Title: A Proteome Analysis of the Cadmium Response in Saccharomyces cerevisiae
Abstract: Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium. Cadmium is very toxic at low concentrations, but the basis for its toxicity is not clearly understood. We analyzed the proteomic response of yeast cells to acute cadmium stress and identified 54 induced and 43 repressed proteins. A striking result is the strong induction of 9 enzymes of the sulfur amino acid biosynthetic pathway. Accordingly, we observed that glutathione synthesis is strongly increased in response to cadmium treatment. Several proteins with antioxidant properties were also induced. The induction of nine proteins is dependent upon the transactivator Yap1p, consistent with the cadmium hypersensitive phenotype of the YAP1-disrupted strain. Most of these proteins are also overexpressed in a strain overexpressing Yap1p, a result that correlates with the cadmium hyper-resistant phenotype of this strain. Two of these Yap1p-dependent proteins, thioredoxin and thioredoxin reductase, play an important role in cadmium tolerance because strains lacking the corresponding genes are hypersensitive to this metal. Altogether, our data indicate that the two cellular thiol redox systems, glutathione and thioredoxin, are essential for cellular defense against cadmium. polymerase chain reaction, WT, wild-type Heavy metals represent major environmental hazards to human health. In particular, cadmium is very toxic and probably carcinogenic at low concentrations. However, the biological effects of this metal and the mechanism of its toxicity are not yet clearly understood. It has been proposed that Cd2+ ions might displace Zn2+ and Fe2+ in proteins (1Stohs S.J. Bagchi D. Free Radic. Biol. Med. 1995; 18: 321-336Crossref PubMed Scopus (3583) Google Scholar), resulting in their inactivation and in the release of free iron, which might generate highly reactive hydroxyl radicals (OH⋅) (2Halliwell B. Gutteridge J.M.C. Biochem. J. 1984; 219: 1-14Crossref PubMed Scopus (4577) Google Scholar). In support of this hypothesis, a major effect of cadmium is oxidative stress (3Brennan R.J. Schiestl R.H. Mutat. Res. 1996; 356: 171-178Crossref PubMed Scopus (165) Google Scholar), particularly lipid peroxidation (1Stohs S.J. Bagchi D. Free Radic. Biol. Med. 1995; 18: 321-336Crossref PubMed Scopus (3583) Google Scholar). However, it is not known whether these effects are responsible for the extreme toxicity of the metal. Living organisms use several mechanisms to counter cadmium toxicity. In bacteria, efflux pumps are able to export toxic ions outside the cell (4Silver S. Phung L.T. Annu. Rev. Microbiol. 1996; 50: 753-789Crossref PubMed Scopus (1063) Google Scholar). In higher eukaryotes, Cd2+ is sequestered by metallothioneins through their high cysteine content (5Hamer D. Annu. Rev. Biochem. 1986; 55: 913-951Crossref PubMed Google Scholar). Cadmium can also be detoxified by chelation to GSH or to phytochelatin, a glutathione polymer of general structure (γ-Glu-Cys)n-Gly synthesized from GSH in plants and in the yeastSchizosaccharomyces pombe. Cd2+-phytochelatin and Cd2+·(GSH)2 complexes are transported into the vacuole by ATP-binding cassette transporters (6Li Z.S. Lu Y.P. Zhen R.G. Szczypka M. Thiele D. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar, 7Ortiz D.F. Ruscitti T. McCue K.F. Ow D.W. J. Biol. Chem. 1995; 270: 4721-4728Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 8Wemmie 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). Yap1p and Skn7p are yeast transcription factors that regulate the adaptive response to oxidative stress (9Lee 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 (466) Google Scholar, 10Morgan 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, 11Toone W.M. Jones N. Curr. Opin. Genet. Dev. 1999; 9: 55-61Crossref PubMed Scopus (100) Google Scholar). Strains lacking either transcription factor are sensitive to H2O2 and are defective in the induction by H2O2 of several enzymes with antioxidant properties (9Lee 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 (466) Google Scholar). Yap1p is also important in cadmium tolerance because yap1-deleted strains are very sensitive to cadmium, and strains overexpressingYAP1 are hyper-resistant to this toxic metal (12Wu A.L. 