Characterization of the Calcium-mediated Response to Alkaline Stress in Saccharomyces cerevisiae*

Exposure of the yeast Saccharomyces cerevisiae to alkaline stress resulted in adaptive changes that involved remodeling the gene expression. Recent evidence suggested that the calcium-activated protein phosphatase calcineurin could play a role in alkaline stress signaling. By using an aequorin luminescence reporter, we showed that alkaline stress resulted in a sharp and transient rise in cytoplasmic calcium. This increase was largely abolished by addition of EGTA to the medium or in cells lacking Mid1 or Cch1, components of the high affinity cell membrane calcium channel. Under these circumstances, the alkaline response of different calcineurin-sensitive transcriptional promoters was also blocked. Therefore, exposure to alkali resulted in entry of calcium from the external medium, and this triggered a calcineurin-mediated response. The involvement of calcineurin and Crz1/Tcn1, the transcription factor activated by the phosphatase, in the transcriptional response triggered by alkalinization has been globally assessed by DNA microarray analysis in a time course experiment using calcineurin-deficient (cnb1) and crz1 mutants. We found that exposure to pH 8.0 increased at least 2-fold the mRNA levels of 266 genes. In many cases (60%) the response was rather early (peak after 10 min). The transcriptional response of 27 induced genes (10%) was reduced or fully abolished in cnb1 cells. In general, the response of crz1 mutants was similar to that of calcineurin-deficient cells. By analysis of a systematic deletion library, we found 48 genes whose mutation resulted in increased sensitivity to the calcineurin inhibitor FK506. Twenty of these mutations (42%) also provoked alkaline pH sensitivity. In conclusion, our results demonstrated that calcium signaling and calcineurin activation represented a significant component of the yeast response to environmental alkalinization.

Calcium-mediated signaling mechanisms are used by virtually every eukaryotic cell to regulate a wide variety or cellular processes, including gene expression. Transient increases in cytosolic calcium results in activation of diverse enzymes, such as the protein phosphatase calcineurin. Calcineurin is a heterodimer of catalytic subunit and regulatory subunits. In the yeast Saccharomyces cerevisiae, the catalytic subunit is encoded by two genes, CNA1 and CNA2 (1), whereas a single gene, CNB1, encodes the regulatory subunit (2). Cells lacking the catalytic subunits, or the regulatory subunit, are deficient in calcineurin activity.
Exposure of yeast cells to a number of signals, such as ␣-factor (3,4), glucose (5), sphingosine (6), and certain stress conditions (7)(8)(9), triggers a rise in cytoplasmic calcium. This increase in calcium can be a consequence of external calcium influx or release from internal stores, such as the vacuole, and results in activation of calcineurin. For instance, hyperosmotic shock has been reported to provoke calcium release from vacuolar stores (8) through Yvc1, a member of the transient receptor potential channel family, and to trigger influx of the cation from the external medium (9) through the Mid1-Cch1 high affinity Ca 2ϩ influx system. Activated calcineurin dephosphorylates and activates the transcriptional factor Crz1/Tcn1 (10,11), which enters the nucleus (12) and, in turn, activates a set of responsive genes by binding to calcineurin-dependent responsive elements (CDRE) 1 (10,13). It has been documented that most of the transcriptional responses driven by activation of calcineurin are mediated by Crz1/Tcn1 (13).
Recent evidence from our laboratory (14) supports early data (15,16) showing that calcineurin might be involved in the tolerance to alkaline pH. We showed (14) that a significant part of the alkaline response of the ENA1 promoter, and most of the PHO89 response, was blocked by the calcineurin inhibitor FK506 as well as by the absence of Cnb1 or Crz1/Tcn1, suggesting that the transcriptional response of certain genes to alkalinization of the medium could be, at least in part, dependent on calcineurin. These findings led us to speculate the possibility that exposure to alkaline pH could trigger a burst in cytoplasmic calcium, which in turn would activate the phosphatase. Here we confirm and expand this hypothesis by showing that alkalinization of the medium provokes entry of calcium from the external medium and by defining, using DNA microarray technology, the subset of genes whose alkaline transcriptional response is mediated by the calcineurin pathway.

MATERIALS AND METHODS
Strains and Growth Conditions-Yeast cells were grown at 28°C in YPD medium (10 g/liter yeast extract, 20 g/liter peptone, and 20 g/liter dextrose) or, when indicated, in synthetic minimal (SD) or complete minimal (CM) medium (17). The relevant genotype of the strains described in this work can be found in Table I.
