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Pkc1 and the Upstream Elements of the Cell Integrity Pathway in Saccharomyces cerevisiae, Rom2 and Mtl1, Are Required for Cellular Responses to Oxidative Stress

2005; Elsevier BV; Volume: 280; Issue: 10 Linguagem: Inglês

10.1074/jbc.m411062200

1083-351X

Felipe Vilella, Enrique Herrero, Jordi Torres‐Rosell, María Ángeles de la Torre-Ruiz,

Plant Gene Expression Analysis

In this study we analyze the participation of the PKC1-MAPK cell integrity pathway in cellular responses to oxidative stress in Saccharomyces cerevisiae. Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidizing agents, diamide and hydrogen peroxide. Mtl1 is characterized for the first time as a cell-wall sensor of oxidative stress. We also show that the actin cytoskeleton is a cellular target for oxidative stress. Both diamide and hydrogen peroxide provoke a marked depolarization of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restore the correct actin organization. Diamide induces the formation of disulfide bonds in newly secreted cell-wall proteins. This mainly provokes structural changes in the cell outer layer, which activate the PKC1-MAPK pathway and hence the protein kinase Slt2. Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the machinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation. In this study we analyze the participation of the PKC1-MAPK cell integrity pathway in cellular responses to oxidative stress in Saccharomyces cerevisiae. Evidence is presented demonstrating that only Pkc1 and the upstream elements of the cell integrity pathway are essential for cell survival upon treatment with two oxidizing agents, diamide and hydrogen peroxide. Mtl1 is characterized for the first time as a cell-wall sensor of oxidative stress. We also show that the actin cytoskeleton is a cellular target for oxidative stress. Both diamide and hydrogen peroxide provoke a marked depolarization of the actin cytoskeleton, being Mtl1, Rom2 and Pkc1 functions all required to restore the correct actin organization. Diamide induces the formation of disulfide bonds in newly secreted cell-wall proteins. This mainly provokes structural changes in the cell outer layer, which activate the PKC1-MAPK pathway and hence the protein kinase Slt2. Our results led us to the conclusion that Pkc1 activity is required to overcome the effects of oxidative stress by: (i) enhancing the machinery required to repair the altered cell wall and (ii) restoring actin cytoskeleton polarity by promoting actin cable formation. Cells are constantly exposed to a series of environmental stresses, which are sensed by complex signal transduction pathways that are responsible for cellular damage repair and adaptation responses. These responses are mediated by (mitogen-activated protein kinase (MAPK) 1The abbreviations used are: MAPK, mitogen-activated protein kinase; PKC, protein kinase C; GFP, green fluorescent protein; HA, hemagglutinin; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol.1The abbreviations used are: MAPK, mitogen-activated protein kinase; PKC, protein kinase C; GFP, green fluorescent protein; HA, hemagglutinin; DTNB, 5,5′-dithiobis(2-nitrobenzoic acid); DTT, dithiothreitol. cascades in eukaryotic cells. In the Saccharomyces cerevisiae, the PKC1-MAPK cell integrity pathway is involved in responses to a wide variety of stresses, including heat-shock (1Kamada Y. Jung U.S. Piotrowski J. Levin D.E. Genes Dev. 1995; 9: 1559-1571Crossref PubMed Scopus (419) Google Scholar), hypoosmotic shock (2Davenport K.R. Sohaskey M. Kamada Y. Levin D.E. Gustin M.C. J. Biol. Chem. 1995; 270: 30157-30161Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar), nutritional stress (3Torres J. Di Como C.J. Herrero E. de la Torre-Ruiz M.A. J. Biol. Chem. 2002; 277: 43495-43504Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar), and those associated with any other injuries that alter the integrity of the outer layer of cells. These environmental signals are generally sensed by Mid2 (4Rajavel M. Philip B. Buehrer B.M. Errede B. Levin D.E. Mol. Cell. Biol. 1999; 19: 3969-3976Crossref PubMed Scopus (173) Google Scholar) and Wsc family (5Verna J. Lodder A. Lee K. Vagts A. Ballester R. Proc. Natl. Acd. Sci. U. S. A. 1997; 94: 13804-13809Crossref PubMed Scopus (315) Google Scholar) cell surface proteins. Mtl1 is a putative cell membrane sensor with 50% homology to Mid2 (6Ketela T. Green R. Bussey H. J. Bacteriol. 