Yeast oxidative stress response Influences of cytosolic thioredoxin peroxidase I and of the mitochondrial functional state Ana P D Demasi1, Goncalo A G Pereira1 and Luis E S Netto2 ¸ ´ Departamento de Genetica e Evolucao – IB – UNICAMP, Campinas, Brazil ¸˜ ´ ´ Departamento de Genetica e Biologia Evolutiva – IB – USP, Sao Paulo, Brazil Keywords hydrogen peroxide; gene expression; mitochondrial dysfunction; oxidative stress; thioredoxin peroxidase Correspondence ´ G Amarante Guimaraes Pereira, Laboratorio ˜ ˆ de Genomica e Expressa – IB UNICAMP, ˜o CP 6109, CEP 13083–970, Campinas-SP, Brazil Fax: + 55 19 37886235 Tel: + 55 19 37886237 ⁄ 6238 E-mail: goncalo@unicamp.br (Received 30 September 2005, revised 12 December 2005, accepted 20 December 2005) doi:10.1111/j.1742-4658.2006.05116.x We investigated the changes in the oxidative stress response of yeast cells suffering mitochondrial dysfunction that could impair their viability First, we demonstrated that cells with this dysfunction rely exclusively on cytosolic thioredoxin peroxidase I (cTPxI) and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2induced death This cTPxI-dependent protection could be related to its dual functions, as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures of cTPxI observed in cells challenged with H2O2 We found that cTPxI deficiency leads to increased basal sulfhydryl levels and transcriptional activation of most of the H2O2-responsive genes, interpreted as an attempt by the cells to improve their antioxidant defense On the other hand, mitochondrial dysfunction, specifically the electron transport blockage, provoked a huge depletion of sulfhydryl groups after H2O2 treatment and reduced the H2O2mediated activation of some genes otherwise observed, impairing cell defense and viability The transcription factors Yap1 and Skn7 are crucial for the antioxidant response of cells under inhibited electron flow condition and probably act in the same pathway of cTPxI to protect cells affected by this disorder Yap1 cellular distribution was not affected by cTpxI deficiency and by mitochondrial dysfunction, in spite of the observed expression alterations of several Yap1-target genes, indicating alternative mechanisms of Yap1 activation ⁄ deactivation Therefore, we propose that cTPxI is specifically important in the protection of yeast with mitochondrial dysfunction due to its functional versatility as an antioxidant, chaperone and modulator of gene expression Aerobic organisms are constantly exposed to reactive oxygen species (ROS), generated as normal metabolism byproducts, especially by respiration [1,2] To modulate ROS concentrations and to counteract their deleterious effects, there are several antioxidant systems with an apparent functional redundancy, which may provide an evolutionary advantage Saccharomyces cerevisiae cells possess multiple H2O2 detoxifying enzymes, such as catalases, cytochrome c peroxidase, glutathione peroxidases, glutaredoxins and peroxiredoxins, described as many isoforms and in distinct cellular compartments [3–6] So far, their specific roles Abbreviations AhpC, alkyl hydroperoxide reductase; cTPxI, cytosolic thioredoxin peroxidase I, which is synonymous with Tsa1 and YML028W; 2-Cys Prx, peroxiredoxins with two conserved cysteines involved in the catalytic mechanism; DTNB, 5,5¢-dithio-bis(2-nitrobenzoic acid); FCCP, p-trifluoromethoxycarbonylcyanide phenylhydrazone; NP-SH, nonprotein sulfhydryl groups; PB-SH, protein bound sulfydryl group; PKA, protein kinase A; Prxs, peroxiredoxins; ROS, reactive oxygen species; t-BOOH, t-butylhydroperoxide; TSA, thiol-specific antioxidant; Ybp1, Yap1-binding protein FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS 805 cTPxI and respiratory function in antioxidant defense A P D Demasi et al have not been well established and are suggested to be related to their differential regulation The transcription factors Yap1, Skn7, Msn2 and Msn4 are the main regulators of S cerevisiae oxidative stress response [7–10] Yap1 confers to cells the ability to tolerate oxidants like H2O2, t-butyl hydroperoxide, diamide, diethylmaleate and cadmium [11] by the activation of the expression of genes encoding most antioxidant enzymes and components of the cellular thiol-reducing pathways, comprising approximately 32 proteins of the H2O2 stimulon [12] Skn7 co-operates in the activation of at least 15 of the Yap1 target proteins in response to H2O2 and t-butyl hydroperoxide, but not to cadmium [7] Contrary to Yap1, this transcriptional regulator does not participate in the regulation of metabolic pathways that regenerate the main cellular reducing power, glutathione and NADPH [7] Msn2 and Msn4 activate genes whose promoters contain the stress responsive element (STRE: CCCCT) after several environmental challenges, including oxidative stress Despite an overlap with the Yap1 regulon (eight proteins), the Msn2 ⁄ regulon comprises a small number of antioxidant enzymes, but several heat-shock proteins, metabolism enzymes and proteases, and is involved with activities of the ubiquitin and proteasome degradation pathways [13] Msn2 ⁄ are negatively regulated by the Ras-cAMP-protein kinase A (PKA) pathway [14], which has been suggested to negatively influence also Yap1regulated transcription [8,15] The involvement of mitochondria in the response of yeast to oxidative stress is not well understood, despite the fact that these organelles are the primary cellular source of ROS In S cerevisiae cells, external mitochondrial NADH dehydrogenases [16], coenzyme Q [17] and succinate dehydrogenase [18] were identified as sites of ROS production in mitochondria Mutations or drugs that block terminal steps of the respiratory chain further increase ROS generation due to the accumulation of reduced electron carriers [19–21] Even so, it was demonstrated that mitochondrial function is required for resistance to oxidative stress [22,23] Peroxiredoxins (Prxs) are abundant, ubiquitously distributed peroxidases that reduce H2O2 and peroxynitrite at the expense of thiol compounds [24–26] They can be divided into 1-Cys and 2-Cys Prxs, based on the number of cysteine residues involved in catalysis It has been shown that 2-Cys Prxs participate in the regulation of H2O2-mediated signal transduction [27– 32] In addition, two recent reports have demonstrated that 2-Cys Prxs can act alternatively as peroxidases and as molecular chaperones [33,34] The peroxidase– chaperone functional switch is established by a shift of 806 the cTPxI oligomeric state from low to high molecular weight complexes, which is triggered by oxidative and heat stress [33,34] Cytosolic thioredoxin peroxidase I (cTPxI), encoded by TSA1, is a 2-Cys Prx and is one of the five Prxs described in S cerevisiae It was shown that cTPxI is essential for the H2O2 defense of yeast with dysfunctional mitochondria [35] Here, we describe results indicating that cells with this dysfunction rely exclusively on cTPxI and its reductant sulfiredoxin, among other antioxidant enzymes tested, to protect them against H2O2-induced death Our results indicated two possibilities (not mutually exclusive) for this cTPxIdependent protection: (a) the dual functions of cTPxI as peroxidase and as molecular chaperone, suggested by mixtures of low and high molecular weight oligomeric structures observed in cells challenged with H2O2 and (b) the capacity of cTPxI to interfere with the expression of various Yap1-target genes Results Effects of gene deletion and mitochondrial perturbation on the oxidative stress response We have previously shown that cTPxI is an important component of the defense of cells with mitochondrial dysfunction against H2O2 [35] Using the same experimental approach, we have compared the sensitivities of tsa1D and other mutants deficient in different antioxidant enzymes to H2O2 when mitochondrial function was affected by antimycin A treatment (inhibitor of complex III) We observed that loss of any of these enzymes, cytosolic thioredoxin peroxidase II, alkyl hydroperoxide reductase, mitochondrial thioredoxin peroxidase I, cytochrome c peroxidase, glutathione reductase, catalase T and catalase A, did not alter the growth, either after the single treatments or after the associations of both treatments (Fig 1) On the other hand, the deficiency of sulfiredoxin, a low molecular weight protein that reduces the overoxidized forms of cTPxI [36], did alter the growth when cells were treated with antimycin A plus H2O2 (Fig 1) Therefore, the response of yeast with dysfunctional mitochondria was very dependent on cTPxI and its reductant sulfiredoxin, among other antioxidants Next, we evaluated the behavior of tsa1D cells and other mutants in response to H2O2 when respiration was altered with p-trifluoromethoxycarbonylcyanide phenylhydrazone (FCCP), an oxidative phosphorylation uncoupler that can carry protons across the mitochondrial inner membrane, thus promoting proton gradient collapse Contrary to antimycin A, FCCP FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS A P D Demasi et al control cTPxI and respiratory function in antioxidant defense H2O2 anti A H2O2 + anti A FCCP H2O2 + FCCP t-BOOH t-BOOH diamide + anti A diamide + anti A WT1 tsa1∆ tsa2∆ ahp1∆ prx1∆ ccp1∆ glr1∆ WT2 ctt1∆ cta1∆ WT1 srx1∆ Fig Comparison of yeast mutants’ sensitivities to several stressful conditions Growth of the strains BY4741, wild-type (WT1), YPH250, wild-type (WT2) and mutants indicated on YPD plates containing no chemicals as a control, 1.2 mM H2O2, 1.2 mM t-BOOH, 1.2 mM diamide, 0.1 lgỈmL)1 antimycin A (anti A), 2.5 lgỈmL)1 FCCP, 5.0 lgỈmL)1 singly or in association For each strain, 12 lL of overnight culture diluted to 0.2 OD600nm units and four subsequent : dilutions were spotted on plates Growth was monitored after days for all plates except for diamide, after days Only the three last dilutions are represented in the figure Oxidation of sulfhydryl groups Sulfhydryl groups, including nonprotein (NP-SH), mostly represented by glutathione, and protein bound (PB-SH), are abundant in cells and can be oxidized by ROS Therefore, they have been widely used as indicators of oxidative stress [41–43] To determine whether TSA1 deletion and the mitochondrial dysfunction can generate stressful conditions, the levels of sulfhydryl groups in the reduced state were evaluated The first remarkable observation was