Exogenous oxidative stress induces Ca 2+ release in the yeast Saccharomyces cerevisiae Claudia-Valentina Popa, Ioana Dumitru, Lavinia L. Ruta, Andrei F. Danet and Ileana C. Farcasanu Faculty of Chemistry, University of Bucharest, Romania Introduction Eukaryotic cells, from yeast to mammals, respond and adapt to environmental stress by evolutionarily con- served multicomponent endogenous systems that utilize a network of signal transduction pathways to regulate the adaptive and protective phenotype. Changes in the chemical or physical conditions of the cell that impose a negative effect on growth demand rapid cellular responses, which are essential for survival. Molecular mechanisms induced upon exposure of cells to such adverse conditions are commonly designated as stress responses. Ca 2+ -mediated signaling of stress conditions is used by virtually every eukaryotic cell to regulate a wide variety of cellular processes through transient increases in cytosolic Ca 2+ . Budding yeast (Saccharomyces cere- visiae) cells use Ca 2+ as a second messenger when they are exposed to various environmental stress conditions, such as hypotonic and cold stress [1,2], hyperosmotic and salt stress [3], b-phenylethylamine-induced intracel- lular H 2 O 2 generation [4], or high pH [5]. The increase in cytosolic Ca 2+ can be a consequence of external Ca 2+ influx via the Cch1p ⁄ Mid1p Ca 2+ channel on the plasma membrane [1,3,6] or release of vacuolar Ca 2+ into the cytosol through the vacuole-located Ca 2+ channel Yvc1p [7,8]. After acting as a second messenger, cytosolic Ca 2+ is restored to the normal very low levels through the action of Ca 2+ pumps and exchangers. Thus, the Ca 2+ -ATPase Pmc1p [9,10] and the vacuolar Ca 2+ ⁄ H + exchanger Vcx1p [11,12] inde- pendently transport cytosolic Ca 2+ into the vacuole, whereas Pmr1p, the secretory Ca 2+ -ATPase, pumps cytosolic Ca 2+ into the endoplasmic reticulum and Keywords aequorin; cytosolic calcium; oxidative stress; Saccharomyces cerevisiae; yeast Correspondence I. C. Farcasanu, University of Bucharest, Faculty of Chemistry, Sos. Panduri 90-92, Bucharest, Romania Fax: +40 021 410 01 40 Tel: +40 721067169 E-mail: farcasanu.ileana@unibuc.ro (Received 16 April 2010, revised 2 July 2010, accepted 28 July 2010) doi:10.1111/j.1742-4658.2010.07794.x The Ca 2+ -dependent response to oxidative stress caused by H 2 O 2 or tert- butylhydroperoxide (tBOOH) was investigated in Saccharomyces cerevisiae cells expressing transgenic cytosolic aequorin, a Ca 2+ -dependent photopro- tein. Both H 2 O 2 and tBOOH induced an immediate and short-duration cytosolic Ca 2+ increase that depended on the concentration of the stres- sors. Sublethal doses of H 2 O 2 induced Ca 2+ entry into the cytosol from both extracellular and vacuolar sources, whereas lethal H 2 O 2 shock mobi- lized predominantly the vacuolar Ca 2+ . Sublethal and lethal tBOOH shocks induced mainly the influx of external Ca 2+ , accompanied by a more modest vacuolar contribution. Ca 2+ transport across the plasma membrane did not necessarily involve the activity of the Cch1p ⁄ Mid1p channel, whereas the release of vacuolar Ca 2+ into the cytosol required the vacuolar channel Yvc1p. In mutants lacking the Ca 2+ transporters, H 2 O 2 or tBOOH sensitivity correlated with cytosolic Ca 2+ overload. Thus, it appears that under H 2 O 2 -induced or tBOOH-induced oxidative stress, Ca 2+ mediates the cytotoxic effect of the stressors and not the adaptation process. Abbreviations BAPTA, 1,2-bis(2-aminophenoxy)ethane-N,N,N ¢,N ¢-tetraacetic acid; tBOOH, tert-butylhydroperoxide; WT, wild-type. FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4027 Golgi, and is responsible for Ca 2+ extrusion from the cell [13,14]. These responses are mediated by the uni- versal Ca 2+ sensor protein calmodulin, which can bind and activate calcineurin; this inhibits, at the post-tran- scriptional level, the function of Vcx1p [11,15,16] and induces the expression of PMC1 and PMR1 genes via activation of the Crz1 transcription factor [15,16]. The release of Ca 2+ from intracellular stores stimulates extracellular Ca 2+ influx, a process known as capacita- tive calcium entry [17]. Conversely, the release of vacu- olar Ca 2+ via Yvc1p can be further stimulated by Ca 2+ from outside the cell as well as that released from the vacuole by Yvc1p itself in a positive feedback process called Ca 2+ -induced Ca 2+ release [8,18–20]. S. cerevisiae is a very useful model and an attractive alternative to mammalian cell lines for studying the effect of oxidative stress on the eukaryotic cells and also an interesting model for studying antioxidants in vivo [21–28]. Living in an oxidant-rich medium under natural conditions, it is expected that yeast cells will respond promptly to variations in the oxidative state of the environment. How fast the cells respond to oxidative stress and whether this type of response is Ca 2+ -mediated are issues that are not clearly understood. In this study, evidence