1999; 181: 3330-3340Crossref PubMed Google Scholar, 4Rajavel M. Philip B. Buehrer B.M. Errede B. Levin D.E. Mol. Cell. Biol. 1999; 19: 3969-3976Crossref PubMed Scopus (173) Google Scholar). Mtl1 has been characterized as a multicopy suppressor of the absence of Rgd1, a protein that functions as a GTPase-activating protein for Rho3 and Rho4 proteins (7De Bettignies G. Thoraval D. Morel C. Peypouquet M.F. Crouzet M. Genetics. 2001; 159: 1435-1448Crossref PubMed Google Scholar) and as a multicopy suppressor of Rho1 functions (8Sekiya-Kawasaki M. Abe M. Saka A. Watanabe D. Kono K. Minemura-Asakawa M. Ishihara S. Watanabe T. Ohya Y. Genetics. 2002; 162: 663-676Crossref PubMed Google Scholar). Cell surface sensors transmit signals to Rom2, a guanine exchange factor protein of the GTP-binding protein Rho1. Rho1 then activates the Pkc1 protein kinase, which in turn activates an MAPK module; Pkc1 phosphorylates Bck1, a MAPK kinase kinase, which transmits the signal to both MAPK kinases, Mkk1 and Mkk2. Those finally activate the last member of the cascade, Slt2/Mpk1, by phosphorylating both the Thr190 and Tyr192 residues of this MAPK (the PKC1-MAPK pathway reviewed in Refs 9Heinisch J.J. Lorberg A. Schmitz H.P. Jacoby J.J. Mol. Microbiol. 1999; 32: 671-680Crossref PubMed Scopus (287) Google Scholar and 10Hohmann S. Mol. Biol. Rev. 2002; 66: 300-372Crossref PubMed Scopus (1261) Google Scholar). Rlm1, a transcription factor involved in the activation of cell wall genes, and Swi6 (a transcription factor involved in cell cycle regulation) are both targets for Slt2. Activation of Slt2 correlates to activation of both downstream events (11Martin-Yken H. Dagkessamanskaia A. Basmaji F. Lagorce A. Francois J. Mol. Microbiol. 2003; 49: 23-35Crossref PubMed Scopus (51) Google Scholar) (Fig. 1). The upper elements of the PKC1-MAPK pathway are involved in the organization of the actin cytoskeleton upon cell wall stress. It has been reported that hyperactivation of either Pkc1 or Rho1 alone causes depolarization of actin cables in patches dispersed throughout the cytoplasm (12Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (241) Google Scholar). In addition, the function of both Rho1 and Pkc1 is necessary for actin to become depolarized following heat stress (12Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (241) Google Scholar). Actin cable assembly relies on the activity of the two redundant formins, Bni1 and Bnr1 (13Evangelista M. Pruyine D. Amberg D.C. Boone C. Bretscher A. Nat. Cell Biol. 2002; 4: 32-41Crossref PubMed Scopus (165) Google Scholar, 14Sagot I. Klee S.K. Pelman D. Nat. Cell Biol. 2002; 4: 42-50Crossref PubMed Scopus (326) Google Scholar). Rho1 is able to localize to actin patches (15Drgonova J. Drgon T. Tanaka K. Kollar R. Chen G.C. Ford R.A. Chan C.S. Takai Y. Cabib E. Science. 1996; 272: 277-279Crossref PubMed Scopus (301) Google Scholar) and is required for the activation of formins at high temperatures through Pkc1 (16Dong Y. Pruyne D. Bretscher A. J. Cell Biol. 2003; 161: 1081-1092Crossref PubMed Scopus (123) Google Scholar). This emphasizes the role of these proteins in polarized growth and morphogenesis. Oxygen is a vital molecule, but at the same time, it is responsible for provoking oxidative stress in cells, and causing cellular damage to various macromolecules (17Ames B. Shigenaga M. Hagen T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5354) Google Scholar). Budding yeast constitutes an optimal eukaryotic model for studying oxidative stress responses (18Toledano M.B. Delaunay A. Biteau B. Spector D. Azevedo D. Hohmann S. Hager P.W.H. Topics in Current Genetics. 