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). The contribution of Skn7p to the cadmium response is more complex, becauseskn7-deleted strains are hyper-resistant to cadmium (9Lee 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 (466) Google Scholar), suggesting that Skn7p is not only dispensable for this response but might also repress genes that are important for cadmium tolerance. Using two-dimensional gel electrophoresis, we have identified several proteins induced by cadmium in Saccharomyces cerevisiae, providing a framework to the mechanisms of Cd2+ toxicity and cellular protection against this toxic metal. In particular, our results highlight the importance of both glutathione and of the thioredoxin/thioredoxin reductase system in the cellular defense against cadmium. Studies were performed with the wild-type strain YPH98 (13Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) (MATa ura3–52lys2–801amber ade2–101ochre trp1Δ1 leu2-Δ1). All disruptants and strains used in this study were isogenic derivatives of YPH98. The strain overexpressing YAP1 and the mutantskn7Δ::TRP1 were previously described (9Lee 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 (466) Google Scholar). Strains tsa1Δ::TRP1,trx1Δ::TRP1,trx2Δ::LEU2,trx1Δ::TRP1-trx2Δ::LEU2and trr1Δ::TRP1 were previously described (14Lee J. Spector D. Godon C. Labarre J. Toledano M.B. J. Biol. Chem. 1999; 274: 4537-4544Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Strains yap1Δ::TRP1,gre2Δ::TRP1,ahp1Δ::TRP1 andgpx3Δ::TRP1 were constructed in this work. The strain gsh1ΔPro2–2 has been described. 1D. Spector, J. Labarre, and M. Toledano (2000)J. Biol. Chem. 276, in press. The strain overexpressing YAP1 (mcYAP1) was obtained by transformation of strain YPH98 by a 2-μm plasmid pRS425 carrying a 2.5-kilobaseEcoRI genomic YAP1 fragment. Cells were grown in liquid medium containing 0.67% yeast nitrogen base without amino acids (YNB, Difco), 2% glucose supplemented with uracil, adenine, lysine, tryptophan, and leucine at a final concentration of 30 mg/liter. TheYAP1-overexpressing strain was grown in the same medium but without leucine. Aliquots from an exponential growth culture (A 600, 0.5) were serially diluted in water (10-fold at each step). Ten μl were spotted onto rich broth (YPD) plates containing different cadmium sulfate concentrations: 0, 25, 50, 75, 100, 150 and 200 μm. Plates were incubated at 30 °C for 2 to 5 days, and the colony-forming units were counted. Ten-ml cultures were inoculated with a colony and cultivated overnight aerobically at 30 °C with shaking. Two-ml aliquots of mid-log phase culture (A 600, 0.4) were withdrawn and treated with 0, 0.5, 1, or 1.5 mm cadmium sulfate. 15 or 45 min after cadmium addition, cells were labeled with [35S]methionine (200 μCi) for 15 min for analysis of protein expression or for 40 min for analysis of glutathione synthesis rate. [35S]methionine-labeled cells (1 ml) were collected by centrifugation, washed in 200 μl of water, and resuspended in 50 μl of water. Cells were then boiled for 5 min to extract metabolites (15Ramos F. Thuriaux P. Wiame J.M. Bechet J. Eur. J. Biochem. 1970; 12: 40-47Crossref PubMed Scopus (71) Google Scholar) and centrifuged to obtain the35S-labeled metabolites in the supernatant. For analysis, the metabolites were oxidized by adding equal volume of supernatant and performic acid (16Kumari K. Khanna P. Ansari N.H. Srivastava S.K. Anal. Biochem. 