that tsa1 mutant presented a pronounced increase in basal sulfhydryl groups compared with the wild-type strain, especially in the NP-SH content (Fig 2) In spite of the high basal sulfhydryl content present in tsa1D cells, exposure to H2O2 promoted a significant loss of these Relative sulphydryl groups (%) treatment accelerates the electron flow through the respiratory chain and diminishes endogenous ROS generation by mitochondria [37–39] No increased sensitivity to H2O2 could be detected in any of the strains treated with FCCP (Fig 1) The phenotype of mutant strains in response to other oxidants besides H2O2 was also investigated All strains presented similar sensitivity to t-butylhydroperoxide (t-BOOH) treatment, but tsa1D was slightly more sensitive than the others to this oxidant when antimycin A was also present in the media (Fig 1) Interestingly, tsa1D cells were not sensitive to diamide, even in the presence of antimycin A (Fig 1) Only glr1D was more sensitive than wild-type cells to diamide and this effect was increased when cells were also exposed to antimycin A (Fig 1) This result was expected, as diamide only induces generation of disulfide bridges [40] and glutathione reductase reduces the disulfide form of glutathione This point will be further explored in the discussion section In summary, the results presented here indicated that cTPxI exhibits a very specific defense of yeast with dysfunctional mitochondrial in situations in which this organelle presents electron transport impediment and ⁄ or produces high levels of superoxide 180 WT tsa1∆ 160 140 120 100 80 60 40 20 NPPB control NPPB H 2O NPPB NPPB NPPB NPPB Anti A A nti A + H2O2 FCCP FCCP + H 2O Fig Comparison of sulfhydryl group levels in wild-type (WT) and tsa1D cells exposed to several stressful conditions Cell protein extracts of strains BY4741 (WT) and tsa1D, obtained after exposition to 1.2 mM H2O2, 0.1 lgỈmL)1 antimycin A (anti A) or 2.5 lgỈmL)1 FCCP, singly or in association, were assayed for thiol groups by spectrophotometric test using DTNB Absorbance was read at 412 nm Results are relative to the concentration of these groups in control cells (100%) that were not exposed to any agent, and represent average of independent experiments PB, protein bound sulfydryl; NP, nonprotein sulfydryl FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS 807 A P D Demasi et al l 2O ro nt H co groups, reaching levels similar to those observed in wild-type cells, much less affected Antimycin A treatment alone caused little increase of sulfhydryl groups in both strains, when compared with the control situation However, the association of antimycin A with H2O2 led to a huge depletion in NP-SH, as well as PB-SH levels, only in cells lacking cTPxI (Fig 2) Only a limited loss of sulfhydryl groups was observed for both strains treated with FCCP alone, and no additional decreases in these levels were found with the addition of H2O2, even in cells lacking cTPxI (Fig 2) These results indicated that tsa1 mutant cells with dysfunctional mitochondria suffered intensive oxidative stress only when this dysfunction is accompanied with electron flow obstruction and ⁄ or increased endogenous ROS production (antimycin A treatment) An ti A An ti FC A + CP H 2O FC CP + H 2O cTPxI and respiratory function in antioxidant defense (kDa) 545 272 132 66 Switching of cTPxI oligomeric states in vivo cTPxI and cTPxII can act as peroxidases and as molecular chaperones, depending on changes of their quaternary structures triggered by oxidative stress and heat shock exposure [33,34] When cTPxI appears mainly as oligomeric protein structures of low molecular weight, this protein possesses mainly peroxidase activity, whereas high molecular weight complexes behave mainly as chaperones [33,34] The specificity of cTPxI in the protection of cells with dysfunctional mitochondria (Fig 1) might be related to the ability of this protein to possess these two activities Therefore, we compared cTPxI oligomeric structures in vivo under situations of normal and inhibited mitochondrial function, as it is hard to measure chaperone activity in vivo Under control conditions, cTPxI appeared as a mixture of complexes with molecular weight below 272 kDa and after treatment of yeast cells with H2O2, a considerable part of these species were converted to HMW complexes of about 500 kDa or even higher (Fig 3), as previously described [33,34] These switches of cTPxI quaternary structures induced by H2O2 were not affected by any of the inhibitors of mitochondrial function (Fig 3) Similar results were obtained with 0.5 mm H2O2 alone or in association with the mitochondrial function inhibitors (not shown) Since it was well demonstrated that the conversion of cTPxI to different oligomerization states is implicated with its chaperone ⁄ peroxidase switching [33,34], we suggest that the chaperone activity of this protein, in addition to its peroxidatic function, is probably involved with its specific role in the antioxidant defense of yeast with mitochondrial dysfunction 808 45 Fig Protein structures of cTPxI in vivo Native-PAGE analysis of crude protein extracts obtained from BY4741 (WT) cultures after exposing cells