1. Springer-Verlag, Berlin, Heidelberg2003: 242-303Google Scholar). However, although in the case of environmental stresses, such as osmotic, nutritional, and heat-shock stresses, there are specific signal transduction pathways that sense and transmit the different signals to specific components in S. cerevisiae (19Banuett F. Microbiol. Mol. Biol. Rev. 1998; 62: 249-274Crossref PubMed Google Scholar), there are no well characterized MAPK pathways for sensing and signaling oxidative stresses to the cytoplasm and nucleus. Nevertheless, there is considerable information in the literature detailing the nuclear transcriptional factors responsible for the induction of specific genes in response to oxidative stress in yeast. These are Yap1, Skn7, and Msn2/4 (for a review, see Ref. 18Toledano M.B. Delaunay A. Biteau B. Spector D. Azevedo D. Hohmann S. Hager P.W.H. Topics in Current Genetics. 1. Springer-Verlag, Berlin, Heidelberg2003: 242-303Google Scholar). In this study, we decided to investigate whether the cell integrity pathway in S. cerevisiae played a role in oxidative stress responses based on the following observations: (i) Skn7 has been shown to interact with the GTPase Rho1 (20Alberts A.S. Bouquin N. Johnston L.H. Treisman R. J. Biol. Chem. 1998; 273: 8616-8622Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar); (ii) it has been reported that Msn2/Msn4 participate with the PKC1-MAPK pathway in the compensatory mechanism that is triggered by cell wall mutations (21Lagorce A. Hauser N.C. Labourdette D. Rodriguez C. Martin-Yken H. Arroyo J. Hoheisel J.D. François J. J. Biol. Chem. 2003; 278: 20345-20357Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar); (iii) genome-wide analysis (22Gash A.P. Spellman P.T. Kao C.M. Carmel-Harel O. Eisen M.B. Storz G. Botstein D. Brown P.O. Mol. Biol. Cell. 2000; 11: 4241-4257Crossref PubMed Scopus (3680) Google Scholar) indicates that the agent diamide induces the activation of genes regulated by Rlm1; and (iv) in human cells, isoforms of PKC are known to be either activated or inactivated by oxidative stress, and this is causally associated with tumorigenesis (for a review see Ref. 23Gopalakrishna R. Jake S. Free Radic. Biol. Med. 2000; 28: 1349-1361Crossref PubMed Scopus (608) Google Scholar). We demonstrate that Pkc1, and the upper elements of the cell integrity pathway, are required for survival and adaptation to the oxidative stress provoked by two different oxidizing agents: hydrogen peroxide and diamide. We also report a new function for Mtl1 as a cell membrane receptor for oxidative stress. We present a detailed study demonstrating that the major effect caused by diamide is the oxidation of cell wall proteins, which activates the cell integrity pathway. Hydrogen peroxide exercises its action at the intracellular level, mainly by affecting a cellular function that is closely related to the polarization of the actin cytoskeleton. Finally, we demonstrate that restoration of cell polarity and recovery from oxidative stress occurs in a Pkc1-dependent manner. Yeast Strains and Gene Disruptions—The yeast strains used in this study are listed in Table I. MTL1 was disrupted by the one-step disruption method using the kanMX4 module (24Wach A. Brachat A. Pohlmann R. Philippsen P. Yeast. 1994; 10: 1793-1808Crossref PubMed Scopus (2218) Google Scholar), whereas the WSC4 gene was disrupted using the natMX4 module (25Goldstein A.L. McCusker J.H. Yeast. 1999; 15: 1541-1553Crossref PubMed Scopus (1341) Google Scholar).Table IYeast strains used in this workStrainRelevant genotypeReferenceAN3-5DMAT?, ura3-52, trp1, leu2-3,113, sec1tsObtained from L. CastilloCML125MATα leu2-3,112 ura3-52 trp1 his4 can1yde la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)CML128MATa as CML125Gallego, et al. (44Gallego C. Gari E. Colomina N. Herrero E. Aldea M. EMBO J. 1997; 16: 7196-7206Crossref PubMed Scopus (136) Google Scholar)CML399MATa slt2::URA3de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)DL2357MATa mtl1::HIS4 mid2::URA3Obtained from D. LevinMML200MATα bck1::kan Mx4de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)MML304MATα pkc1::LEU2(pBCK1-20)de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)MML344MATa