction by oxidants, we have used a panel of isogenic flr1Δ and FLR1 strains overexpressing or not YAP1. These strains have proven useful in addressing the role of Yap1p in the tolerance of cells to a given compound and evaluating the contribution of Flr1p to this tolerance (15Alarco A.M. Balan I. Talibi D. Mainville N. Raymond M. J. Biol. Chem. 1997; 272: 19304-19313Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In parallel, we also tested a strain overexpressing the FLR1 ORF under the control of a strong constitutive promoter. Our results show that FLR1overexpression, achieved either from Yap1p overexpression or from the heterologous promoter, confers resistance to DA, MD, and to a lesser extent, DEM (Fig. 5). It had been shown that a yap1 deletion confers hypersensitivity to DA and MD and that YAP1overexpression confers resistance to DA (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar, 45Stephen D.W. Rivers S.L. Jamieson D.J. Mol. Microbiol. 1995; 16: 415-423Crossref PubMed Scopus (176) Google Scholar), but the targets of Yap1p mediating these phenotypes were not known. The results presented here clearly demonstrate that FLR1 is the major target of Yap1p mediating resistance to both compounds in S. cerevisiae. Moreover, our results also show that YAP1overexpression confers resistance to DEM and that FLR1 is one of the Yap1p targets mediating this resistance. These results constitute, to our knowledge, the first demonstration that a major facilitator behaves as a determinant of resistance to oxidants. Whether Flr1p functions by mediating the direct extracellular transport of these compounds, either unmodified or coupled to glutathione (47Kosower N.S. Kosower E.M. Methods Enzymol. 1995; 251: 123-133Crossref PubMed Scopus (284) Google Scholar, 48Zadzinski R. Fortuniak A. Bilinski T. Grey M. Bartosz G. Biochem. Mol. Biol. Int. 1998; 44: 747-759PubMed Google Scholar, 49Meister A. Methods Enzymol. 1985; 113: 571-585Crossref PubMed Scopus (134) Google Scholar), by indirectly regulating the function of other proteins, including transporters, or by introducing changes in the plasma membrane properties affecting cell tolerance to these compounds remains to be elucidated.The ability of DA, DEM, MMS, and BN to induce FLR1transcription was found to correlate with the ability of the Flr1p transporter to protect the cells from the cytotoxic effects of these compounds (Fig. 5 and data not shown) (27Broco N. Tenreiro S. Viegas C.A. Sa-Correia I. Yeast. 1999; 15: 1595-1608Crossref PubMed Google Scholar). However, MD and fluconazole, which both belong to the spectrum of compounds to which Flr1p confers resistance, are unable to induce FLR1transcription, indicating that Flr1p substrates do not necessarily function as transcriptional inducers. Conversely, it has been shown in the case of the MDR transporter P-glycoprotein that compounds that are not substrates can nevertheless induce transcription of the mdr1 gene (50Santoni-Rugiu E. Silverman J.A. Carcinogenesis. 1997; 18: 2255-2263Crossref PubMed Scopus (21) Google Scholar). Taken together, these observations suggest that there is no strict correlation between transporter substrates and inducers.Finally, we find that FLR1 overexpression confers hypersensitivity to H2O2 (Fig. 5). This phenotype is not only caused by FLR1 overexpression since deletion of the gene causes the cells to be more tolerant to H2O2, indicating that the endogenous levels of FLR1 are sufficient to sensitize the cells to this compound. Yap1p-mediated tolerance to H2O2 has been attributed to a number of genes, including TRX2,GLR1, and TSA1 (5Kuge S. Jones N. EMBO J. 1994; 13: 655-664Crossref PubMed Scopus (385) Google Scholar, 9Grant C.M. Collinson L.P. Roe J.-H. Dawes I.W. Mol. Microbiol. 1996; 21: 171-179Crossref PubMed Scopus (212) Google Scholar, 10Inoue Y. Matsuda T. Sugiyama K. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (221) Google Scholar, 12Lee 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). Our results suggest that Yap1p-mediated response to H2O2represents the sum of opposing phenotypes (namely H2O2 resistance conferred by TRX2/GLR1/TSA1 and H2O2 hypersensitivity conferred by FLR1) with the contribution of the targets conferring resistance exceeding that of FLR1 such that tolerance is the net result. However, we find that overexpression of YAP1results in H2O2 hypersensitivity, suggesting that, under such conditions resulting in strong FLR1transactivation (Fig. 2), the contribution of FLR1 exceeds that of the other Yap1p targets. This proposition is supported by the finding that YAP1 overexpression in an flr1Δ background results in H2O2 resistance rather than hypersensitivity (Fig. 5 A). Moreover, constitutively active mutants of Yap1p lacking the CRD have also been shown to confer resistance to diamide but hypersensitivity to H2O2, although the same mutants were transcriptionally competent in the presence of either diamide or H2O2 (24Coleman S.T. Epping E.A. Steggerda S.M. Moye-Rowley W.S. Mol. Cell. Biol. 1999; 19: 8302-8313Crossref PubMed Scopus (128) Google Scholar). Given our results, it is possible that the H2O2 hypersensitivity observed for these mutants results from the transactivation of FLR1. The mechanism by which FLR1 expression sensitizes the cells to H2O2 is not known. However, this phenomenon is not restricted to major facilitators since overexpression of the CDR2 gene, which codes for a transporter of the ATP binding cassette family in Candida albicans, was also found to confer diamide resistance but H2O2hypersensitivity. 2S. Weber, C. Gauthier, A.-M. Alarco, R. Daoud, E. Georges, and M. Raymond, submitted for publication. It remains to be seen if transporter-mediated H2O2hypersensitivity is specific for FLR1 or also extends to other transporters regulated by Yap1p such as YCF1 and ATR1 (13Wemmie 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, 14Coleman S.T. Tseng E. Moye-Rowley W.S. J. Biol. Chem. 1997; 272: 23224-23230Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). The Saccharomyces cerevisiae transc
Referência(s)