1994; 220: 374-376Crossref PubMed Scopus (14) Google Scholar). This treatment converts both oxidized and reduced forms of glutathione to glutathione-sulfonic acid. Samples (0.1 μl) were then applied on cellulose thin layer chromatography in the following solvent system: butanol-1/acetic acid/water (90:15:33). The 35S-labeled metabolites were quantified by phosphor technology (Phosphor-Imager, Molecular Dynamics). Protein extraction and two-dimensional gel electrophoresis were performed as previously described (17Maillet I. Lagniel G. Perrot M. Boucherie H. Labarre J. J. Biol. Chem. 1996; 271: 10263-10270Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar) with a Millipore Investigator apparatus. The radioactive gels were recorded by PhosphorImager and were analyzed with two-dimensional gel analysis software (Melanie II, Bio-Rad). The spot intensities were obtained in pixel units and normalized to the total radioactivity of the gel. The cadmium stimulation index was calculated as the ratio of spot intensity between cadmium and standard conditions. Proteins with a stimulation index of higher than 1.8 are reported in Table I. Proteins with a stimulation index lower than 0.60 (repressed proteins) are: ACS2, ADH1, ADH2, ADK1, ALD6, ARG1, BEL1, COF1, CPA2, EFB1, EFT1, EGD1, EGD2, FBA1, GDH1, GLN1, GPP1, HIS4, HOM2, ILV2, ILV3, ILV5, KRS1, LYS9, LYS20, PDC1, PUB1, PRO2, RPA0, RPA2, RPL45, RPS5, SSB1, SSB2, TDH2, TIF1, TIF45, TIF51A, TPI1, VMA1, YEF3, YJL200C, and YKL056C. The other analyzed proteins with stimulation indices lower than 1.8 and higher than 0.60 are not reported in Table I (with the exception of CTT1, TRR1, and SOD1). Mutant and wild-type strains were compared by their ratio of spot intensities under different cadmium conditions. The experiments were performed at least twice with similar results, and the mean value was reported here. The S.D. of the analysis ranged from 20 to 25%.Table IIdentification of proteins induced by 1 mm Cd and effects of Yap1p and Skn7p on its inductionProtein classGene nameStimulation index1-aThe stimulation index is the ratio of protein expression in cadmium versus standard conditions.Protein functionRatio of expression level in 1 mM Cd2+conditionyap1/WTskn7/WTmcYAP1/WTEnzymes with antioxidant propertiesAHP13.0alkyl hydroperoxide reductase0.470.81.6CCP12.6cytochrome c peroxidase0.951.62.1CTT11-bResults for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by cadmium.1.0catalase T0.92.32.4TRR11-bResults for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by cadmium.0.9thioredoxin reductase0.340.81.2TRX23.3thioredoxin0.631.31.4TSA13.1thioperoxidase0.280.81.7PRX11-cProteins identified in this work by mass spectrometry.>5thioperoxidase, homolog to TSA1nd1-fnd, not determined.nd>5SOD11-bResults for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by cadmium.0.7Cu/Zn superoxide dismutase0.851.81.8SOD23.8Mn superoxide dismutase0.481.25.5Heat shock proteins and chaperonesHSP122.0heat shock protein1.11.41.1HSP261-dHsp26p does not contain methionine or cysteine residues, and the induction was qualitatively observed by Coomassie Blue staining.>3.0heat shock proteinndndndHSP42>10heat shock protein1.21.12.2HSP825.1heat shock protein1.0ndndHSP104>10heat shock protein1.052.43.7KAR2>20heat shock protein0.91.12.4SSA14.8heat shock protein1.81.42.9SSA26.9heat shock protein0.721.41.8SSA4>10heat shock protein1.823.76.3STI15.1heat shock protein2.01.41.5ProteasesHSP786.8mitochondrial protease2.31.11.8PRE13.1proteasome subunit1.21.91.7PUP25.4proteasome subunitnd0.91.43MPR13.8proteasome subunit1.15.06.1UBA15.4ubiquitin activating enzyme0.850.60.4YTA11-cProteins identified in this work by mass spectrometry.3.0proteasome subunitnd1.21.5Sulfur amino acid and glutathione biosynthesis enzymesCYS314.3cystathionine-gamma lyase0.880.81.05CYS42.2cystathionine-beta-synthase0.720.90.6GSH11-eGsh1p has not been identified on two-dimensional gels, and the induction was measured at the mRNA level by RT-PCR analysis.