pkc1::LEU2de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)MML357MATa wsc1::kanMx4de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)MML363MATα rom2::kanMx4This workMML384MATa wsc2::natMx4This workMML387MATa mid2::kanMx4de la Torre, et al. (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar)MML392MATa wscs1::URA wsc2::natMx4This workMML393MATa mid2::kanMx4 wsc1::URAThis workMML411MATa Galpkc1::kanMx4This workMML429MATa wsc3::kanMx4This workMML431MATa wsc4::natMx4This workMML513MATa mtl1::kanMx4This workMML550GFPPkc1This workX2180-1AMATa SUC2 gal2 CUP1 ma10ATCC 204504 Open table in a new tab GFP-Pkc1 was constructed as follows: a 1-kb XhoI-KpnI fragment from pRS416-sGFP (26Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (345) Google Scholar) was cloned at the SmaI site of the pFA6a plasmid containing the kanMX4 cassette. The resulting plasmid, pCYC86, was provided by Dr. Marti Aldea's laboratory. We then used PCR to amplify the sGFP-kanMX4 module from pCYC86. We did this by using oligonucleotides designed to insert the product in-frame, just before the stop codon of the genomic PKC1 sequence to obtain the fusion protein Pkc1-GFP. These were: MMO218, 5′-gcaagaagagtttagaggatttt-CCTTTATGCCAGATGATTTGGATTTACCAGCTGAAGCTTCGTACGC; MMO219, 5′-CCGCTTAGATGTTTTATATAAAATTAAATAAATCATGGCATGACCTTTTCTgcataggccactagtggatc, where the use of lowercase letters represents the sequence used to amplify the kanMX4 cassette, and the use of capital letters represents the sequence homologous to the C-terminal domain of PKC1 designed for recombinational integration. Plasmids—Plasmid pMM126 contains PKC1 under the tetO7 promoter and is tagged with the HA epitope, as described before (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To detect the total amount of Slt2 protein in protein extracts, we used yeast strains transformed with a YEP352 plasmid derivative containing the Slt2 open reading frame under its own promoter and tagged with the HA epitope in C-terminal (a gift from Dr. Maria Molina). All the strains used in this report were transformed with this plasmid, and the total amount of Slt2 was determined by using the anti-hemagglutinin monoclonal antibody (right panel), which was established as a loading control throughout this study. Media and Growth Conditions—Yeasts were grown in SC (0.67% yeast nitrogen base, 2% glucose, and auxotrophic requirements) or SD (SC plus drop-out mixture) media (28Kaiser C. Michaelis S. Mitchel A. Methods in Yeast Genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1994Google Scholar) plus the required amino acids. Where needed, sorbitol was added to a final concentration of 0.8 m final concentration. Diamide (Sigma) was prepared in dimethyl sulfoxide, whereas hydrogen peroxide (Sigma) was diluted with sterile Milli Q water. Yeast Extracts and Immunoblot Analyses and Conditions for the Use of the Anti-phospho-p44/42 and Anti-Swi6—These analyses were performed as described previously (27de la Torre-Ruiz M.A. Torres J. Ariño J. Herrero E. J. Biol. Chem. 2002; 277: 33468-33476Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). The anti-HA antibody was used at a dilution of 1:1000 in TBST buffer (20 mm Tris-HCl, pH 8, 0.125 m NaCl, 0.1% tween 20) in the presence of 0.25% milk fat, and the corresponding horseradish peroxidase-linked anti-mouse secondary antibody, at a dilution of 1:10.000 in TBST containing 0.25% milk fat. Sulfhydryl Determination with DTNB (Ellman's Reagent)—Yeast cells were grown in SC medium at 25 °C (to an initial A600 = 0.6). The assay was essentially conducted as described before (29de Nobel J.G. Klis F.M. Munnik T. Priem J. van den Ende H. Yeast. 