is presented that the cytotoxic effect of the exogenous oxidative stress induced by H 2 O 2 or by the less hydrophilic oxidant tert-butylhydr- operoxide (tBOOH) is triggered by transient elevations in cytosolic Ca 2+ as a result of both rapid influx from outside the cell and of release from vacuolar stores. Results H 2 O 2 induces a transient increase in cytoplasmic Ca 2+ As a free radical generator, H 2 O 2 is known to have deleterious effects on cell growth, and exposure to high concentrations requires an immediate response for cell survival. To determine whether the cell response to high concentrations of H 2 O 2 is mediated by Ca 2+ in S. cerevisiae, the wild-type (WT) strain BY4741 was transformed with a plasmid expressing the luminescent Ca 2+ reporter apoaequorin from a constitutive pro- moter. After reconstitution of functional aequorin by addition of its cofactor coelenterazine, cells were exposed to H 2 O 2 shock directly in the luminometer tube. The cells responded promptly, as H 2 O 2 initiated immediate and transient luminescence peaks, indicating sudden elevations in the cytosolic Ca 2+ .Ca 2+ pulses could be recorded for H 2 O 2 concentrations as low as 0.5 mm, and a sharp rise followed by a rapid fall in the cytosolic Ca 2+ -caused luminescence was noted for concentrations of 2 mm and over (Fig. 1A). The pulse amplitude increased with H 2 O 2 concentration (Fig. 1B). H 2 O 2 treatment of the cells transformed with the empty vector under the same conditions did not elicit any luminescence response (data not shown). The luminescence spectra were reproducible for the range of H 2 O 2 concentrations used (0.5–10 mm), which covered toxic but nonlethal concentrations (0.5–2 mm) and lethal concentrations (3–10 mm). AB Fig. 1. Changes in cytosolic Ca 2+ upon exposure to H 2 O 2 . (A) Effect of H 2 O 2 on the intensity of the Ca 2+ -dependent cell luminescence. WT BY4147 cells were transformed with the plasmid pYX212–cytAEQ. The cells expressing coelenterazine-reconstituted aequorin were exposed to various H 2 O 2 concentrations directly in the luminometer tube, as described in Experimental procedures. The arrow indicates the addition of H 2 O 2 . (B) Maximal response of H 2 O 2 -treated cells. The WT cells transformed with pYX212–cytAEQ were subjected to H 2 O 2 shocks as in (A) (closed circles). The data are presented as the ratio of the maximum relative light units (RLU) to the average RLU recorded before H 2 O 2 shock. Closed diamonds correspond to the response of control cultures not expressing aequorin (transformed with the empty vector pYX212). Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05). Oxidative stress and Ca 2+ C V. Popa et al. 4028 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS Under H 2 O 2 stress, Ca 2+ is mobilized from both external and internal sources To further characterize the Ca 2+ response to exoge- nous H 2 O 2 , we examined whether the Ca 2+ flux comes from an external or an internal source. In the WT cells, H 2 O 2 exposure induced sharp and transient lumi- nescence peaks caused by the increase in cytosolic Ca 2+ (Fig. 2A). The peak intensity was attenuated by the presence of the membrane-impermeable Ca 2+ che- lator 1,2-bis(2-aminophenoxy)ethane-N,N,N¢,N¢-tetra- acetic acid (BAPTA) (Fig. 2D), suggesting that, at least in part, the Ca 2+ flux comes from outside the cells. Following the addition of BAPTA, the pattern of the luminescence spectrum recorded for the WT strain changed from a short-duration Ca 2+ pulse (Fig. 2A) to a prolonged elevation of cytosolic Ca 2+ , which gradually diminished within 2–3 min (Fig. 2D). Never- theless, addition of BAPTA did not completely sup- press the cytosolic Ca 2+ elevation, suggesting that, on exposure to H 2 O 2 , internal stores are also mobilized. To test this possibility, the cytosolic Ca 2+ burst was also monitored in cells lacking the genes encoding the components of the main plasma membrane Ca 2+ channel, Cch1p ⁄ Mid1p. It was found that, upon expo- sure to H 2 O 2 , the cch1D and mid1D null mutants exhibited robust, albeit different, responses. Thus, whereas the luminescence peak caused by the Ca 2+ flux decreased to approximately half in the cch1D cells (Fig. 2B), in the mid1D mutant it was (surprisingly) slightly higher than in the WT cells (Fig. 2C). This observation suggested that, if involved, Cch1p alone, independently of Mid1p, may be partially responsible for the Ca 2+ influx under H 2 O 2 stress. As the differ- ence between the WT and mid1D cells was small, the tests described above were repeated on seven different days, with no obvious variations being seen from day to day. Statistical analysis showed that mid1D cells exhibited luminescence maximum peaks that were con- stantly higher than that recorded for the WT cells, with an average that was 10 ± 1.6% higher than the normal luminescence maximum intensity (P < 0.05). In the parallel study, cch1D cells exhibited lumines- cence maximum peaks that were constantly lower