Plasmids and Gene Disruptions-Strains carrying deletions of the MID1 and CCH1 genes were generated as follows. Deletion cassettes were amplified by PCR from genomic DNA prepared from mid1::KanMX and cch1::KanMX deletion mutants in the BY4741 background (18). The oligonucleotides used spanned nucleotides Ϫ489 to ϩ2021 (MID1) and Ϫ532 to ϩ6708 (CCH1). Positions are relative to the initiating ATG codon. The amplification fragments were purified and used to transform the DBY746 wild type strain.
A deletion cassette used to disrupt the YVC1 gene (p⌬YVC1_Wa) was generated as follows. The region from Ϫ1118 to ϩ3010 relative to the ATG of the YVC1 locus was amplified by PCR from genomic DNA prepared from the DBY746 wild type strain by using oligonucleotides 5Ј-GCTCTAGAAGCTTCATGGATACTTC-3Ј (which contains an artificial XbaI site) and 5Ј-GCGGTACCACCATTGGTGCAGCATTC-3Ј (which has an added KpnI restriction site). This fragment was cloned into the same sites of plasmid pBluescript and was digested with EcoRI to remove a 1.8-kbp internal region (from nucleotide Ϫ41 to ϩ1780). To generate p⌬YVC1_Wa, this region was replaced by an EcoRI fragment, containing the TRP1 marker, isolated from plasmid pUC19_W_2RI. This construct was made by cloning into pBluescript the TRP1-containing SmaI-NheI fragment of YDpW (19), to yield pBS_W, and recovering the marker as an HincII-SacI fragment, for final cloning into pUC19. Strain RSC46 (yvc1::TRP1), was then generated by transformation with the 2.1-kbp SpeI-HpaI fragment isolated from p⌬YVC1_Wa. All gene disruptions were verified by PCR.
The reporter plasmid pPRX1-LacZ was generated as follows. The PRX1 upstream DNA region enclosed between Ϫ1288 and ϩ33 (relative to the starting ATG) was amplified by PCR with added EcoRI/HindIII restriction sites and was cloned into the same sites of YEp357 (20). Reporter plasmids pPHO84-LacZ, pMRK212, and pPHO89-LacZ were described previously in Ref. 14 and plasmid pAMS366 in Ref. 10.
Calcium Measurements-Evaluation of cytoplasmic calcium burst was carried out essentially as described previously (8). Briefly, yeast strains were transformed with plasmid pEVP11/AEQ (7) and grown in SD medium lacking leucine to an A 660 of 1.8. Cells were pelleted and resuspended in 250 l of fresh medium containing 2 M coelenterazine (Sigma) and incubated for 5.5 h in the dark. The culture was centrifuged, and cells were resuspended in 173 l of fresh medium (which included 10 mM EGTA when chelating external calcium was desired). Three l of the culture was removed, and cells were fixed by addition of a formaldehyde solution (7% v/v final concentration) and kept at 4°C for cell counting. The remaining culture was transferred to luminometer tubes after 15 min (to ensure that the transient calcium increase induced by the glucose present in the fresh medium has vanished), and then the luminescence was integrated every second by using a Berthold LB9507 luminometer. Calcium shock was performed by adding 30 l of a 1.33 M calcium chloride solution. Alkaline shock was accomplished by adding 30 l of 100 mM KOH (which raises the pH to ϳ8.2). Potassium chloride was added as a negative control at the same concentration as KOH. In all experiments, the luminescence values were corrected according to the number of cells present in the sample.
Screen of a Systematic Deletion Library for FK506 Sensitivity-A systematic kanMX deletion library constructed in the BY4741 genetic background (21) was grown in YPD medium supplemented with 150 g/ml G418 up to saturation (3-4 days). The cultures were replicated by using a stainless steel 96-pin replicator (Nalge Nunc International) into duplicate 96-well plates. One set of plates contained 200 l/well YPD (control plates) and the other YPD plus 1.5 g/ml FK506 (Fujisawa Co.). Growth was monitored by using a Labsystem multiwell photometer at A 620 after 18, 20, 22, 24, and 48 h of incubation at 28°C. For each clone, the ratio between the absorbance of the culture in the absence and in the presence of the drug was calculated. This value was divided by the average of the ratios for the entire plate (to normalize for hypothetical differences in the inoculation step). Clones giving a growth ratio equal or higher than 2.0 were initially considered positive. These clones were diluted until the A 620 was 0.005 and re-tested for FK506 sensitivity at four different concentrations of the drug (0.5, 1.0, 1.5, and 2.0 g/ml) at different growth times. Clones consistently yielding a ratio of at least 1.2 were considered as sensitive to FK506. The ratio of absorbance of cultures in the absence and in the presence of each concentration of the drug was calculated, and the highest value obtained was considered as an indicator of the sensitivity of the strain.
