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 identified in this by to not for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by proteins and not or cysteine and the induction was observed by identified in this by amino acid and glutathione has not been identified on two-dimensional and the induction was at the level by cysteine reductase to response to of to to identified in this by The stimulation index is the ratio of protein expression in cadmium versus standard for Ctt1p, Sod1p, and Trr1p were reported though these enzymes were not induced by Proteins identified in this by not or cysteine and the induction was observed by has not been identified on two-dimensional and the induction was at the level by not in a Proteins induced by cadmium were identified by two-dimensional of cell with a gel containing more than 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, H. F. I. Labarre J. Perrot M. 1996; PubMed Scopus (66) Google C. Lagniel G. J. J.M. S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) with the exception of previously proteins were identified by a of A. M. M. Anal. Chem. 1996; PubMed Scopus Google Scholar). and were used J. T. Molecular a Scholar). Strains andgpx3Δ::TRP1 were by a that the or by the A. A. F. C. Res. 1993; PubMed Scopus Google Scholar). The was by a C. G.R. PubMed Scopus Google Scholar), which the YAP1 from to the to the and it with the The strain used in the was by of the previously described (14Lee J. Spector D. Godon C. Labarre J. Toledano M.B. J. Biol. 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Full Text Full Text PDF PubMed Scopus Google were used as We analyzed the proteomic response to cadmium to which be important for the to this toxic cells were or treated with 1 mm cadmium for with and analyzed by two-dimensional gel electrophoresis The in spot intensity between and treated cells were quantified by and software analysis and than 50 proteins were induced by a factor than 2 after cadmium treatment 40 other proteins were A lower of cadmium or of treatment a similar of protein but with lower induction Proteins induced by cadmium enzymes with antioxidant proteins, enzymes of the sulfur amino acid and other proteins or with proteins repressed by cadmium were of the and The particularly high induction level of enzymes of the sulfur amino acid an increased synthesis of cysteine and of which is essential in the cellular of cadmium. this hypothesis, we GSH synthesis by after [35S]methionine cadmium the synthesis of GSH was increased more than 1 in from cells the of GSH GSH was and The of was also in response to cadmium but we not this synthesis is increased after cadmium treatment. of thin layer chromatography of metabolites from [35S]methionine-labeled A and the wild-type and the strains were or treated with for 30 min and labeled with [35S]methionine for 40 Cells were as described under and The radioactive observed on the are the acid forms of cysteine and The stimulation of GSH with a more than of level after 1 of cadmium treatment and D.W. D.J. Microbiol. 1997; PubMed Scopus Google Scholar). metallothioneins are and important for cadmium we also analyzed and increased expression of this in response to cadmium, but a results strongly that the cysteine was induced to glutathione Strains lacking the Yap1p are very sensitive to cadmium and strains overexpressing this 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 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 analyzed the cadmium proteomic response in strains lacking either YAP1 or or overexpressing The of the proteomic response between these and the wild-type cells is in Table I. The strain a induction of 9 of the proteins, by a factor of 2 or antioxidant defense proteins the the thioredoxin and the and other proteins and for these proteins were overexpressed in response to cadmium in of the proteins and were also overexpressed in In only two proteins and were not induced by cadmium. However, Ctt1p, Sod1p, and several proteins and were overexpressed in response to cadmium. the importance of GSH in cellular cadmium we also the of GSH in these as we in the wild-type strain. The GSH was in and in However, GSH was not in Proteins with both a defective cadmium induction in and an increased expression in be important for Cd2+ 5 of 9 proteins this were for their role in Cd2+ tolerance the thioredoxin reductase or of and in a tolerance to cadmium However, the of either thioredoxin not the tolerance to cadmium, that these two proteins can for each other in this In strains carrying a of either a wild-type tolerance to cadmium not that the strain has a wild-type tolerance an (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 is sensitive to this metal. However, the cadmium phenotype of this strain was to an not of the Cd2+ toxic effects is to be the of toxic lipid It was to that the strain of the an important hydroperoxide reductase, is not defective in cadmium tolerance. the glutathione is also against T. S. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar), we analyzed the cadmium of the strain. this is clearly sensitive to hydroperoxide T. S. A. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google and data not it a cadmium into the biological effects of to to its we have analyzed the proteomic response to this toxic metal. proteins were to be induced by enzymes of the cysteine and glutathione and proteins with antioxidant In addition, the induction of several of these proteins is by the transcription factor The proteomic response to cadmium has a strong induction of enzymes of the sulfur amino acid and GSH particularly the of the cysteine and the of glutathione This increased expression was with a strong stimulation of GSH results are consistent with the that GSH as a of defense against cadmium toxicity by chelation and of the toxic metal. In cadmium is sequestered in the vacuole upon of Cd2+·(GSH)2 complexes by the ATP-binding cassette (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 system is by the hypersensitive phenotype resulting from the of either M.S. Wemmie J.A. Moye-Rowley W.S. Thiele D.J. J. Biol. Chem. 1994; 269: Full Text PDF PubMed Google Scholar) A.L. Moye-Rowley W.S. Biol. 1994; PubMed Google Scholar). The cysteine biosynthetic is also important as by the cadmium hypersensitive phenotype of strains for not This is probably essential for the increased of GSH under these stress conditions. The of expression with enzymes of the sulfur amino acids that a mechanism is in these with this a J. J. D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) has that the by cadmium is dependent upon the transcription factors and which to the of It was also that the of genes J. J. D.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the sulfur amino acid is not induced but repressed under of the other stress the oxidative stress C. Lagniel G. J. J.M. S. Perrot M. Boucherie H. Toledano M.B. Labarre J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar), the stress J. A. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar), and the response Lagniel G. Perrot M. F. A. M. Labarre J. Microbiol. 1999; PubMed Scopus Google Scholar). it is that the enzymes in amino acid only of the sulfur amino acid biosynthetic are which is consistent with a very Cd2+ is not a redox metal However, it oxidative stress and lipid by this to for the This is on an in lipid peroxidation after to Cd2+ (1Stohs S.J. Bagchi D. Free Radic. Biol. Med. 1995; 18: 321-336Crossref PubMed Scopus (3583) Google Scholar, S. M. Srivastava PubMed Scopus Google Scholar, Microbiol. 1997; PubMed Scopus Google Scholar, Microbiol. 1997; PubMed Google Scholar, S. P. D. PubMed Scopus Google Scholar). In addition, yeast strains for and and and are hypersensitive to cadmium (3Brennan R.J. Schiestl R.H. Mutat. Res. 1996; 356: 171-178Crossref PubMed Scopus (165) Google Scholar). The that several as and are induced by cadmium is consistent with this We also that strains for which two are not sensitive to cadmium, which not support the that cadmium toxicity is to the cellular of lipid peroxidation However, we the that yeast tolerance to lipid peroxidation other than or which have not yet been It is also that cadmium to oxidative stress by the cellular thiol redox We that is induced by cadmium and that strains for both and or for are hypersensitive to this metal. In support of a a has that cadmium thioredoxin and glutathione D.W. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the essential of these Biol. 1996; PubMed Scopus Google Scholar), thiol inactivation be the effect of cadmium. In addition, the inactivation of the thiol also result in an in cellular lipid peroxidation as a of their of the for the thiol and GSH The that 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, 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 A.L. Moye-Rowley W.S. Biol. 1994; PubMed Google Scholar), that this might genes important for cadmium and are two of A.L. Moye-Rowley W.S. Biol. 1994; 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). the importance of the these two genes are probably the by which Yap1p a of cadmium tolerance. In with this we that GSH synthesis is by We also that nine other genes the of Yap1p for their induction by cadmium. antioxidant defense genes also induced by H2O2 in a (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 and were induced by Cd2+ in Table suggesting the of other for the cadmium This not the Yap1p factor because a strain for both YAP1 the same proteomic response to cadmium than strain of the Yap1p-dependent proteins were also overexpressed in strain after cadmium treatment. which was not observed in the J. J. Labarre, and M. with the of the strain Cd2+ but not Skn7p is an important of the H2O2 response that with Yap1p to 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, is also important for the cadmium the of Skn7p in this response is more because 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 phenotype not be by a defective Cd2+ or by the of metallothioneins not or glutathione However, this be by the that several proteins of the cadmium were in the the that Skn7p to repress these genes upon cadmium treatment. This was particularly striking for proteins and for Yap1p-dependent proteins of or the YAP1 strain the same of proteins, suggesting a between the cadmium phenotype and the of these proteins. of these proteins, yet to be might be important for cadmium We A. for and D. for providing J. for analysis, and J. Garin and S. for