than DE GH F AB C 50 s Fig. 2. Effect of mutations affecting Ca 2+ fluxes in response to H 2 O 2 . Isogenic strains expressing coelenterazine-reconstituted cytAEQ were exposed to H 2 O 2 (2 mM; a toxic but sublethal concentration) directly in the luminometer tube. When used, the membrane-impermeant Ca 2+ chelator BAPTA was added (5 mM final concentration) 1 min before the H 2 O 2 shock. The arrows indicate the addition of H 2 O 2 . Each determi- nation was repeated at least three times on different days, with no significant variations being seen (P < 0.05). One typical luminescence spectrum is presented for each strain. (A) WT strain. (B) Null mutant cch1D strain. (C) Null mutant mid1D strain. (D) WT strain pretreated with BAPTA. (E) Null mutant cch1D strain pretreated with BAPTA. (F) Null mutant mid1D strain pretreated with BAPTA. (G) Null mutant yvc1D strain. (H) Null mutant yvc1D strain pretreated with BAPTA. C V. Popa et al. Oxidative stress and Ca 2+ FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4029 that recorded for the WT cells, with an average that was 33.5 ± 2.2% smaller than the normal lumines- cence maximum intensity (P < 0.05). Interestingly, BAPTA treatment of both cch1D (Fig. 2E) and mid1D (Fig. 2F) cells attenuated the Ca 2+ influx to levels comparable to those detected for the BAPTA-treated WT cells (Fig. 2D), indicating that, under H 2 O 2 stress, external Ca 2+ may enter the cytosol via a transporter other than the Cch1p ⁄ Mid1p channel. Similar results were obtained when BAPTA was replaced with another Ca 2+ chelator, EGTA (data not shown). The addition of BAPTA or other Ca 2+ chelators to the medium equalized the H 2 O 2 -induced cytosolic Ca 2+ peak in WT, cch1D and mid1D cells (Fig. 2D–F), but it still allowed a robust Ca 2+ burst, suggesting that, on exposure to the oxidant, internal stores are also mobilized. In yeast, the vacuole is the main stor- age location for intracellular Ca 2+ , so the H 2 O 2 - induced Ca 2+ flux was monitored in cells lacking Yvc1p, the vacuolar membrane channel responsible for release of Ca 2+ from the vacuole to the cytosol. The YVC1 gene deletion drastically reduced the amplitude of the Ca 2+ burst, indicating that most of the Ca 2+ released into the cytosol after H 2 O 2 exposure comes from the vacuole via the Yvc1p channel (approxi- mately 93%). However, a luminescence peak was still noticeable in the yvc1D cells, reaching approximately 1 ⁄ 15 of the normal value (Fig. 2G). This residual peak was practically abolished by the addition of BAPTA (Fig. 2H), suggesting that the small Ca 2+ burst seen in yvc1D cells was the result of Ca 2+ influx from outside the cell. As BAPTA addition removes  50% of the signal in the case of WT cells (Fig. 2A,D), it seems probable that, in the presence of H 2 O 2 , the vacuolar Yvc1p channel is activated by cytoplasmic Ca 2+ that may come from outside the cell or be released from the vacuole by Yvc1p itself in a positive feedback pro- cess, as previously reported [8,18–20]. Thus, by dele- tion of YVC1, both the H 2 O 2 -induced component and the Ca 2+ -induced component are removed. If this is so, the signal that disappears in BAPTA-treated WT cells (Fig. 2D) may be interpreted as the signal through Yvc1p secondarily induced by external Ca 2+ . Adaptation to H 2 O 2 -induced oxidative stress is favored under low-Ca 2+ conditions As the elevation of cytosolic Ca 2+ in response to oxidative stress was different in the mutant cells used, growth in media supplemented with H 2 O 2 was investigated. It was found that the cch1D and yvc1D cells were more tolerant to H 2 O 2 than the WT cells, suggesting that the lower level of cytosolic Ca 2+ burst achieved during oxidative shock may result in a pro- tective effect on cell growth. In this regard, mid1D cells, which exhibited the highest Ca 2+ burst when exposed to H 2 O 2 shock, were slightly more sensitive to H 2 O 2 than WT cells and considerably more sensitive than cch1D and yvc1D cells (Fig. 3A, upper right). At the same time, supplementation of the medium with the Ca 2+ chelator EGTA augmented cell tolerance to H 2 O 2 (Fig. 3A, middle), whereas supplementary Ca 2+ augmented sensitivity to H 2 O 2 (Fig. 3A, bottom right). Paradoxically, yvc1D cells, which exhibited the lowest cytosolic Ca 2+ burst, were the most tolerant to exoge- nous H 2 O 2 (Fig. 3B), suggesting that lower cytosolic Ca 2+ bursts during oxidative shocks may actually increase the chances of cell survival. To check this pos- sibility, transgenic cells expressing the YVC1 gene from a galactose-inducible promoter were monitored in terms of sensitivity to oxidative stress. WT cells over- expressing YVC1 are known to release more Ca 2+ into the cytosol [7] and, indeed, such cells were more sensi- tive to H 2 O 2 than cells expressing the control vector (Fig. 3C). Moreover, sensitivity increased with induc- tion time, probably because of increasing gene expres- sion induced by activation of the GAL1 promoter by galactose. In