RNA Preparation and Northern Blot Analysis-For RNA preparation, yeast cells were grown in YPD to an optical density of 0.8 and split into aliquots. Cells were centrifuged and resuspended either in fresh YPD (nonstressed cells, pH 6.2) or YPD containing 50 mM TAPS (stressed cells, pH 8.0) for different periods. Cultures were then centrifuged for 5 min at 3000 rpm, and total RNA was extracted by using hot phenol and glass beads as described (22). Total RNA (15 g/lane) was transferred to filters and probed as described (14) with a 0.65-kbp fragment encompassing the entire coding region of gene PNC1.
DNA Microchips Analysis-Fifteen g of total RNA was used for cDNA synthesis and labeling, using either the direct (CyScribe first strand labeling kit) or indirect (CyScribe post-labeling kit) labeling kits, in conjunction with Cy3-dUTP and Cy5-dUTP fluorescent nucleotides from Amersham Biosciences. Fluorescently labeled cDNAs were combined and hybridized to yeast genomic microchips constructed in our laboratory by arraying 6014 different PCR-amplified open reading frames from S. cerevisiae on CMT-Ultragaps (Corning) and Genorama SA-1 (Asper Biotech) amino-coated glass slides, using a MicroGrid™ II (BioRobotics) machine. Details on the construction of the slides will be provided elsewhere (23). In addition, the slides included 286 duplicated genes, corresponding to genes responsive under a variety of stress conditions, plus a number of positive and negative control spots. Dyes were swapped for wild type and mutant cells to avoid dye-specific bias. Prehybridization, hybridization, and washing conditions were essentially as described previously (24). Slides were scanned with a ScanArray 4000 fluorescence scanner and analyzed using both commercial (QuantArray) and in-house developed software. The data from different experiments (5 slides/time point for wild type and cnb1 cells and 3 slides/time point for crz1/tcn1 cells) were combined, and the median was calculated. A given gene was considered to be induced or repressed when the ratio stressed/nonstressed was higher than 2.0 or lower than 0.5, respectively, in at least three (for wild type and cnb1 cells) or two experiments (for crz1 cells). Expression ratios were logarithm (base 2)-transformed for statistical analysis. Statistical significance of both gene induction and repression was assessed by testing the null hypothesis log ratio equal to zero under the Student's t test. A p value lower than 0.1 was considered significant for initial gene selection. The differences between the three strains (wild type, cnb1, and crz1) for each selected gene was analyzed by a two-way (slide/time, strain) analysis of variance. The post hoc Tukey's test was used for multiple comparisons between strains (25) with statistical significance set at a p value lower than 0.05. Data analysis was carried out with the SAS/STAT® release 8.01 statistical package (SAS Institute Inc., Cary, NC).
Quantitative Real Time PCR-First strand cDNAs were synthesized from 1 g of total RNA, using the Reverse Transcription System (In-  Two different extractions of total RNA were analyzed, at least by duplicate PCRs. The results presented are the mean of the two different RNA extractions. Basic analysis was performed by using SDS software (Applied Biosystems). The 2 Ϫ⌬⌬Ct method (26) was used for relative quantification using the cDNA from the noninduced sample of each strain as calibrator and RPL28 as the endogenous control.
Evaluation of Oxidative Stress after High pH Exposure-Exponentially growing wild type DBY746 cells (A 660 0.5) in YPD medium were incubated for 1 h with dihydrorhodamine 123 (D1054, Sigma) at 2.5 g/ml essentially as described previously (27). Aliquots were taken and made 30 mM KOH (which raised the pH to 8.1) or 2 mM hydrogen peroxide and further incubated with shaking for 30 and 90 min, respectively. Cultures were centrifuged, and cells were resuspended in phosphate-buffered saline solution and examined through a fluorescein filter using a Nikon Eclipse E800 fluorescence microscope. The percentage of cells showing fluorescence was determined after scoring ϳ2000 cells for each condition.