>10gamma glutamyl cysteine synthase<0.21.11.05MET165.2PAPS reductase1.11.50.5MET253.5homocysteine synthase1.00.850.8MET36.3ATP sulfurylase2.02.1ndECM172.5sulfite reductase (beta subunit)ndndndSAM14.4S-adenosylmethionine synthetase0.50.70.4SAM22.1S-adenosylmethionine synthetase0.731.01.1Other metabolism enzymesADE32.2C1-tetrahydrofolate synthase1.50.8ndSHM22.7serine hydroxymethyltransferase1.030.60.14ENO12.5enolase1.11.051.5DAK12.9dihydroxyacetone kinase0.81.82.0TPS12.1trehalose-6-phosphate synthase1.071.31.1TAL110.7transaldolase0.961.651.6ZWF13.2glucose 6-phosphate dehydrogenase1.10.80.3HXK12.1hexokinasendndndPGK11.8phosphoglycerate kinase0.90.850.85IPP11.8inorganic pyrophosphatase1.21.051.05Unclassified proteinsBMH21.8homolog to 14–3-3 protein0.840.91.0CDC482.3ATPases family0.680.60.75DDR487.0DNA damage response protein0.70.81.3GRE21.6similar to dihydroflavonol-4-reductase0.531.42.1OYE36.1NADPH-dehydrogenase0.382.35.8RNR45.6ribonucleotide reductase2.31.80.9WTM12.0transcriptional regulator1.541.41.2YST24.1ribosomal protein0.90.60.5TFS12.4suppressor of CDC25 mutation1.071.52.2YLR179C3.0similar to Tfs1p0.86nd0.5YNL274C3.5similar to alpha-ketoisocaproate reductase0.450.852.3YKL117W2.1Ste5p-associated protein1.011.11.3YGL037C2.2unknown function1.21.151.4YNL134C3.1unknown function0.321.82.4YOR007C 1-cProteins identified in this work by mass spectrometry.2.3unknown function0.931.11.31-a The stimulation index is the ratio of protein expression in cadmium versus standard conditions.1-b Results for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by cadmium.1-c Proteins identified in this work by mass spectrometry.1-d Hsp26p does not contain methionine or cysteine residues, and the induction was qualitatively observed by Coomassie Blue staining.1-e Gsh1p has not been identified on two-dimensional gels, and the induction was measured at the mRNA level by RT-PCR analysis.1-f nd, not determined. Open table in a new tab Proteins induced by cadmium were all identified by matching two-dimensional maps of cadmium-treated cell extracts with a reference gel containing more than 450 previously identified proteins (17Maillet I. Lagniel G. Perrot M. Boucherie H. Labarre J. J. Biol. Chem. 1996; 271: 10263-10270Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 18Boucherie H. Sagliocco F. Joubert R. Maillet I. Labarre J. Perrot M. Electrophoresis. 1996; 17: 1683-1699Crossref PubMed Scopus (66) Google Scholar,19Godon 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 (499) Google Scholar) with the exception of 3 new spots. These previously uncharacterized proteins were identified by a peptide mass mapping approach using matrix-assisted laser desorption ionization-time of flight mass spectrometry (20Shevchenko A. Wilm M. Vorm O. Mann M. Anal. Chem. 1996; 68: 850-858Crossref PubMed Scopus (7831) Google Scholar). Standard protocols and buffers were used (21Sambrook J. Fritsch E.F. Maniatis T. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). Strains gre2Δ::TRP1 andgpx3Δ::TRP1 were prepared by a one step PCR2 amplification protocol that replaced the entire GRE2 or GPX3open-reading frame by the TRP1 gene (22Baudin A. Ozier-Kalogeropoulos O. Danouel A. Lacroute F. Cullin C. Nucleic Acids Res. 1993; 21: 3329-3330Crossref PubMed Scopus (1127) Google Scholar). The mutantyap1Δ was created by a one-step gene disruption technique (23Guthrie C. Fink G.R. Methods Enzymol. 1991; 194: 273-362Crossref PubMed Scopus (163) Google Scholar), which removed the YAP1 coding sequence from theBamHI site (+186) to the KpnI site (+1650) relative to the ATG and replaced it with the TRP1 gene. The ahp1Δ::TRP1 strain used in the present work was generated by one back-cross of the previously described ahp1Δ::TRP1 (14Lee J. Spector D. Godon C. Labarre J. Toledano M.B. J. Biol. Chem. 1999; 274: 4537-4544Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) with a wild-type strain. Quantitative reverse transcriptase-PCR analysis was performed essentially as previously described (19Godon 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 (499) Google Scholar).ACT1 reverse transcriptase-PCR products were used as internal standards. We analyzed the proteomic response to cadmium to identify activities, which could be potentially important for the resistance to this toxic compound. Exponentially growing cells were untreated or treated with 1 mm cadmium for 60 min, pulse-labeled with [35S]methionine, and analyzed by comparative two-dimensional