1990; 6: 483-490Crossref PubMed Scopus (125) Google Scholar). Aliquots of 30 ml of culture were used for each treatment. They were centrifuged at 3,000 rpm for 5 min and resuspended in 2 ml of Tris-HCl buffer (10 mm, pH 7.4), and then treated and incubated at 4 °C for 30 min as follows: (i) control without treatment; (ii) 20 mm dithiothreitol (DTT); (iii) 1 mm H2O2; (iv) 2 mm H2O2; and (v) 4 mm diamide. After that, samples were washed three times with the same buffer. Next, they were resuspended in 1 ml of 50 mm potassium phosphate buffer, pH 7.4, containing 20 μm 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB, Ellman's reagent), and incubated for 10 min at room temperature. Finally, samples were centrifuged at 10,000 rpm for 3 min, and the supernatants were used to measure the optical density at 412 nm. The amount of free SH residues was quantified at this wavelength. The reducing agent DTT was used as a control to quantify the maximum amount of free SH residues on cell surfaces. Osmotic stabilization was achieved in pkc1 and wild type cell cultures by adding sorbitol to a final concentration of 0.8 m. Zymolyase Digestions—Cells were exponentially grown in SC medium at 25 °C and subsequently treated with 4 mm diamide and 1 mm hydrogen peroxide for 30 and 60 min, respectively. Mock treated cells were used as controls. After the treatments, 10 ml of each cell culture were centrifuged at 2,500 rpm for 5 min. The resulting pellets were washed first with 10 ml of sterile Milli Q water and then with 5 ml of Tris-HCl (50 mm, pH 9.4). The final pellet was resuspended in the last buffer. Samples of 1 ml of volume of each culture were added to 0.2 or 0.5 units zymolyase. Cell lysis was spectrophotometrically quantified at 600 nm. Actin Staining—Cells growing exponentially at 25 °C were stained with rhodamine-phalloidin as described before (3Torres J. Di Como C.J. Herrero E. de la Torre-Ruiz M.A. J. Biol. Chem. 2002; 277: 43495-43504Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar). Electron Microscopy—Log phase cells were treated, or mock treated, with 1 mm diamide for 9 h, then fixed with 2.5% glutaraldehyde for 1 h in 0.1 m phosphate buffer (pH 7.4) at 4 °C. Samples were then washed three times in the same buffer followed by a second fixation in osmium tetraoxide 1% in H2O for 2 h at 4 °C. After washing, the pellets were processed by dehydration using a series of acetones from 30–100% followed by propylene oxide treatment. Samples were then incubated in a resin (Durcupan ACM Epoxy Resin, Fluka): propylene oxide 3:1 for 45 min, 1:1 for 45 min, and 1:3 for 45 min, before embedding in resin and curing. Sections were cut and treated with lead citrate before viewing under a Zeiss 910 transmission electron microscope. Oxidative Stress Provoked by Diamide and Hydrogen Peroxide Activates the Cell Integrity Pathway—Given the role that the cell integrity pathway plays in the response to a wide variety of stresses, we decided to investigate whether this pathway was also involved in the oxidative stress response. Exponentially growing cells were treated either with diamide or hydrogen peroxide for the indicated times (Fig. 2A). Samples were taken for Western blot to detect activated Slt2, using the anti-phospho-p44/42 MAPK antibody raised against dually phosphorylated Thr202/Tyr204-p44/42 MAPK. It is generally accepted that detection of the doubly phosphorylated form of Slt2 provides a measure of kinase activation. The concentrations of the two oxidizing agents chosen for this study were sublethal and did not cause cell death during the course of the experiment. We had previously tested the following concentrations for the two agents: 0.5, 1, 2, 2.5, 3, and 5 mm in the case of hydrogen peroxide, and 1, 2, and 4 mm for diamide (not shown). During the first 3 h of treatment, we observed that concentrations of hydrogen peroxide of 2 mm caused no detectable activation whatsoever. Increasing the concentrations of diamide had a different effect in Slt2 activity. Greater concentrations of diamide provoked higher inductions of Slt2 double phosphorylation, although these were concomitant with an increase in cell lethality (results not shown in this study). Diamide and peroxide treatments both significantly induced Slt2 dual phosphorylation, although the kinetics of activation differed, with activation occurring considerably sooner with diamide (Fig. 2A). The addition of the osmotic stabilizer sorbitol almost totally abolished Slt2-dependent diamide