Exposure of Yeast Cells to Alkaline pH Results in a Transient
Increase in Cytoplasmic Calcium Levels-To evaluate the changes in cytoplasmic calcium after exposure of yeast cells to alkalinization of the medium, the wild type strain JA100 was transformed with a plasmid expressing the luminescent Ca ϩ2 reporter aequorin and then challenged with 15 mM potassium hydroxide, to raise the pH to 8.1. This treatment resulted in a sharp increase in luminescence, which peaked about 15-20 s after the treatment and declined to base-line levels after approximately 1 min, indicating a transient increase in cytosolic calcium level (Fig. 1A). Treatment of the cells with KCl under the same conditions, which does not result in pH change, did not elicit this response. Exposure of the cells to 0.2 M CaCl 2 resulted in a sharp increase in luminescence, with kinetics very similar to the calcium burst induced by exposure to alkali. The intracellular calcium increase observed in response to alkali FIG. 2. Effect of mutations affecting calcium fluxes in the response to alkaline pH. Wild type strain DBY746 and its isogenic derivatives RSC46 (yvc1⌬), RSC28 (mid1⌬), and RSC31 (cch1⌬) were subjected to alkaline shock (pH 8.2), and intracellular calcium was measured as described for Fig. 1A. When included, EGTA was present in the medium at 10 mM.
FIG. 1. Changes in cytoplasmic calcium concentration upon exposure of cells to alkaline pH. A, wild type strain JA100 was transformed with plasmid pEVP11/AEQ and incubated with coelenterazine for 5.5 h. Cells were centrifuged and resuspended in 173 l of fresh SD medium (lacking leucine). Three l of the culture were removed, and cells were fixed by adding a formaldehyde solution (7% final concentration) and kept at 4°C for cell counting. After 15 min, the culture was transferred to luminometer tubes, and the appropriate solution was injected at zero time (indicated by the dotted arrow). The luminescence was integrated every second and is expressed as relative luminescence units (RLU) per 10 6 cells. Treatments are as follows: 0.2 M calcium chloride (solid line), 15 mM KOH (pH 8.1, dotted line), and 15 mM potassium chloride (dashed line). B, the wild type strain DBY746 was subjected to alkaline shock by injection of different concentrations of KOH to produce the indicated pH in the culture, and the intracellular calcium peak was measured as described for the A (closed circles). The data are presented as the percentage of the peak for a given pH divided by the maximal response obtained. Open circles correspond to the response of control cultures in which the equivalent concentrations of potassium chloride were injected. was confirmed in three different wild type strains available in our laboratory, although the intensity of the response varies from strain to strain. In fact, in strain DBY746 the calcium burst triggered by alkali was particularly intense, even more than that observed in response to 0.2 M CaCl 2 (compare Figs. 1 and 2). For this reason we selected this strain for further studies. The effect of pH on cytoplasmic calcium increase was dose-dependent. As shown in Fig. 1B, the increase becomes noticeable when cells are exposed to pH 7.5 and maximal at pH 8.2. These results indicate that budding yeast responds to alkaline stress with an intense and transient calcium burst.
To characterize the origin of the calcium, we repeated these experiments under different conditions by using mutants in genes involved in calcium homeostasis. Most interestingly, we observed that addition of the calcium chelating EGTA to the medium resulted in an almost negligible increase in intracellular calcium. As this could indicate that the main source for intracellular calcium may be of extracellular origin, we evaluated the intensity of the calcium burst in cells lacking genes encoding components of the main calcium channel, such as mid1 and cch1 mutants. As shown in Fig. 2, the calcium burst in response to alkalinization was largely abolished in both cases. In contrast, mutation of YVC1, encoding a vacuolar membrane channel responsible for release of calcium from the vacuole to the cytosol, resulted in a calcium burst identical to that of the wild type strain. Therefore, exposure of yeast to alkali appears to trigger entry of calcium from the extracellular medium.
We considered that if alkaline stress is followed by a rise in cytosolic calcium, this should result in activation of a number of calcium-responsive promoters. To test this possibility, we transformed wild type cells as well as ycv1, mid1, and cch1 mutants with constructs containing different CDRE fused to ␤-galactosidase as a reporter gene, and we tested their response to alkali. As shown in Fig. 3, alkaline induction observed in wild type cells transformed with plasmid pAMS366, which contains a tandem of four synthetic CDRE from the FKS2 gene, is fully blocked by the presence of EGTA in the external medium. Most interestingly, although the response of the yvc1 mutant mimics that of the wild type strain, the reporter gene was not induced by alkaline stress in mid1 and cch1 cells. When the different strains were transformed with plasmids pPHO89-lacZ (which carries the entire PHO89 promoter fused to ␤-galactosidase) or plasmid pMRK212, containing a region of the ENA1 promoter which includes a CDRE, the response to alkali was essentially lost in the presence of external EGTA and largely reduced in mid1 and cch1 mutants. These results demonstrate that exposure to alkali stress results in a burst of calcium that triggers a calcineurin-mediated transcriptional response.