during 40 to no agent as a control, 1.2 mM H2O2, 0.1 lgỈmL)1 antimycin A (anti A), 1.2 mM H2O2 plus 0.1 lgỈmL)1 antimycin A, 2.5 lgỈmL)1 FCCP and 2.5 lgỈmL)1 FCCP plus 1.2 mM H2O2, were separated on 9% native-PAGE and subjected to western blotting with a polyclonal anti-cTPxI IgG cTPxI influences the expression of genes involved in yeast oxidative stress response: mitochondrial function contribution cTPxI participates of H2O2-mediated signaling processes, including regulation of gene expression [27–30] Therefore, we have evaluated possible influences of cTPxI and of the functional state of the mitochondria in the expression of selected yeast antioxidant genes In this manner, we expected to obtain some clues to better understand cTPxI importance in the response of cells with mitochondrial dysfunction to oxidative stress It could be readily observed that the expression levels of several genes were increased in cells lacking cTPxI (Fig 4) Four gene subsets could be delineated: (a) genes with increased basal expression levels in tsa1D cells: GSH1, GSH2, GLR1, PRX1, SOD1, GPX2, AHP1, TRR1, SSA1; (b) genes with increased H2O2induced expression levels in tsa1D cells: CCP1, CTT1, TRX2, TRX3, SOD2, GRX5, POS5; (c) genes without expression alteration in tsa1D cells: ZWF1, IDP1, GPX3, HSP104 (not shown); and (d) genes without FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS A P D Demasi et al cTPxI and respiratory function in antioxidant defense Protein function Gene anti A H2O2 Actin Increased basal expression levels in tsa1∆ cells TSA1 γ-glutamyl GSH1 cysteine synthase Glutathione GSH2 synthetase Glutathione GLR1 reductase Mitochondrial PRX1 thioredoxin peroxidase I Coper/zinc superoxide SOD1 dismutase Alkyl hydroperoxide reductase Thioredoxin reductase I Heat shock protein of HSP70 family Yap1 - Yap1, Msn2/4 + Yap1, Skn7 - Yap1 +/- AHP1 Yap1, Skn7 - TRR1 Yap1, Skn7 +/- SSA1 Yap1, Skn7 + Yap1, Skn7 + Yap1, Skn7, Msn2/4 + Yap1, Skn7 + Cytochrome c peroxidase CCP1 Increased (mitochondrial) H2O2induced CTT1 Catalase T expression levels in TRX2 Thioredoxin II tsa1∆ cells Thioredoxin III (mitochondrial) Manganese superoxide dismutase (mitochondrial) Glutaredoxin (mitochondrial) Mitochondrial NADH kinase TRX3 + SOD2 Yap1, Skn7 +/- GRX5 + POS5 +/1 b + - - + + - - + + - + - + - + - + Glutathione peroxidase II GPX2 a - tsa1∆ ACT1 Cytosolic thioredoxin peroxidase I Mitochondrial function influenceb Yap1 WT Regulationa Yap1, Skn7 Expression levels Data from [7] and [13] In H2O2- induced expression in tsa1 mutant cells (compare lanes and 8) Fig Expression of genes in wild-type and tsa1D cells exposed to several stressful conditions Northern blot analysis of RNA isolated by the hot acid phenol method from yeast strains BY4741 (WT) and tsa1D grown on YPD to mid-log phase, treated during 40 with no agent as a control or with 1.2 mM H2O2 and 0.1 lgỈmL)1 antimycin A (anti A), singly or in association, as indicated in the figure The symbols –, + or ± in the last column denote absence, strong or mild influence of mitochondrial function on gene expression (comparison of band intensities between lanes and 8) FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS 809 cTPxI and respiratory function in antioxidant defense control A P D Demasi et al H2O2 anti A H2O2 + anti A FCCP H2O2 + FCCP WT yap1∆ skn7∆ yap1∆ skn7 ∆ msn2∆ msn4 ∆ Fig Sensitivities of mutants lacking transcription factors to several stressful conditions Growth of the strains BY4741 (WT), and mutants indicated on YPD plates containing no chemicals as a control, 0.8 mM H2O2, 0.1 lgỈmL)1 antimycin A (anti A), and 2.5 lgỈmL)1 FCCP singly or in association Proceedings were performed as described in Fig detectable expression in both wild-type and tsa1D cells: TSA2, CTA1, DOT5, TRX1, TTR1 (not shown) Genes that belong to subsets (c) and (d) encode glucose-6phosphate dehydrogenase, mitochondrial isocitrate dehydrogenase, glutathione peroxidase III, heat shock protein 104, thioredoxin peroxidase II, catalase A, nuclear thioredoxin peroxidase, thioredoxin I and glutaredoxin II, respectively Among genes described in subset (a), GSH1, GPX2, AHP1 and TRR1 expression levels were further induced by H2O2 (Fig 4) In addition, most if not all genes that presented altered expression in tsa1 mutant are regulated by Yap1 (Fig 4), indicating that cTPxI could affect Yap1 activity Furthermore, Skn7 co-operates in the control of many of these genes (Fig 4) and constitute another transcription factor whose activation might be influenced by cTPxI It is worth noting the influence of the functional state of mitochondria in the H2O2-induced expression levels of various genes, at least in tsa1 mutant (Fig 4, compare lanes and 8), suggesting that respiratorycompromised cells fail to activate some H2O2 responsive genes transcription at the same degree of respiratory-competent ones The H2O2-induced expression levels of GSH1, PRX1, CCP1, CTT1, TRR1 were affected the most by the defective mitochondria, while those of GPX2, TRX2, TRX3, SOD2 GRX5, POS5 were influenced at a lower level The treatment with antimycin A alone, in all cases, led to expression levels resembling those observed in control cells (Fig 4) These results are in agreement with genome-wide studies that did not find significant differences in the expression of antioxidant genes in cells with mitochondrial dysfunction [44,45] Participation of transcription factors in the antioxidant defense of cells with normal or impaired mitochondrial function In order to identify transcription factors involved in the response of cells with dysfunctional mitochondria 810 to oxidative stress, we evaluated H2O2 sensitivity of deletion mutants for the regulators most frequently associated with oxidative stress response: Yap1, Skn7, Msn2 and Msn4 Single or double mutants for Yap1 and Skn7 were very sensitive to H2O2, although deletion of YAP1 gene appeared to be more deleterious than the SKN7 gene deletion (Fig 5) This high sensitivity was already expected given the diversity of antioxidant enzymes regulated by these factors [7] The association of H2O2 with antimycin A totally inhibited growth of these mutants In contrast, no further growth inhibition of yap1D and skn7D was achieved by the association of FCCP with H2O2, relative to H2O2 alone (Fig 5) No significant growth retardation of yap1D and skn7D relative to wild-type counterparts was observed when these cells were treated with antimycin A alone or with FCCP alone Therefore, the phenotypes of yap1D and skn7D, were similar to those of tsa1D described in Fig 1, suggesting that Yap1, Skn7 and cTPxI act in the same pathway in the response of yeast with dysfunctional mitochondria to oxidative stress Because cell growth was more affected by the deletion of YAP1 and SKN7 genes than by deletion of cTPxI (Fig 5), we suggest that other enzymes whose genes are regulated by these factors could also be involved in the antioxidant defense of respiratory-incompetent cells On the other hand, Msn2 and Msn4 appear to not play significant role in the response of cells to H2O2 or to either of the compounds that interfere with mitochondrial function (antimycin A or FCCP), as no considerable growth alterations for their deletion mutants were detected (Fig 5) Since Ras-cAMP-PKA pathway inhibits Msn2 ⁄ under catabolic repressing conditions [14], the sensitivity of msn2Dmsn4D was also evaluated in the absence of glucose In this case, cells were grown in raffinose medium Again, these mutants grew similarly to the wild-type cells (data not shown), dismissing the involvement of Msn2 ⁄ in the response of respiratory incompetent cells to oxidative stress FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS A P D Demasi et al cTPxI and respiratory function in antioxidant defense Yap1 cellular distribution It is well known that Yap1 is accumulated in the nucleus of cells exposed to oxidative stress and, as a consequence, the expression of its target genes is activated [11,46,47] We observed that mitochondrial dysfunction negatively affects the H2O2-induced expression levels of various Yap1-target genes in tsa1 mutant cells (Fig 4, compare lanes and 8), which could account for the decreased capacity of yeast to cope oxidative stress (Fig 1) To check this possibility, we examined the distribution of Yap1 in the cells expressing GFPYap1 fusion protein No significant difference in the cellular GFP-Yap1 distribution was observed between the wild-type and tsa1 mutant cells in all of the conditions tested (Fig 6) In spite of the increased basal expression levels of a variety of genes in tsa1 cells, we did not observe GFP-Yap1 accumulation in the nucleus of these cells, corroborating results previously obtained [27] Therefore, GFP-Yap1 is located in nucleus and cytoplasm of both wild type and tsa1D cells in control conditions (Fig 6) Antimycin A treatment alone did not lead to a nuclear accumulation of GFP-Yap1 (Fig 6), which is in agreement with the similar expression levels of genes from control and antimycin A-treated (Fig 4, compare lanes with and with 7) Antimycin A treatment did not alter the nuclear Yap1 accumulation induced by H2O2, in neither the wild-type nor in tsa1D cells (Fig 6) Hence, the dimin- ished H2O2-induced expression levels of some Yap1target genes observed in cells with impaired mitochondrial function can not be attributed to alteration in Yap1 cellular distribution Probably other factors, such as ability of Yap1 to bind DNA [51], are also involved in the activation of genes involved in the response of yeast to oxidative stress These possibilities are further discussed below Discussion It was previously demonstrated that cTPxI is essential for the antioxidant defense of cells with mitochondrial dysfunction [35] Remarkably, we have shown here that cTPxI is very specific among several other antioxidants in the protection of cells with respiratory incompetence against peroxides (Fig 1) The protective action of cTPxI was prominent in situations of electron flow impediment This was demonstrated by the severe growth retardation (Fig 1) and by the large depletion of sulfydryl content (Fig 2) of tsa1 mutant treated with H2O2 in association with antimycin A, effects that were not observed when these cells were exposed to H2O2 + FCCP As it has long been shown, while antimycin A augments ROS generation by defective mitochondria [19–21], FCCP diminishes it [37–39] Therefore, it is possible that cTPxI could be specifically important when internal ROS production is elevated In agreement, it was demonstrated that a bacterial peroxiredoxin, alkyl hydroperoxide reductase WT tsa1∆ control Fig Cellular distribution of GFP-tagged Yap1 Cells of the strains JD7–7C (WT) and tsa1D carrying expression plasmids for the GFP-YAP1 fusion gene were exposed to 1.2 mM H2O2 and 0.1 lgỈmL)1 antimycin A (anti A), separately or in association, and confocal laser scanning microscopy was carried out as described under ‘Experimental procedures’ The left panels show the fluorescent images and the right panels show the transmission images All of the data shown are representative of at least three independent experiments, all of which gave similar results H2O2 Anti A Anti A +H2O2 FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS 811 cTPxI and respiratory function in antioxidant defense A P D Demasi et al (AhpC), is the primary scavenger of endogenous H2O2 [48] Altered ATP levels not appear to influence respiratory deficient yeast antioxidant defenses, since cells treated with FCCP, which leads to a more pronounced energy limitation due to the higher cytoplasmic ATP hydrolysis rate [37] did not present alteration in H2O2 sensitivity, even for the tsa1 mutant (Fig 1) The peroxidatic function of cTPxI probably overlaps to some extent with other H2O2 detoxifying enzymes, but the recent finding that this protein possesses chaperone activity under stressful conditions provides a very tempting explanation for the distinctive role of cTPxI in protecting cells with mitochondrial dysfunction against oxidative stress Indeed, our data showed that cTPxI appears not only as low molecular weight, but mostly as high molecular weight complexes in cells exposed to H2O2 alone or in association with antimycin A (Fig 3), suggesting that it may be acting as peroxidase and as chaperone under these conditions, as this functional and structural correlation was already well demonstrated [33,34] Its high molecular weight form with chaperone activity could protect essential proteins from denaturation or could mediate activation of downstream defense signaling cascades that prevent H2O2-induced cell death Actually, it was demonstrated that oxidant-mediated proper folding of Yap1 is required for transcriptional activation and for the nuclear accumulation of this regulator during stress [49] Another phenomenon that could be related to the unique phenotype of tsa1 mutant cells is that most thiol proteins are inactivated when oxidized to sulfinic acids, because this oxidation state of cysteine residues is not reducible by classical reducing agents such as glutathione and thioredoxin In contrast, the sulfinic acid form of cTPxI can be specifically reduced by sulfiredoxin [36] In fact, srx1D presented similar phenotype of tsa1D cells (Fig 1) The fact that antimycin A, but not diamide, increased the sensitivity of tsa1 mutant cells to peroxides (Fig 1) gave support to the notion that higher oxidation states of cysteines might be taking place in the cTPxI dependent response to oxidative stress This is because diamide is an oxidant that gives rise only to disulfides [40], whereas peroxides generate disulfides as well as sulfenates (Cys-SOH), sulfinates (Cys-SO2H) and sulfonates (Cys-SO3H) The hypotheses raised here to explain the unique tsa1D phenotype are not mutually exclusive Actually, sulfinic acid formation in cTPxI by H2O2 was suggested as a trigger event for the switch of this peroxiredoxin from a peroxidase to a chaperone enzyme [33] 2-Cys Prxs have been implicated in the regulation of stress-induced gene expression [27–32] A previous 812 work has shown that expression levels of GSH1, GLR1 and GPX2 were increased in a tsa1 mutant and this effect was dependent on Yap1, since transcriptional activation of these genes were not observed in tsa1D ⁄ yap1D double mutants [27] Our data confirmed these results, but indicate that there may be more mechanisms involved in this TSA1-dependent regulation For some genes, the transcription increase seemed to be stress-independent, while for others, it was dependent on H2O2 (Fig 4) It is most relevant that, in several cases, the H2O2 induction was reduced by the presence of antimycin A, suggesting that the functional state of the mitochondria is somehow sensed by regulatory mechanisms Perhaps the decreased levels of these enzymes, and other yet not detected, could be responsible for the reduced viability of tsa1D cells grown in the presence of H2O2 plus antimycin A (Fig 1) The existence of a connection between cTPxI and Yap1 is becoming evident, although its molecular basis is not yet clear It was demonstrated that Yap1 is retained in the nucleus in the presence of H2O2 and thereby it interacts with the target genes [11,46,47] This retention is dependent of the oxidation of Yap1 cysteine residues by H2O2, that modifies its conformation and hinders its interaction with Crm1, which otherwise would export this factor to cytoplasm H2O2 oxidizes Yap1 in a process mediated by Gpx3 ⁄ Orp1, a glutathione peroxidase homologue with thioredoxin peroxidase activity [11], with