contrast, cells overexpressing the PMC1 gene from a galactose-inducible promoter became more tolerant to H 2 O 2 than cells expressing the control vector (Fig. 3C). Pmc1p transports Ca 2+ to the vacu- ole, and its overproduction probably results in faster restoration of cytosolic Ca 2+ . Other mutations that alter cytosolic Ca 2+ were also investigated. For example, whereas pmc1D and vcx1D null mutants were both more tolerant than the wild type, the double mutant pmc1D vcx1D was found to be hyper- sensitive to H 2 O 2 (Fig. 3B). This double mutant lacks Ca 2+ in the vacuole, and therefore cannot release Ca 2+ via Yvc1p. Nevertheless, this strain is deficient in restor- ing cytosolic Ca 2+ levels after a stress burst [12], and is consequently less tolerant to exogenous oxidants. Cytosolic Ca 2+ was reported to upregulate the calci- neurin-dependent degradation of Yap1p [29], the main transcription factor inducing gene expression in response to oxidative stress [19,20,30]. If the high cyto- solic Ca 2+ achieved by cells when they are exposed to H 2 O 2 can result in calcineurin-dependent Yap1p degradation and hence sensitivity to oxidants, cells lacking functional calcineurin should gain a certain tolerance to H 2 O 2 . To check this possibility, the growth of cnb1D cells (which lack the gene encoding the regulatory subunit of calcineurin) under oxidative stress was tested. It was noted that CNB1 deletion resulted in an increase of tolerance to H 2 O 2 (Fig. 3B), Oxidative stress and Ca 2+ C V. Popa et al. 4030 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS suggesting that the Ca 2+ -mediated toxicity of H 2 O 2 may be also the result of Ca 2+ -mediated degradation of Yap1p. Exposure to lethal H 2 O 2 concentrations results in Ca 2+ mobilization predominantly from the vacuole In the experiments described above, Ca 2+ appearance in the cytosol was monitored in cells exposed to toxic, but nonlethal, concentrations (as seen from the cell viability following exposure; data not shown) of H 2 O 2 . To determine whether cells behaved differently when subjected to stronger oxidative insults, WT cells were exposed to a lethal concentration of H 2 O 2 (10 mm). In this case, it was found that BAPTA pretreatment of cells did not attenuate the Ca 2+ burst (Fig. 4A,B), indicating that, under lethal oxidative conditions, cells rely mainly on internal Ca 2+ stores. Under the same stress conditions, yvc1D cells exhibited only faint Ca 2+ -mediated luminescence (Fig. 4A,B), suggesting that when cells are confronted with higher, lethal con- centrations of H 2 O 2 , they utilize the vacuole as the main source for Ca 2+ to be directed to the cytosol. Alkylhydroperoxide also induces a transient increase in cytosolic Ca 2+ To determine whether the Ca 2+ -mediated response to exogenous H 2 O 2 is part of a more general oxidative stress response mechanism, a less hydrophilic oxidant, tBOOH, was considered. This substance is largely used A B C Fig. 3. Effect of H 2 O 2 -induced oxidative stress on the cell growth of Ca 2+ channel mutants. (A) Growth properties of Ca 2+ channel mutants. Isogenic strains (WT, cch1D, mid1 and yvc1D) were spotted (approximately 4 lL) in 10-fold serial dilutions (from 10 7 cellsÆmL )1 , left, to 10 3 cellsÆmL )1 , right) onto YPD plates supplemented with the indicated chemicals (4 m M H 2 O 2 ,20mM EGTA, 10 mM CaCl 2 ). Early exponential growth phase cultures were used to prepare the diluted cultures, which were spotted on plates by means of a replicator. Cells were photographed after 3 days of incubation at 28 °C. The concen- trations used were 4 m M H 2 O 2 ,20mM EGTA and 10 mM CaCl 2 . (B) Effect of YVC1 and PMC1 overexpression on sensitivity to oxidative stress. WT cells transformed with plasmids pGREG506DSalI (empty vector, with fragment SalI–SalI excised), pGAL11–YVC1 and pGAL11–PMC1 were shifted from a fresh preculture to SG-Ura for galactose induction of the genes, 6 or 16 h before being stamped [approximately 4 lL, in 10-fold serial dilutions from 10 7 cellsÆmL )1 (left) to 10 3 cellsÆmL )1 (right)] onto SG-Ura agar plates containing H 2 O 2 . Time variation was not observed in the case of the PMC1 galactose-induced phenotype. Similar results were obtained for tBOOH (1.5 and 2 m M, data not shown). (C) H 2 O 2 effect on growth of mutants with altered Ca 2+ -related phenotype. The strains were spotted (approximately 4 lL, 10 6 cellsÆmL )1 ) onto YPD plates containing the indicated concentrations of H 2 O 2 . C V. Popa et al. Oxidative stress and Ca 2+ FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4031 to mimic the oxidative stress that causes formation of alkyl hydroperoxides (e.g. oxidation of unsaturated lipid chains). As the minimal inhibitory concentration for the WT strain is approximately 1 mm,Ca 2+ pulses were recorded in aequorin-expressing WT cells exposed to 0.25–1.5 mm tBOOH (Fig. 5A). The pulse ampli- tude increased with tBOOH concentration (Fig. 5B). When exposed to toxic, but