Identification of Mutations That Result in Sensitivity to the Calcineurin Inhibitory Drug FK506 -The observations that alkaline stress results in a burst of calcium and that calcineurin mutants were sensitive to high pH prompted us to investigate whether it could be some significant overlap between the set of mutations that results in sensitivity to high pH and those that present a synthetic phenotype with the loss FIG. 3. Alkaline pH induction of CDRE-containing promoters is reduced in cch1⌬ and mid1⌬ mutants. Wild type strain DBY746 and its isogenic derivatives RSC46 (yvc1⌬), RSC28 (mid1⌬), and RSC31 (cch1⌬) were transformed with plasmid pAMS366, containing four copies in tandem of the CDRE from the FKS2 promoter (10), plasmid pPHO89-lacZ, or plasmid pMRK212 (14). ␤-Galactosidase activity was determined in cells incubated for 60 min (pAMS366 and pMRK212) or 90 min (pPHO89-LacZ) in YPD medium at pH 6.2 (open bars), YPD-50 mM TAPS at pH 8.0 (hatched bars), or the same alkaline medium containing 10 mM EGTA (crossed bars). Data are mean Ϯ S.E. from six independent transformants.

TABLE II
Mutations that result in growth defects in the presence of the calcineurin inhibitor FK506 Numbers in parentheses indicate the level of sensitivity to the drug (expressed as the ratio between of the absorbance of the culture in the absence and in the presence of the drug, see text for details). Genes in boldface type correspond to those identified in a recent report (28) as conferring a growth defect at alkaline pH when deleted. Underlines indicate that the gene was also identified in a recent survey for FK506-sensitive mutations on semi-solid media (48).
TABLE III Genes induced at least 2-fold after alkaline stress Expression data correspond to the maximal induction observed. The Time column indicates the kinetics of the response. Genes showing the highest induction at 10 min are denoted by "E" (early). "I" (intermediate) indicates a peak at 20 min, and "L" (late) corresponds to genes that show the highest induction after 45 min at alkaline pH. Functional categories were assigned based on information provided by MIPS and SGD (29,37). Genes in boldface type were found to also be induced at mild pH stress in a previous report (14). ORF indicates open reading frame.
of calcineurin function. To this end, we monitored the growth of roughly 4800 haploid mutants in the presence of 1.5 g/ml of the calcineurin inhibitor FK506. A total number of 241 putative positive clones were isolated. Upon retesting these clones at different FK506 concentrations, we obtained 48 mutants presenting a measurable growth defect in the presence of (at least) one concentration of the drug. As shown in Table II, more than one-half of these mutations affected genes involved in either vacuolar organization and biogenesis or cell polarization and cell wall organization. This second functional subset of genes presented rather intense phenotypes and included FKS1 (encoding a ␤-1,3-glucan synthase), GAS1 (a ␤-1.3-glucanosyltransferase), and KRE6 (required for ␤-1,6-glucan biosynthesis). Most interestingly, 20 genes presented in Table II (42% of the total number of genes, denoted in boldface type) were also identified in a recent search carried out in our laboratory that yielded 118 gene deletions producing hypersensitivity to high pH (28). Global Analysis of the Transcriptional Response to Alkaline pH Mediated by Calcineurin-The experiments described above raised the possibility that calcium and calcineurin could represent important components of the transcriptional response to alkaline pH. To evaluate this possibility, we have performed DNA microarray experiments in which wild type cells, calcineurin-deficient mutants (cnb1), and cells lacking the calcineurin-activated Crz1/Tcn1 transcription factor (crz1) were challenged with high pH (8.0) for different times (10, 20, and 45 min). Exposure of wild type cells to pH 8.0 resulted in at least a 2-fold increase in the mRNA level of 266 genes (Table  III). Among these genes, 159 showed a peak at 10 min (early response), 86 peaked 20 min after alkaline challenge (intermediate), and 21 showed a late response (peak after 45 min). They belong to diverse functional categories, according to the "Saccharomyces Genome Data base" classification (29). It is remarkable that 77 genes (29% of the total number of induced genes) can be listed as involved in metabolic pathways, and 37 of those correspond to carbohydrate metabolism or energygenerating pathways. A substantial number of genes related to stress response are also activated in response to high pH. Remarkably, the list contains several genes known to be involved in the response to oxidative stress, such as GRX1, TRX2, PRX1, and others. Exposure to alkali resulted in a decrease in mRNA levels of at least 2-fold for 157 genes. It is remarkable that a substantial number (33 genes) corresponds to ribosomal proteins or to proteins related to ribosome assembly. Additional data on induced and repressed genes can be found in the supplemental Tables I and II, respectively. DNA microarray experiments were conducted in cells lacking calcineurin or the Crz1 transcription factor. The ratio between the responses of cnb1 cells and wild type cells was calculated, and different levels of calcineurin dependence were established. Thus, a cnb1/wild type ratio of Ͻ0.67 was considered the threshold for defining the dependence of calcineurin. Genes showing a ratio 0.67 Ͼ X Ͼ 0.50 were considered "weakly dependent"; those with a ratio 0.50 Ͼ X Ͼ 0.25 were defined as "strongly dependent," and those with a ratio Ͻ0.25 were ranked as "totally dependent." Genes induced more than 2.5fold in wild type cells and considered not induced (ratio high pH/low pH Ͻ1.3) in cnb1 cells were also considered as totally dependent. Twenty-seven genes showed some level of calcineurin dependence at its peak response (Table IV). The induction of 21 (78%) of these genes was totally or strongly dependent on the presence of Cnb1, indicating that calcineurin signaling is a major component of its transcriptional response to alkaline pH. Most calcineurin-dependent genes (74%) corresponded to early responsive genes, and three additional genes showed very similar induction 10 and 20 min after stress.