activation, suggesting that diamide might affect the cell wall. In contrast, sorbitol did not significantly affect Slt2 induction of activity provoked by peroxide. This seems to indicate that the mechanism that regulates the induction of the PKC1-MAPK pathway mediated by peroxide takes place inside the cells. Interestingly, when we used other peroxides such as t-butyl hydroperoxide we obtained similar results to those observed with H2O2 (not shown). Activation of the PKC1-MAPK pathway and the Slt2 kinase was correlated to two different processes: phosphorylation of Swi6 and transcriptional induction of genes regulated by Rlm1. To check whether the increase in Slt2 phosphorylation dependent on oxidative stress could be correlated with the activity of the kinase and the pathway, we tested both processes in cells treated with either diamide or hydrogen peroxide. We thereby confirmed this hypothesis by means of two results. One consisted on the observation that Swi6 became hyperphosphorylated upon both peroxide or diamide treatments as shown in Fig. 2B. In this figure we observe a slower mobility hyperphosphorylated Swi6 band detected with a polyclonal antibody (Dr. Noel Lowndes gift). And the second that the transcriptional levels of SLT2 and PST1 (both genes are transcriptionally regulated by Rlm1) increased concomitantly with increases in Slt2 phosphorylation (Fig. 2B). From these results we conclude that when cells sense the oxidative stress provoked by diamide and hydrogen peroxide, Slt2 becomes activated. Mtl1 Senses and Transduces the Oxidative Effect Caused by Diamide—We tested mid2, mtl1, wsc1, wsc2, wsc3, and wsc4 single mutants together with wsc1wsc2, mid2wsc1, and mtl1mid2 double mutants for Slt2 phosphorylation due to the addition of diamide or hydrogen peroxide to check whether any of the known cell receptors related to the cell integrity pathway were involved in sensing and signaling the oxidative stress input. In this study we decided to perform a quantitative analysis of Slt2 activation by using the following criteria: we calculated the value called induction -fold, as the ratio between quantitative levels of Slt2 phosphorylation at either 30 or 60 min and Slt2 phosphorylation level measured at time 0 (untreated cells). All of the single quantified values were also normalized with respect to their respective loading controls (Fig. 2C). Therefore, the numerical values shown in this study correspond to relative levels of Slt2 activation in samples treated with oxidative agents after different times of exposure. In some of the mutants used in this study the basal levels of Slt2 activity were significantly lower than those estimated for wild type cells. For this reason and to avoid possible misinterpretations we show the numerical values corresponding to Slt2 relative induction for each experiment. Relative induction of Slt2 phosphorylation was partly defective in mid2 mutant cells treated with diamide, whereas it was almost completely eliminated in mtl1 cells (Fig. 2C). The latter result led us to consider the possibility of Mtl1 and Mid2 being partly redundant for sensing the oxidative effect caused by diamide. To test this, we checked the status of Slt2 activity in the mtl1mid2 double mutant and obtained that for this function Mid2 and Mtl1 did not constitute additive sensors of oxidative stress. The other cell wall receptor mutants assayed displayed no significant differences with respect to their respective single mutants and wild type cells (not shown). However, different results were obtained for hydrogen peroxide: none of the mutants showed marked deficiency in the relative induction of Slt2 phosphorylation with respect to wild type levels, although a partial reduction in Slt2 phosphorylation was observed in mid2 and mid2mtl1 mutants upon 2 h of exposure to hydrogen peroxide (Fig. 2C). To further characterize the role of Mtl1 and Mid2 as sensors of oxidative stress caused by diamide, we tested cell viability in the presence of diamide. We observed that only the absence of Mtl1 significantly impaired cell viability in response to