a still unclear participation of Ybp1 [50] Interestingly, it was demonstrated in cells with a truncated form of Ybp1, that cTPx1 can replace Gpx3 ⁄ Orp1 in the oxidation of Yap1 thus promoting its nuclear retention [30] Moreover, in Schizosaccharomyces pombe, a 2-Cys Prx, but not a GPx, directly oxidizes Pap1 (a Yap1 homologue) provoking the nuclear retention of this transcription factor [51] Despite the observed alterations in the expression of Yap1-target genes, the cellular localization of this transcription factor was not altered by either the TSA1 deletion or by the inhibition of mitochondrial function (Fig 6) Inoue et al [27] have also demonstrated that the expression of a reporter gene fused to a Yap1-dependent promoter was significantly increased in the absence of cTPxI by a mechanism independent of Yap1 nuclear retention There is a precedent showing that Yap1 binding activity may be affected It was shown that the accessibility of Yap1 to the GSH1 promoter could be repressed by Cbf1, a DNA-binding protein that binds to elements in the vicinity of Yap1 binding site [52] Alternatively, the increased reducing power of tsa1 mutant, achieved by the expression elevation of GSH1, GSH2, GLR1 and TRR1 and detected by our analysis (Fig 2), could FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS A P D Demasi et al contribute to the reduction of Yap1, hence diminishing the oxidized Yap1 ‘lifetime’, thus avoiding its accumulation in the nucleus In fact, Wiatrowski and Carlson [53] did not observe Yap1 accumulation in the nucleus of cells shifted from glucose to glycerol, otherwise observed, in the presence of glutathione externally added Another striking point in the adaptation of cells to H2O2 is the redirection of carbohydrate flux from hexose phosphate pool (glycolysis) to the pentose phosphate pathway to the regeneration of NADPH [12] which, in turn, is responsible for the maintenance of both thioredoxin and glutathione in their reduced states As cells treated with antimycin A rely only on glycolysis to produce ATP, the carbohydrate metabolism redirection after H2O2 treatment would be affected and the generation of NADPH would be diminished The depletion of sulfhydryl groups occurred in tsa1 cells treated with antimycin A plus H2O2 (Fig 2) could corroborate with this hypothesis These multiple activities of cTPxI (peroxidase, chaperone and redox signaling) might be related to the central roles of this protein in prevention of yeast against genotoxic processes [54,55] Here, we have shown some alterations that could impair the oxidative stress response of yeast cells with mitochondrial dysfunction and that cTPxI is specifically important in their antioxidant defense Although the mitochondrial inhibition procedures used here were extreme, these approaches have been largely employed in bioenergetics studies and have provided valuable information Moreover, the nonphysiological doses of peroxides used here were due to the high redundancy of the yeast antioxidant systems Nevertheless, our results suggest that peroxiredoxins, especially those with high similarity to the yeast cTPxI could exert a decisive role in the establishment of mitochondrial dysfunction-related diseases, although further studies are necessary to ascertain this relationship In support of this hypothesis, peroxiredoxins have been implicated in the development of different kinds of cancer [56–60] and neurodegenerative diseases [61,62] Experimental procedures Yeast strains and growth conditions The following S cerevisiae strains were used in this study: JD7–7C (MATa ura3–52 leu2 trpA K + and tsa1D (MATa ) ura3–52 leu2 trpA K + tsa1D::LEU2) were obtained from Chae [63] (National Institute of Health, Bethseda, MD, USA); BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0), tsa1D (MATa; his3D1; leu2D0; met15D0; ura3D0; tsa1D:: Kan Mx4), prx1D (MATa; his3D1; leu2D0; met15D0; cTPxI and respiratory function in antioxidant defense ura3D0; prx1D::Kan Mx4) tsa2D (MATa; his3D1; leu2D0; met15D0; ura3D0; tsa2D::Kan Mx4), ahp1D (MATa; his3D1; leu2D0; met15D0; ura3D0; ahp1D::Kan Mx4), ccp1D (MATa; his3D1; leu2D0; met15D0; ura3D0; ccp1D::Kan Mx4), glr1D (MATa; his3D1; leu2D0; met15D0; ura3D0; glr1D::Kan Mx4), yap1D (MATa; his3D1; leu2D0; met15D0; ura3D0; yap1D::Kan Mx4), skn7D (MATa; his3D1; leu2D0; met15D0; ura3D0; skn7D::Kan Mx4) were obtained from EUROSCARF (University of Frankfurt, Germany); YPH250 (MATa trp-D1 his3-D200 lys2–801 leu2-D1 ade2– 101 ura3–52), ctt1D (MATa trp-D1 his3-D200 lys2–801 leu2D1 ade2–101 ctt1::URA3), cta1D (MATa his3-D200 lys2– 801 leu2-D1 ade2–101 ura3–52 cta1::TRP1) were obtained from Izawa [64] (Kyoto University, Japan), and W303–1a (MATa, ade2, can1, his3, leu2, trp1, ura3) and msn2Dmsn4D (MATa, ade2, can1, his3, leu2, trp1, ura3, msn2::HIS3, msn4::TRP1) were obtained from Boy-Marcotte [65] (Universite Paris-Sud, France) Cells were grown at 30 °C on YPD medium (1% yeast extract, 2% bacto-peptone, 2% glucose) For most analysis, cells were harvested by centrifugation at mid-log phase, usually at an OD600nm between 0.8 and 1.4 Determination of tolerance to different oxidants Spot test: cells were first grown in YPD media until