Similar experiments were carried out by using crz1/tcn1 cells, defective in the transcription factor known to be activated by calcineurin. In this case, 21 genes were found to be sensitive in response to the absence of the transcription factor using the criteria employed to define calcineurin sensitivity. As shown in Table IV, most genes whose induction is affected by the cnb1 mutation are also affected by the absence of Crz1, and in many cases, the intensity of the effect of both mutations is similar. The existence of experimental data regarding the sequences that could be recognized by Crz1 in responsive promoters allowed us to search within both strands of the Ϫ800/Ϫ1 upstream regions of the calcineurin-sensitive genes for putative Crz1-binding sites (a GAGGCT core sequence plus some variants such as GNGGCKCA and GWGGCTG) by using the DNA pattern algorithm (30) available at the "Regulatory Sequence Analysis Tools" website (rsat.ulb.ac.be/rsat/). We found 16 sequences in 11 of the calcineurin-sensitive genes (41%). A similar analysis performed with 400 randomly selected genes in- dicated that only 28% of the genes contained one or more copies of the query sequences. The relationship between the effect of the cnb1 and crz1 mutations in the response to alkaline pH can be deduced from Fig. 4. In this figure, the ratio between the induction level in cnb1 or crz1 mutants and the wild type cells has been calculated and their logarithms plotted. Analysis of the data reveals that in most cases a good correlation is found (r ϭ 0.81). Most interestingly, four genes (YAL061w, PNC1, SPI1, and PRB1) lie clearly outside the main data and appear as dependent on Crz1 but not on calcineurin. Northern blot analysis of one of these genes (PNC1 (see inset in Fig. 4)) and quantitative real time PCR analysis ( Table V) of three of them (PNC1, PRB1, and SPI1) confirmed the DNA microarray data. A computer analysis of the Ϫ800/Ϫ1 upstream region revealed that all four genes contain one (YAL061w and SPI1) or two (PNC1 and PRB1) putative Crz1-binding sequences. PRB1 was also induced at 10 min, and at this time the effect was strongly dependent on calcineurin (Table IV).
Exposure to High pH May Result in Oxidative Stress-The observation that a number of genes functionally related to oxidative stress were induced after alkaline stress or were required to display normal sensitivity to high pH (28) prompted us to investigate whether alkalinization of the media could somehow result in oxidative stress. To this end, cells were loaded with dihydrorhodamine 123 (which is commonly used as a probe for oxidative stress because it can be oxidized inside the cells to the fluorescent derivative rhodamine 123) and exposed to high pH or to the oxidizing agent hydrogen peroxide. As it can be observed in Fig. 5A, microscopic examination revealed that a number of cells exposed for 30 min to alkaline pH presented fluorescence. The percentage of fluorescent cells was around 12% of the total population, and fluorescence was maintained (although somewhat less intense) after 60 min of exposure to alkali (not shown).