diamide (Fig. 2D). Moreover, overexpression of the constitutive active Pkc1 allele under the Gal1 promoter (named GalPkc1*, a gift from Dr. M. Hall, for details see Ref. 12Delley P.A. Hall M.N. J. Cell Biol. 1999; 147: 163-174Crossref PubMed Scopus (241) Google Scholar) rescued both cell viability in the mtl1 mutant (Fig. 2E). These results indicate that Mtl1 functions in the cell integrity pathway by sensing and transmitting the oxidative signal. Our results indicate that both Mtl1 and, at least in part, Mid2 are sensors for the diamide-mediated oxidative effect at the cell surface and play a role in the cell integrity pathway for this response. These findings are also consistent with the results shown above and suggest that hydrogen peroxide activates the pathway at the intracellular level. Pkc1, Rom2, and Mtl1 Are Required for Transmitting the Oxidative Signal to Slt2 and for Cell Viability in Response to Oxidative Stress—Once demonstrated that oxidative stress activates Slt2 mediated by cell wall receptors, we investigated which elements of the cell integrity pathway were involved in this activation. To do this, we treated rom2 mutant cells with either diamide or hydrogen peroxide, and upon both treatments a marked defect in Slt2 phosphorylation was observed, with respect to wild type cells (Fig. 2C). We also confirmed that Slt2 activation was totally eliminated in both pkc1 and bck1 mutants following treatments with both peroxide and diamide (Fig. 2C). These results demonstrate that upstream Pkc1 elements are required to transduce the activating signal to Slt2 upon oxidative treatment. However, at present we still cannot rule out the possibility of oxidants directly activating elements downstream of Pkc1. We tested cell viability in various mutants after diamide and peroxide treatment to gain an understanding of the physiological consequences of the involvement of the cell integrity pathway in oxidative stress response (Fig. 3). We observed that rom2 and pkc1 deletions were significantly sensitive to the oxidizing agents, whereas bck1 and mpk1 cell growth was indistinguishable from that of wild type cells (Fig. 3, A and B). It is relevant to note that we obtained the same results (qualitatively speaking) when checking with different concentrations of the two agents: 0.5, 1, 1.5, 1.5, 2, 4, and 6 mm in the case of hydrogen peroxide, and 0.5, 0.75, 1, 1.5, 2, 3, 4, and 6 mm in the case of the diamide (data not shown). It is important to note that lack of viability of pkc1 due to the presence of the oxidizing agents was not totally rescued by sorbitol (Fig. 3B). This strongly suggests that Pkc1 function is essential for cell viability under oxidative conditions on despite of the presence of sorbitol as osmotic stabilizer and cell wall protective agent. We next performed cell viability studies in a rom2 mutant that had been transformed using the plasmid GalPkc1*. We observed that its overexpression compensated for rom2 cell death caused by diamide and peroxide (Fig. 3C). These data support that the Rom2 and Pkc1 functions are needed for cell survival against oxidation. The MAPK module downstream of Pkc1 (see Fig. 4A, bck1 and mpk1 viability) is dispensable for this cellular response.Fig. 4Diamide modifies the cell outer layer structure conferring greater resistance to zymolyase digestion and oxidating free-SH residues in cell-wall proteins.A, exponentially growing pkc1 and slt2 mutant cells in SC medium at 25 °C were treated with either diamide 4 mm for 30 min or with H2O2 1 mm for 1 h. Subsequently, 10 ml of culture at A600 of 0.6 was first washed with H2O and then with 50 mm Tris-HCl, pH 9.4. Cell pellets were then resuspended in 1 ml of this buffer prior to the addition of either 0.2 or 0.5 units zymolyase. Cell lysis was estimated by measuring at A600 for the indicated periods. The pkc1 mutant was grown in the presence of the osmotic stabilizer sorbitol 0.8 m. B, when quantifying free-SH residues with DTNB (see "Materials and Methods"), the A412 was determined in intact cells treated with DTT, diamide, or hydrogen peroxide.View Large