gel electrophoresis (Fig.1). The changes in spot intensity between untreated and treated cells were quantified by phosphorimager and software analysis (see "Materials and Methods"). More than 50 proteins were induced by a factor greater than 2 after cadmium treatment (Table I). Concomitantly, about 40 other proteins were significantly repressed. A lower dose of cadmium (0.5 mm) or shorter period of treatment (15 min) gave a similar pattern of protein expression, but with lower induction levels. Proteins induced by cadmium included enzymes with antioxidant properties, heat shock proteins, proteases, enzymes of the sulfur amino acid biosynthesis pathway, carbohydrate metabolism enzymes, and other unclassified proteins or with unknown function. Conversely, proteins repressed by cadmium were mainly components of the translational apparatus and metabolic enzymes. The particularly high induction level of enzymes of the sulfur amino acid pathway (Fig.2) suggested an increased synthesis of cysteine and perhaps of GSH, which is essential in the cellular detoxification of cadmium. To test this hypothesis, we directly measured GSH synthesis rate by TLC after [35S]methionine pulse labeling. After cadmium treatment, the synthesis rate of GSH was increased more than 4-fold (Fig.3 A, lanes 1 and2). As expected, in extracts from cells lackingGSH1 (encoding the rate-limiting enzyme of GSH biosynthesis), no GSH was produced (Fig. 3 A, lanes 3 and 4). The production of another35S-labeled compound was also augmented in response to cadmium treatment, but we could not identify this molecule.Figure 3Glutathione synthesis rate is increased after cadmium treatment. Autoradiograms of thin layer chromatography of intracellular metabolites from [35S]methionine-labeled cells. A and B, the wild-type and the mutant strains were left untreated (−) or treated with 0.5 mmCd2+ for 30 min (+) and labeled with [35S]methionine for 40 min. Cells were further processed as described under "Materials and Methods." The radioactive spots observed on the figure are the sulfonic acid forms of cysteine and glutathione.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The stimulation of GSH biosynthesis correlated with a more than 10-fold increase of GSH1 mRNA level after 1 h of cadmium treatment (not shown and Ref. 24Stephen D.W. Jamieson D.J. Mol. Microbiol. 1997; 23: 203-210Crossref PubMed Scopus (61) Google Scholar). As metallothioneins are cysteine-rich and potentially important for cadmium detoxification, we also analyzed CUP1 mRNA levels and found no increased expression of this gene in response to cadmium, but rather a slight repression. These results strongly suggest that the cysteine biosynthesis pathway was induced to allow enhanced glutathione synthesis. Strains lacking the transcriptional activator Yap1p are very sensitive to cadmium and conversely, strains overexpressing this regulator are hyper-resistant (12Wu A.L. 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). However, strains lacking the transcriptional activator Skn7p are hyper-resistant to this toxic metal (9Lee 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 (466) Google Scholar). We therefore analyzed the cadmium proteomic response in strains lacking either YAP1 (yap1Δ) or SKN7(skn7Δ) or overexpressing YAP1(mcYAP1) (Fig. 4). The comparative measure of the proteomic response between these mutants and the wild-type cells is given in Table I. The yap1Δ strain had a weaker induction of 9 of the 57 responsive proteins, by a factor of 2 or more. These included four antioxidant defense proteins (the superoxide dismutase Sod2p, the alkyl hydroperoxidase Ahp1p, the thioredoxin Trx2p, and the thioperoxidase Tsa1p) and five other proteins (Sam1p, Oye3p, Gre2p, YNL134C, and YNL274C). Except for Sam1p, all these proteins were significantly overexpressed in response to cadmium in mcYAP1. Additionally, most of the heat shock proteins and proteasome subunits were also overexpressed in mcYAP1 (Table I). In skn7Δ, only two proteins (Cdc48p and Shm2p) were not properly