We then considered the possibility that if exposure to high pH involved oxidative stress to some extent, then the enhanced expression of certain genes as a result of alkaline stress could be mediated, at least in part, by well known oxidative stressresponse mechanisms. To this end, we tested the response to high pH and hydrogen peroxide of PRX1, which encodes a thioredoxin peroxidase known to be induced upon oxidative stress, by using a lacZ translational fusion. As shown in Fig.  5B, exposure to high pH or to hydrogen peroxide increased the activity of the promoter, and in both cases, the increase was abolished in the absence of the Yap1 transcriptional promoter. Deletion of the SKN7 gene affected only partially the response FIG. 4. Relationship between calcineurin dependence and CRZ1 dependence in the transcriptional response to alkaline stress. The scatter plot represents the log 2 of the degree of dependence of the response to alkaline pH in calcineurin (cnb1/WT) and crz1 mutants (crz1/WT) for genes whose response is significantly affected in at least one of these mutant strains after 10 (circles), 20 (squares), or 45 min (triangles) of stress. The dotted ellipse includes the genes sensitive to the absence of Crz1 but unaffected in the calcineurin mutant. The inset shows the Northern blot analysis of one example (PNC1). Methylene blue staining of the rRNA is included as reference for loading and transfer efficiency. NI, noninduced wild-type cells. of the PRX1 promoter to high pH. Under the same conditions, the alkaline-responsive PHO84 promoter (14) did not respond to oxidative stress, and its pH-dependent activation was not affected by deletion of YAP1 or SKN7.

DISCUSSION
In a recent report (14), we described a genome-wide analysis of the transcriptional response of yeast cells to mild alkaline stress, and we presented evidence that in specific cases (such as ENA1 or PHO89) the signaling pathway involving calcineurin and the calcineurin-activated transcription factor Crz1/Tcn1 was necessary for partial or even full response to this specific stress condition. Activation of calcineurin has been commonly considered as the result of an increase of cytosolic calcium levels (31). However, recent evidence indicates that the functional regulation of the phosphatase can be quite complex. For instance, calcineurin activity can be regulated by members of the RCN (regulators of calcineurin) family, also termed calcipressins (32)(33)(34), represented in the budding yeast by the RCN1 gene (32). Most interestingly, the effect of Rcn1 on calcineurin activity depends on its phosphorylation state, and recent data points to Mck1, a member of the glycogen synthase kinase 3 family, as the major Rcn1 kinase in vivo (35). Furthermore, the subcellular location and function of Crz1/Tcn1 is also regulated by phosphorylation (12,36). Therefore, the calcineurin pathway probably integrates diverse signals at different levels. Consequently, we considered it necessary to test directly in vivo whether exposure to alkali would represent a TABLE V Evaluation by DNA microarray and quantitative real time PCR of the alkaline pH induction of genes PCN1, PRB1, and SPI1 Numbers indicate fold of induction for strain DBY746 (wild type), RSC21 (cnb1), and EDN92 (crz1), as well as the ratio of induction calculated for strains EDN92 and RSC21 (crz1/cnb1). Microarray data correspond to the mean of three different microchip experiments (from two independent RNA preparations), and real time PCR results are the means from these two RNA preparations, determined in duplicate. Note that in this table  microarray data for the wild type strain may differ from Table III 5. Alkaline stress may result in oxidative stress. A, DBY746 wild type cells were loaded with dihydrorhodamine 123 and exposed to pH 8.1 for 30 min or to 2 mM hydrogen peroxide for 90 min, as indicated. Reactive oxygen species oxidizes dihydrorhodamine 123 to the fluorescent derivative rhodamine 123. B, the wild type strain BY4741 and its isogenic yap1 and skn7 derivatives were transformed with the indicated reporter plasmids, and cells were subjected to high pH stress (pH 8.1) or oxidative stress (0.25 mM hydrogen peroxide). Cells were collected, and ␤-galactosidase activity was determined. Data are mean Ϯ S.E. from three independent clones. rise in the level of cytosolic calcium and, if so, to evaluate the kinetics of the process and to investigate the origin of the cation.
To this end, we used a reporter system based in the expression of apoaequorin (5), a method employed by several laboratories to monitor in vivo changes in calcium concentration under a number of circumstances. Our results indicate that alkali stress triggers a very rapid and transient burst of calcium, very similar to what has been observed for hypotonic or saline stress (7)(8)(9), and much faster than that described for sphingosine treatment (6). This increase is only apparent when cells are exposed at pH 7.5 (Fig. 1B) and increases until pH 8.2 is reached. This profile fits well with the dose response for the alkaline pH induction of PHO89, a gene known to be activated by alkaline pH through the calcineurin pathway, whose activation becomes significant around pH 7.4 -7.5 (14). In contrast, activation by alkaline pH of a related gene, PHO84, which requires a lower pH, is independent of calcineurin (14).