nonlethal, concentra- tions of tBOOH, WT cells promptly responded with a transient elevation in cytosolic Ca 2+ , as seen by the luminescence spectrum of the aequorin-expressing cells (Fig. 6A). This observation suggested that, as in the response to H 2 O 2 , cells utilize Ca 2+ -mediated signaling to initiate the defense mechanisms. CCH1 deletion lowered the amplitude of the Ca 2+ burst to approxi- mately 75% of the normal peak (Fig. 6B), but MID1 deletion resulted in a Ca 2+ cytosolic burst that was slightly higher than in the WT cells (Fig. 6C); this was paralleled by an increased sensitivity of the mid1D mutant to tBOOH (Fig. 7A,C). As the difference between WT and mid1D cells was small, the tests described above were repeated on seven different days, with no major variations being seen from day to day. Statistical analysis showed that mid1D cells exhibited luminescence maximum peaks that were consistently greater than that recorded for WT cells, with an aver- age that was 12.14 ± 0.8% higher than the normal luminescence maximum intensity (P < 0.05). In the parallel study, cch1D cells exhibited luminescence maxi- mum peaks that were consistently lower than that recorded for WT cells, with an average that was 25.56 ± 1.2% lower than the normal luminescence maximum intensity (P < 0.05). Surprisingly, BAPTA addition prior to exposure to tBOOH strongly attenuated the Ca 2+ burst to less than one-quarter of the normal level, both in WT and mutant cch1D and mid1D cells (Fig. 6D–F). This obser- vation suggested that, under tBOOH stress, external Ca 2+ may be predominantly responsible for the cyto- solic bursts, but that the major route of entry into the cell may, again, not be through the Cch1p ⁄ Mid1p channel. The contribution of vacuolar stores could not, however, be ruled out, because although the Ca 2+ -dependent luminescence was drastically reduced by the presence of BAPTA, it still peaked at approxi- mately one-fifth to one-quarter of the normal A B Fig. 4. Ca 2+ mobilization under lethal H 2 O 2 conditions. Isogenic strains (WT and yvc1D) expressing coelenterazine-reconstituted cytAEQ were exposed to 10 m M H 2 O 2 (lethal) in the absence (A) or presence (B) of 5 m M BAPTA directly in the luminometer tube. The arrow indicates the addition of H 2 O 2 . Each determination was repeated at least three times on different days, with no significant variations (P < 0.05) being seen. One typical luminescence spectrum is presented for each type of experiment. RLU, relative light units. AB Fig. 5. Changes in cytosolic Ca 2+ concentration upon exposure to tBOOH. (A) Effect of tBOOH on the intensity of the Ca 2+ -dependent cell luminescence. WT cells expressing coelenterazine-reconstituted cytAEQ were exposed to various tBOOH concentrations directly in the lumi- nometer tube. The arrow indicates the addition of tBOOH. (B) Maximal response of tBOOH-treated cells. The WT cells transformed with pYX212–cytAEQ were subjected to tBOOH shocks as in (A) (closed squares). The data are presented as ratio of the maximum relative light units (RLU) to the average RLU recorded before tBOOH shock. Closed diamonds correspond to the response of control cultures not expressing aequorin (transformed with the empty vector pYX212). Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05). Oxidative stress and Ca 2+ C V. Popa et al. 4032 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS maximum, and this was followed by a steady state, which was observed throughout the recording time. To determine whether the Ca 2+ also comes from the vacu- ole, the cytosolic Ca 2+ release under tBOOH stress was monitored in yvc1D cells. It was seen that, as com- pared with WT cells, the Ca 2+ burst was attenuated in yvc1D cells (towards approximately one-quarter of the normal peak; Fig. 6G), suggesting that the vacuolar stores may also contribute to the total cytosolic pool. The burst was completely abolished when yvc1 D cells were preincubated with BAPTA (Fig. 6H), indicating that, under tBOOH shock also, the transient elevation in cytosolic Ca 2+ is the result of cooperative influx from both outside the cell and from the vacuole, even though, in this case, the vacuolar contribution seems to be lower. In terms of growth, cch1D and yvc1D cells were more tolerant to tBOOH than WT cells, whereas mid1D cells were more sensitive to tBOOH than WT cells (Fig. 7A,C). Supplementation of the medium with the Ca 2+ chelator EGTA slightly augmented cell toler- ance to tBOOH (Fig. 7A, right), but external Ca 2+ had no obvious effect on cell growth (data not shown). It was found that the Ca 2+ -mediated cytosol lumi- nescence increased when the tBOOH concentration was varied from 0.75 mm (toxic, but nonlethal) to 1.5 mm (lethal) (Fig. 7B, dotted lines), but did not change when the same conditions were applied to BAPTA-pretreated cells (Fig. 7B, solid line). This observation indicated that, unlike the case with H 2 O 2 , the extent of vacuolar Ca 2+ mobilization may not be dependent on tBOOH