induced by cadmium. However, surprisingly, Ctt1p, Ccp1p, Sod1p, Oye3p, Gre2p, YNL134C, and several heat shock proteins and proteasome subunits were overexpressed in response to cadmium. Given the importance of GSH in cellular cadmium protection, we also measured the biosynthesis of GSH in these mutants as we did in the wild-type strain. The GSH biosynthesis rate was significantly decreased in yap1Δ and augmented in mcYAP1(Fig.3 B, lanes 5–7). However, GSH biosynthesis rate was not modified in skn7Δ. Proteins with both a defective cadmium induction in yap1Δ and an increased expression in mcYAP1 could be important for Cd2+ protection. 5 of 9 proteins matching this criteria were therefore evaluated for their role in Cd2+ tolerance (Fig.5 A). Deletion ofTRR1 encoding the thioredoxin reductase or of bothTRX1 and TRX2 resulted in a significant decreased tolerance to cadmium (Fig. 5). However, the deletion of either thioredoxin does not affect the tolerance to cadmium, showing that these two proteins can substitute for each other in this function. In contrast, strains carrying a deletion of either GRE2, TSA1,or AHP1 had a wild-type tolerance to cadmium (data not shown). Note that the ahp1Δ strain has a wild-type tolerance contradicting an earlier report (14Lee J. Spector D. Godon C. Labarre J. Toledano M.B. J. Biol. Chem. 1999; 274: 4537-4544Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar) that demonstratesahp1Δ is sensitive to this metal. However, the cadmium phenotype of this strain was shown related to an additional unidentified mutation (data not shown). One of the main Cd2+ toxic effects is believed to be the generation of toxic lipid peroxides. It was therefore surprising to find that the strain deleted of AHP1, the gene encoding an important alkyl hydroperoxide reductase, is not defective in cadmium tolerance. Because another enzyme, the glutathione peroxidase Gpx3p, is also active against organic peroxides (25Inoue Y. Matsuda T. Sugiyama K.-i. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar), we analyzed the cadmium resistance of the GPX3-disrupted strain. Although this mutant is clearly sensitive to tert-butyl hydroperoxide (t-BOOH, Ref. 25Inoue Y. Matsuda T. Sugiyama K.-i. Izawa S. Kimura A. J. Biol. Chem. 1999; 274: 27002-27009Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar and data not shown), it showed a normal cadmium resistance (Fig. 5). To gain insight into the biological effects of Cd2+and to identify activities relevant to its detoxification, we have analyzed the proteomic response to this toxic metal. Fifty-seven proteins were found to be induced by Cd2+, including enzymes of the cysteine and glutathione biosynthesis pathway and proteins with antioxidant properties. In addition, the induction of several of these proteins is controlled by the transcription factor Yap1p. The proteomic response to cadmium has revealed a strong induction of eight enzymes of the sulfur amino acid and GSH biosynthesis pathways, particularly the last enzyme of the cysteine biosynthesis pathway, Cys3p, and the rate-limiting enzyme of glutathione biosynthesis, GSH1. This increased gene expression was correlated with a strong stimulation of GSH biosynthesis. These results are consistent with the notion that GSH acts as a first line of defense against cadmium toxicity by chelation and sequestration of the toxic metal. In yeast, cadmium is sequestered in the vacuole upon transport of Cd2+·(GSH)2 complexes by the membrane ATP-binding cassette Ycf1 (6Li Z.S. Lu Y.P. Zhen R.G. Szczypka M. Thiele D. Rea P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 42-47Crossref PubMed Scopus (507) Google Scholar). The importance of this detoxification system is demonstrated by the hypersensitive phenotype resulting from the deletion of either YCF1 (26Szczypka M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: 22853-22857Abstract Full Text PDF PubMed Google Scholar) orGSH1 (27Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar). The cysteine biosynthetic pathway is also important as