a concentration of approximately 107 cellsỈmL)1, and then diluted to OD600nm ¼ 0.2 Four subsequent : dilutions of these cell suspensions were realized and a 12 lL droplet of each was plated on YPD-agar medium containing 1.2 mm H2O2, or 1.2 mm t-BOOH, 1.2 mm diamide, 0.1 lgỈmL)1 antimycin A, or 2.0 lgỈmL)1 FCCP, separately or in association Plates were then incubated days Only the three highest dilutions were represented in the figures Determination of sulfhydryl groups PB-SH levels were measured according to the method of Sedlak and Lindsay [66], by subtracting the NP-SH content from the total sulfhydryl (T-SH) content Cells of the strains JD7–7C and tsa1D were grown on YPD and, after treatments with 1.2 mm H2O2, 0.1 lgỈmL)1 antimycin A and 2.0 lgỈmL)1 FCCP, separately or in association (as described in the figure), approximately · 106 cells from each culture were collected Protein extracts were obtained in 0.02 m EDTA pH 4.7 with glass beads addition followed by centrifugation at 17 900 g for 15 The T-SH concentrations were determined by absorption levels at 412 nm after incubating 200 lL aliquots of protein extracts supernatants with 780 lL 0.2 m Tris pH 8.2 and 20 lL mm DTNB for 30 The NP-SH contents were determined in the supernatant, after proteins precipitation with 5% trichloroacetic acid (final concentration) by incubating 450 lL supernatant, 900 lL 0.4 m Tris pH 8.9 and 26 lL mm DTNB for Absorption levels were measured at 412 nm FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS 813 cTPxI and respiratory function in antioxidant defense A P D Demasi et al Acknowledgements Determination of the switching of cTPxI structures in vivo Cells grown on YPD were treated during 40 with 1.2 mm H2O2, 0.1 lgỈmL)1 antimycin A and 2.0 lgỈmL)1 FCCP, separately or in association The corresponding whole cell extracts, obtained as described by Ausubel et al [67], were separated by 9% native-PAGE (15 · 15 cm gels, overnight running) and subjected to immunoblotting with an anti-cTPxI antibody The nondenatured protein molecular weight marker kit was purchased from Sigma As positive control, recombinant cTPxI was also present in the gels We thank Dr Shusuke Kuge for providing strains and plasmids We also thank Hugo Metz for technical assistance with the confocal laser scanning microscopy analysis and Lyndel Meinhardt for his comments on the manuscript Special thanks to Vasco dos Santos Dias (in memoriam) This work was supported by grants from the Brazilian Agencies FAPESP and CNPq References DNA manipulation To generate the probes for northern blot analysis, the DNA sequences of the selected antioxidant genes were PCR amplified from the collection ExClonesTM, Yeast ORF Expression Clones, Research Genetics (Invitrogen, Madison, WI, USA) The clones containing the expression plasmids corresponding to the ORFs of interest (YPL091W, YJL101C, YML028W, YLR109W, YDR353W, YDR453C, YDR513W, YNL241C, YCL035C, YFL039C, YHR008C, YIL010W, YLR043C, YGR088W, YJR104C, YBL064C, YDR256C, YDL066W, YPL188W, YOL049W, YAL005C, YLL026W and YIR037W) were grown separately on YPD medium, and DNA of each clone was extracted as described by Ausubel et al [67] PCR was carried out using the following primers: 5¢-GAATTCCAGCTGACCACC-3¢ and 5¢GATCCCCGGGAATTGCCAT-3¢ The resulting PCR products were purified and the sequences were confirmed previously to the probes preparation RNA isolation and analysis Total yeast RNA was extracted by the method of hot acid phenol method and northern blotting was performed as previously described [67] The 32P-labeled probes were prepared by random primed synthesis [67] Actin was used as loading control and no significant difference was found relative to ribosomal RNA (not shown) Localization of GFP-tagged Yap1 The expression plasmids for the green fluorescent protein (GFP) fused to Yap1 were kindly provided by Kuge [68] They were transferred to cells of the strains JD7–7C and tsa1D Cells were grown on YPD to mid-log phase, concentrated into 25 lL of medium, treated with 1.2 mm H2O2 and 0.1 lgỈmL)1 antimycin A separately or in association, and lL of each culture were 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192–205 67 Ausubel FM, Brent R, Kingstone RE, Moore DD, Seidman JA, Smith JA & Struhl K (1994) Ch 13 Saccharomyces cerevisiae In Current Protocols in Molecular Biology John Wiley and Sons, Inc, Chichester, UK 68 Kuge S, Jones N & Nemoto A (1997) Regulation of yAP-1 nuclear localization in response to oxidative stress EMBO J 16, 1710–1720 FEBS Journal 273 (2006) 805–816 ª 2006 The Authors Journal compilation ª 2006 FEBS ... any of the inhibitors of mitochondrial function (Fig 3) Similar results were obtained with 0.5 mm H2O2 alone or in association with the mitochondrial function inhibitors (not shown) Since it was... expression of genes involved in yeast oxidative stress response: mitochondrial function contribution cTPxI participates of H2O2-mediated signaling processes, including regulation of gene expression... that the conversion of cTPxI to different oligomerization states is implicated with its chaperone ⁄ peroxidase switching [33,34], we suggest that the chaperone activity of this protein, in addition