concentration. The susceptibility to tBOOH of other transgenic strains with altered Ca 2+ homeostasis was tested. Simi- lar to what was seen with H 2 O 2 , YAP1 overexpression reduced, whereas PMC1 overexpression augmented, the tolerance of WT cells to tBOOH (data not shown). Other mutations that alter cytosolic Ca 2+ were also investigated, and the response to tBOOH paralleled the response to H 2 O 2 . For example, whereas pmc1D and vcx1D null mutants were both more tolerant than the wild type, the double mutant pmc1D vcx1D was found to be hypersensitive to tBOOH (Fig. 7C). Also, CNB1 deletion resulted in an increase in tolerance to tBOOH (Fig. 7C). Thus, it seems that Ca 2+ mediates ABC DEF GH Fig. 6. Effect of mutations affecting Ca 2+ fluxes in response to tBOOH. Isogenic strains expressing coelenterazin-reconstituted cytAEQ were exposed to tBOOH (0.5 m M, a toxic but sublethal concentration) directly in the luminometer tube. When used, the membrane-imper- meant Ca 2+ chelator BAPTA was added (5 mM final concentration) 1 min before the tBOOH shock. The arrows indicate the addition of tBOOH. Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05). One typical luminescence spectrum is presented for each strain. (A) WT strain. (B) Null mutant cch1D strain. (C) Null mutant mid1D strain. (D) WT strain pretreated with BAPTA. (E) Null mutant cch1D strain pretreated with BAPTA. (F) Null mutant mid1D strain pretreated with BAPTA. (G) Null mutant yvc1D strain. (H) Null mutant yvc1D strain pretreated with BAPTA. RLU, relative light units. C V. Popa et al. Oxidative stress and Ca 2+ FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4033 the cytotoxic effect of exogenous oxidants rather than the adaptative process. Discussion Aerobic cells are continuously exposed to oxidative insults that have to be perceived and scavenged rapidly to allow normal growth and development. Studies con- cerning the antioxidant arsenal of the cell are numer- ous, and many defense mechanisms have been elucidated, but how exactly the cell senses the threat of oxidative species that are above the non-noxious thresholds is still not clearly understood. Ca 2+ is an important second messenger in the eukaryotic cell, and there is increasing evidence that cytosolic Ca 2+ entry in yeast is critical for survival under a variety of stress conditions, including hypo-osmotic or hyperosmotic shock [1,3,5,7], protein-unfolding agents [30], or anti- fungal drugs [31,32]. The results presented in this study provide evidence that exogenous oxidative stress induces a transient increase in cytosolic Ca 2+ that originates from both outside the cell and from the A B C Fig. 7. (A) Effect of tBOOH-induced oxida- tive stress on cell growth. Isogenic strains (WT, cch1D, mid1D and yvc1D) were spotted (approximately 4 lL) in 10-fold serial dilutions [from 10 7 cellsÆmL )1 (left) to 10 3 cellsÆmL )1 (right)] onto YPD plates supplemented with the indicated chemicals (0.75 m M tBOOH, 20 mM EGTA). Early exponential growth phase cultures were used to prepare the diluted cultures, which were spotted on plates by means of a repli- cator. Cells were photographed after 3 days of incubation at 28 °C. (B) Ca 2+ mobilization under lethal tBOOH concentrations. WT cells expressing coelenterazine-reconstituted cytAEQ were exposed to toxic but nonlethal (0.75 m M, left) or lethal (1.5 mM, right) concentrations of tBOOH directly in the luminometer tube. The arrow indicates the addition of tBOOH. When used, BAPTA was added to a final concentration of 5 m M. Each determination was repeated at least three times on different days, with no significant variations being seen (P < 0.05). One typical luminescence spectrum is pre- sented for each type of experiment. (C) The effect of tBOOH on growth of mutants with altered Ca 2+ -related phenotype. The strains were spotted (approximately 4 lL, 10 6 cellsÆmL )1 ) onto YPD plates containing the indicated concentrations of tBOOH. Oxidative stress and Ca 2+ C V. Popa et al. 4034 FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS vacuole. Toxic, but sublethal, concentrations of exoge- nous H 2 O 2 (1–2 mm) induced very rapid and transient Ca 2+ bursts. Cytosolic Ca 2+ elevations dropped to roughly half the normal value when cells were pretreat- ed with Ca 2+ chelators (BAPTA or EGTA) shortly before the oxidative shock, so part of the Ca 2+ must come from outside the cell. However, the external Ca 2+ seems to be taken up through a system that is different from the Cch1p ⁄ Mid1p channel, as Ca 2+ bursts were still clearly detectable in cch1D cells and were higher than normal in mid1D mutants. At the same time, the process seems to be assisted by the Yvc1 vacuolar channel, as the H 2 O 2 -triggered cytosolic Ca 2+ elevation was rather modest in the yvc1D mutant. The residual Ca 2+ increase observed in the yvc1D mutant could be explained by the existence of a low-affinity Ca 2+ system of unknown identity [30,33]. Nevertheless, as the H 2 O 2 concentration increased