demonstrated by the cadmium hypersensitive phenotype of strains deleted for CYS4 (data not shown). This pathway is probably essential for the increased production of GSH under these stress conditions. The coordinated regulation of GSH1 expression with enzymes of the sulfur amino acids pathway may suggest that a common transcriptional mechanism is involved in these inductions. Consistent with this idea, a recent work (28Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellot J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar) has shown that the GSH1induction by cadmium is dependent upon the transcription factors Met4p, Met31p, Met32p, and Cbf1p, which belong to the transcriptional complex of MET genes. It was also found that the GSH1promotor contains functional elements typical of MET genes (28Dormer U.H. Westwater J. McLaren N.F. Kent N.A. Mellot J. Jamieson D.J. J. Biol. Chem. 2000; 275: 32611-32616Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). Interestingly, the sulfur amino acid pathway is not induced but rather repressed under most of the other stress conditions examined, including the oxidative stress (19Godon 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 (499) Google Scholar), the osmotic stress (29Norbeck J. Blomberg A. J. Biol. Chem. 1997; 272: 5544-5554Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar), and the heat shock response (30Boy-Marcotte E. Lagniel G. Perrot M. Bussereau F. Boudsocq A. Jacquet M. Labarre J. Mol. Microbiol. 1999; 33: 274-283Crossref PubMed Scopus (140) Google Scholar). Furthermore, it is remarkable that among the enzymes involved in amino acid metabolism, only those of the sulfur amino acid biosynthetic pathway are induced, which is consistent with a very specific control mechanism. Cd2+ is not a redox active metal ion. However, it could cause oxidative stress and lipid peroxidation, perhaps by displacing protein-bound Fe2+, allowing this ion to become available for the Fenton reaction. This hypothesis is based on previous studies showing an increase in lipid peroxidation products after exposure to Cd2+ (1Stohs S.J. Bagchi D. Free Radic. Biol. Med. 1995; 18: 321-336Crossref PubMed Scopus (3583) Google Scholar, 31Gupta S. Athar M. Behari J.R. Srivastava R.C. Ind. Health. 1991; 29: 1-9Crossref PubMed Scopus (50) Google Scholar, 32Howlett N.G. Avery S.V. Appl. Microbiol. Biotechnol. 1997; 48: 539-545Crossref PubMed Scopus (55) Google Scholar, 33Howlett N.G. Avery S.V. Appl. Environ. Microbiol. 1997; 63: 2971-2976Crossref PubMed Google Scholar, 34Sarkar S. Yadav P. Bhatnagar D. BioMetals. 1998; 11: 153-157Crossref PubMed Scopus (123) Google Scholar). In addition, yeast strains deleted for cytosolic copper and zinc and mitochondrial manganese superoxide dismutases (SOD1 and SOD2) are hypersensitive to cadmium (3Brennan R.J. Schiestl R.H. Mutat. Res. 1996; 356: 171-178Crossref PubMed Scopus (165) Google Scholar). The observation that several antioxidants such as Ahp1p, Ccp1p, Tsa1p, and Sod2p are induced by cadmium is consistent with this hypothesis. We also found that strains deleted for AHP1 orGPX3, which encode two main organic peroxide-scavenging activities, are not sensitive to cadmium, which does not support the idea that cadmium toxicity is related to the cellular generation of lipid peroxidation products. However, we cannot rigorously rule out the possibility that yeast tolerance to cadmium-generated lipid peroxidation products involves specific activities other than Ahp1p or Gpx3p, which have not yet been elucidated. It is also possible that cadmium indirectly contributes to oxidative stress by affecting the cellular thiol redox balance. We indeed found that Trx2p is significantly induced by cadmium and that strains deleted for both TRX1 and TRX2 or for TRR1 are hypersensitive to this metal. In support of such a mechanism, a recent work has shown that cadmium inactivates thioredoxins, thioredoxin reductases, and glutathione reductases (35Chrestensen C.A. Starke D.W. Mieyal J.J. J. Biol. Chem. 2000; 275: 