from very toxic to lethal doses (3–10 m m), the cells gradually turned to internal vacuolar stores rather than external Ca 2+ . The tolerance to high H 2 O 2 correlated inversely with the amount of cytosolic Ca 2+ achieved during oxida- tive shock. Thus, the mid1D mutant, which exhibited the highest cytosolic Ca 2+ burst, was the least tolerant, and the yvc1D mutant, with the lowest Ca 2+ load, was the most tolerant. Excessive or unregulated levels of Ca 2+ in the cytosol can lead to cell death [34] and, indeed, there was a good correlation between hyper- sensitivity to H 2 O 2 or tBOOH and excessive cytosolic Ca 2+ elevation. The triad Ca 2+ ⁄ oxidative stress ⁄ cell death has already been reported for other systems. In mammals, for instance, Ca 2+ overload apparently leads to mitochondrial dysfunction, reactive oxygen species production and cell death [35]. However, Ca 2+ has also been reported to upregulate the calcineurin- dependent degradation of Yap1p [29], the transcription factor that regulates gene expression in response to oxidative stress in yeast [24,25,36]. In this respect, the high cytosolic Ca 2+ achieved by the cells when exposed to lethal concentrations may result in Yap1p degradation, a situation in which the cells become hypersensitive to oxidative stress. The cell response to high H 2 O 2 was paralleled by the response to the less hydrophilic oxidant tBOOH. Here also, the cytosolic Ca 2+ transients could be detected upon exposure, with both external and vacuo- lar contributions. Again, the mid1D mutant exhibited the highest Ca 2+ load and the lowest tolerance, whereas the yvc1D mutant exhibited the lowest Ca 2+ burst, correlating with the highest tolerance to tBOOH. The only difference seemed to reside in the prevalence of the external Ca 2+ contribution to the cytosolic pool, as seen by the low Ca 2+ burst in BAP- TA-pretreated cells. Release of vacuolar Ca 2+ through Yvc1p requires activation by Ca 2+ on the cytosolic side of the vacuolar membrane [8], and the low level of cytosolic Ca 2+ achieved in BAPTA-pretreated cells might be accounted for by the inactive Yvc1p. One can also speculate that tBOOH, being less hydrophilic than H 2 O 2 , diffuses more slowly in the yeast aqueous environment, exerting a milder signaling effect. On the other hand, being unable to synthesize polyunsaturated fatty acids, the yeast has not evolved special protective mechanisms against lipid peroxidation, and is less pre- pared to respond to a stressor that mimics lipid hydro- peroxides. Special attention deserves to be paid to the discrep- ancy between the mid1D and cch1 D mutants. Although Mid1p and Cch1p cooperate to form a high-affinity influx system, differences between mid1D and cch1D mutants have already been reported [4,37]. Among other things, Mid1p is known to localize not only in the plasma membrane, but also in the endoplasmic reticulum membrane [38]. Recently, MID1 mutations have been reported to cause a defect in the cell wall structure [39] that might render mid1D cells more per- meable to Ca 2+ or more susceptible to stressors such as H 2 O 2 or tBOOH. The data above suggest that Ca 2+ pulses serve fre- quently, but not invariably, to transduce an oxidative burst signal. However, Ca 2+ seems unlikely to be involved in the adaptation to oxidative stress. Yap1p, the major yeast transcription factor responsible for the transcriptional regulation of many genes involved in the response to oxidative stress, is upregulated directly by the presence of oxidants [40–43], whereas Ca 2+ mediates its calcineurin-dependent degradation [29]. Thus, it is probable that, under strong oxidative condi- tions, the cytosolic Ca 2+ elevations actually signal the ultimate possible way out, which is cell death. This would explain why, under extreme H 2 O 2 stress, the cell blocks the import of external Ca 2+ and turns in a last effort to internal stores. Experimental procedures Strains and culture conditions The S. cerevisiae strains used in this study were isogenic with the WT parental strain BY4741 (MATa; his3D1; leu2D0; met15D0; ura3D0) [44]. The deletion mutant strains used were Y04847 (BY4741, cch1::kanMX4), Y01153 (BY4741, mid1::kanMX4), Y01863 (BY4741, yvc1::kan- MX4), Y04374 (BY4741, pmc1::kanMX4), Y03825 (BY4741, vcx1::kanMX4), Y13825 (MATa; his3D1; leu2D0; C V. Popa et al. Oxidative stress and Ca 2+ FEBS Journal 277 (2010) 4027–4038 ª 2010 The Authors Journal compilation ª 2010 FEBS 4035 lysD0; ura3D0 vcx1::kanMX4) and Y05040 (BY4741, cnb1::kanMX4). All strains were obtained from EURO- SCARF (European S. cerevisiae Archive for Functional Analysis, Institute of Molecular Biosciences Johann Wolf- gang Goethe-University Frankfurt, Germany). Strain VP012 (BY4741, pmc1::kanMX4 vcx1::kanMX4) was obtained by crossing of strains Y04374 and Y13825 fol- lowed by diploid sporulation and random spore analysis. The absence of PMC1 and VCX1 from VP102 cells was checked by colony PCR with primers specific for PMC1 (forward primer, 5¢-ATGTCTAGACAAGACGAAAAT-3¢; reverse primer, 5¢-GAAATGACATCACCGACTAA-3¢) and for VCX1 (forward primer, 5¢-ATGGATGCAACTA- CCCCACT-3¢; reverse primer, 5¢-GGATAACTCCAATAT- TTTTC-3¢). Internal control primers for ACT1 (forward primer, 5¢-GAGGTTGCTGCTTTGGTTAT-3¢; reverse primer, 5¢-GCGGTTTGCATTTCTTGT-3¢) were included in all PCR reactions. Cell growth and manipulation were performed as previously described [45]. Strains were grown in standard YPD, SD or SG supplemented with the neces- sary amino acids. For solid media, 2% agar was used. Plasmid construction The expression vectors harboring PMC1 or YVC1 were obtained as follows. The corresponding ORFs were ampli- fied from the yeast genome by PCR, and were subsequently cloned into vector pGREG506 [46], which allows for galac- tose-inducible expression via the GAL1 promoter. Plasmid pGREG506 was purchased from EUROSCARF. The fol- lowing primers were used: for PMC1,5¢-CATT GGAT CCATGTCTAGACAAGACGAAAAT-3¢ (forward primer, introducing the BamHI site, underlined) and 5¢-TACT GTC GACTTAATAAAAGGCGGTGGACT-3¢ (reverse primer, introducing the SalI site, underlined); and for YVC1,5¢-TA CT GTCGACATGGTATCAGCCAACGGCGA-3¢ (forward primer, introducing the SalI site, underlined) and 5¢-GAG A CTCGAGTTACTCTTTCTTATCCTTTA-3¢ (reverse pri- mer, introducing the XhoI site, underlined). The PMC1 and YVC1 ORFs obtained by PCR were purified with a Wizard SV gel and PCR Clean-up System (Promega), cut with the appropriate restriction enzymes, and cloned into the BamHI–SalI and SalI–XhoI sites of pGREG506, respec- tively, to generate pGAL1–PMC1 and pGAL1–YVC1.As control vector, pGREG506 with the SalI–SalI fragment deleted was constructed (pGREG506DSalI). This deletion removes the HIS3 gene used as a counterselective marker of pGREG vectors for the in vivo plasmid recombination [46]. Growth assessment Overnight precultures were inoculated in fresh media at a density of 10 5 cellsÆmL )1 , and cells were then incubated with shaking (200 r.p.m.) at 28 °C for 2 h before H 2 O 2 , tBOOH, BAPTA or EGTA was added from sterile stocks prepared in 0.1 m Mes ⁄ Tris (pH 6.5). The influence of stressors on cell growth was monitored at time intervals by determining the attenuance (D) of a cell suspension at 600 nm (Shimadzu UV–visible spectrophotometer, UV mini 1240). For spot assays, fresh cell cultures of D 600 nm  1 were diluted 10-fold, 100-fold, 1000-fold and 10 000-fold, and stamped on agar plates with a replicator. In vivo monitoring of oxidative stress-induced Ca 2+ pulse Monitoring of cytosolic Ca 2+ was performed with an apo- aequorin cDNA expression system [47]. Yeast strains were transformed with the plasmid pYX212–cytAEQ, containing the apoaequorin gene under the control of the TPI pro- moter, generously provided by E. Martegani (Department of Biotechnology and Biosciences, University of Milano- Bicocca, Milan, Italy) [48]. Yeast transformation was per- formed with a modified lithium acetate method [49], and Ura + transformants were selected for growth on SD med- ium lacking uracil. For luminescence assays, overnight pre- cultures of cells expressing the apoaequorin gene were diluted in fresh SD-Ura medium to a density of 5 · 10 6 cellsÆmL )1 . The cells were incubated with shaking for another 2 h at 28 °C, before being harvested by centri- fugation. The cells were washed three times (by centrifuga- tion at 9167 g, 1 min) and resuspended at a density of approximately 10 9 cellsÆmL )1 in 0.1 m Mes ⁄ Tris buffer (pH 6.5). To reconstitute functional aequorin, 50 lm native coelenterazine (Sigma; stock solution of 1 lgÆlL )1 in meth- anol) was added to the cell suspension, and the cells were incubated for 2 h at 28 °C in the dark. Aliquots containing approximately 10 7 cells were harvested, and excess coelen- terazine was removed by centrifuging three times (9167 g, 30 seconds). The cells were resuspended in 0.1 m Mes ⁄ Tris buffer (pH 6.5) and transferred to the luminometer tube. A cellular luminescence baseline was determined by 1 min of recording at 1 s intervals. The exogenous oxidants H 2 O 2 and tBOOH were added from sterile stocks to give the final concentrations indicated, and light emission was monitored with a single-tube luminometer (Turner Biosystems, 20 n ⁄ 20). The light emission was monitored for at least 5 min after the stimulus at 1 s intervals, and reported as relative luminescence units ⁄ s. When used, the calcium che- lator BAPTA was added to the cell suspension to the final concentration of 5 mm 1 min before application of the stress. To ensure that total reconstituted aequorin was not limiting in our assay, at the end of each experiment aequorin expression and activity were checked by lysing cells with 10% Triton X-100, and only the cells with con- siderable residual luminescence were considered. Multiple tests, on different days (a minimum of three for large differ- ences between transgenic lines; a maximum of seven for Oxidative stress and Ca 2+ C V. 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