26556-26565Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). Therefore, given the essential nature of these systems (36Muller E.G.D. Mol. Biol. Cell. 1996; 7: 1805-1813Crossref PubMed Scopus (166) Google Scholar), thiol transferase inactivation could be the primary deleterious effect of cadmium. In addition, the inactivation of the thiol transferase pathway could also result in an increase in cellular lipid peroxidation products as a consequence of their unavailability of the main electron donors for the thiol and GSH peroxidases. The observation that YAP1-deleted strains were hypersensitive to cadmium (9Lee 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 (466) Google Scholar, 12Wu A.L. 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) and strains overexpressing YAP1 were hyper-resistant (27Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar), suggested that this transcriptional activator might control genes important for cadmium tolerance.YCF1 and GSH1 are two of them (27Wu A.L. Moye-Rowley W.S. Mol. Cell. Biol. 1994; 14: 5832-5839Crossref PubMed Google Scholar, 8Wemmie 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). Given the importance of the Ycf1p-GSH detoxification system, these two genes are probably the primary targets by which Yap1p exerts a control of cadmium tolerance. In accord with this notion, we found that cadmium-induced GSH synthesis is controlled by Yap1p. We also discovered that nine other genes required the presence of Yap1p for their proper induction by cadmium. These mostly include antioxidant defense genes also induced by H2O2 in a Yap1-dependent manner (9Lee 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 (466) Google Scholar). Several other proteins of the Yap1p H2O2-inducible regulon (Ccp1p, Ssa1p, Hsp78p, Hsp82p, Mpr1p, Uba1p, Cys3p, Tal1p, Zwf1p, Dak1p, and Tps1p) were still induced by Cd2+ in yap1Δ (see Table I), suggesting the existence of other control pathways for the cadmium response. This pathway does not involve the Yap1p homologous factor Yap2p, because a strain deleted for both YAP1 andYAP2 had the same proteomic response to cadmium than theyap1Δ strain (not shown). Interestingly, most of the cadmium-inducible Yap1p-dependent proteins were also overexpressed in aYAP1 multicopy strain after cadmium treatment. ThisYAP1 gene dosage effect, which was not observed in the H2O2response 3C. Godon, J. Lee, J. Labarre, and M. Toledano, unpublished results. correlate well with the hyper-resistance of the YAP1-overexpressing strain toward Cd2+ but not toward H2O2. Skn7p is an important regulator of the H2O2 response that cooperates with Yap1p to activate the expression of several genes in response to H2O2 (9Lee 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 (466) Google Scholar). However, although Yap1 is also important for the cadmium response, the function of Skn7p in this response is more complex because skn7Δ is hyper-resistant to this metal (9Lee 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 (466) Google Scholar). This resistance phenotype could not be explained by a defective Cd2+ intracellular transport or alternatively, by the overproduction of metallothioneins (data not shown) or glutathione (this work). However, this could be explained by the observation that several proteins of the cadmium stimulon were superinduced in the skn7Δ mutant, supporting the idea that Skn7p may act to repress these genes upon cadmium treatment. This was particularly striking for heat shock proteins and for some Yap1p-dependent proteins of unknown function (YNL134C, Gre2p, or Oye3p). Interestingly, the YAP1 multicopy strain overexpresses nearly the same set of proteins, suggesting a correlation between the cadmium hyper-resistance phenotype and the overexpression of these proteins. Some of these proteins, yet to be studied, might be important for cadmium resistance. We thank A. Sentenac for encouragement and critical discussions, D. Thomas for providing cys4Δstrain, J. Lee for mRNA analysis, and J. Garin and S. Kieffer for mass spectrometry analysis.