An increase in cytosolic calcium can result from entry of the cation from the external medium or by release from internal stores, thus being the vacuole of the major calcium storage site in yeast (38,39). For instance, the initial increase of intracellular calcium upon hypotonic shock has been attributed to release from intracellular stores, whereas the sustained one would have an extracellular origin (7). There is some controversy regarding the origin of cytosolic calcium upon saline stress. Thus, it has been defined as being of vacuolar origin and mediated by the Yvc1 channel (8) or of extracellular origin and imported into the cells through the Mid1-Cch1 channel (9). In our case, the absence of Yvc1 did not result in a significant decrease in the peak of calcium, suggesting that the contribution of the vacuole to this burst is negligible. In contrast, our data are consistent with the notion that the increase in cytosolic calcium observed after alkaline stress is the result of an enhanced influx from the external medium and that the vast majority of this effect requires the presence of both the Cch1 and Mid1 proteins. This is important to note because, although there is plenty of biochemical and genetic evidence that both Cch1 and Mid1 work together as a high affinity calcium influx system (4, 9, 40 -42), recent evidence indicates that in some cases, such as the amiodarone-induced transient cytosolic calcium burst, Mid1 but not Cch1 is required (43). The residual calcium increase observed in mid1 and cch1 cells could be explained by the existence of a recently identified low affinity calcium influx system (4,44,45).
Our data also indicate that the transient increase in intracellular calcium triggered by alkaline stress can result in functional changes at the transcriptional levels. We show (Fig. 3) that several transcriptional elements containing known CDRE do respond to increases in external pH in a fashion that closely agrees with the profiles of the observed calcium burst. For instance, the lack of Yvc1 does not affect the response of any of the calcium-responsive reporters tested. In contrast, addition of EGTA or deletion of either MID1 or CCH1 fully blocks the response from reporter pAMS366, which contains a tandem of four CDRE from the FKS2 gene (10), or largely abolishes the response from a fragment of the ENA1 promoter, which contains a CDRE (pMRK212), as well as from the entire PHO89 promoter. The partial response to high pH observed with reporter pMRK212 could be because of the residual calcium entry observed in such mutants (Fig. 1) and/or to the existence of additional alkaline pH-responsive elements. In this regard, it is worth noting the existence of an Nrg1 binding site at positions Ϫ730/Ϫ724 of the ENA1 promoter, which is included in pMRK212 and largely overlaps the CDRE. NrgI is a transcriptional repressor that has been recently shown to be the target of Rim101, a C 2 H 2 zinc finger protein important for a subset of transcriptional alkaline pH responses (46,47).
Our systematic screening for mutations conferring sensitivity to the calcineurin inhibitor FK506 yielded 48 genes. In a recent report, 35 mutations were defined as FK506-sensitive by growing the cells in semi-solid (agar) medium (48). Eighteen of these mutations were also found in our screening (see Table II). It must be noted that most mutations reported here and missed in Ref. 48 correspond to relatively weak phenotypes, suggesting that our screen was probably more sensitive. On the other hand, most mutations reported in Ref. 48 that are not present in our Table II correspond to strains that show rather slow growth and, consequently, presented unreliable absorbance values even after the largest incubation period (48 h). Therefore, both sets of data can be considered as complementary. In any case, we show that 20 mutations (42%) that confer sensitivity to FK506 (Table II) also resulted in sensitivity to high pH in a recent screen of the same library (28). It is worth noting, given a total number of 4825 common deletions analyzed in both screens and assuming that FK506 sensitivity and high pH sensitivity were independent events, that one should expect only one gene sharing both phenotypes. This large overlap probably reflects the importance of a functional calcineurin pathway in the response to high pH stress.
We have tested the transcriptional response of wild type yeast cells 10, 20, and 45 min after a shift from standard growth conditions (pH 6.2) to pH 8.0, and we found that 266 genes were induced at least 2-fold and at least one of the time points was investigated. We carried out a similar time course experiment some time ago (14) aiming to identify genes responsive to a milder pH stress (pH 7.6). By using the same wild type genetic background, this experiment resulted in the induction of only 150 genes. Comparison of both sets of data reveals that 46 genes are also induced when cells are challenged at pH 8.0. Therefore, different stress intensity is able to elicit a different transcriptional response, indicating that some promoters are more sensitive to alkaline pH conditions than others. This is in agreement with the results obtained in our previous work in which four pH-responsive promoters were analyzed under different alkaline pH conditions (14). The intensity of the pH stress used in the present work resembles quite closely the conditions studied by Causton et al. (49), in which cells were switched from pH 6.0 to 7.9. This resulted in 463 genes with at least a 3-fold increase in expression from 10 to 60 min. In our case, we detect 113 overlapping genes (43%), a figure much higher than that previously observed in wild type cells shifted to pH 7.6 (33 genes, 22%). In any case, it should be noted that the different technological platforms used in both experiments make it difficult to carry out direct comparison of the results.
As can be deduced from Table III, the genes induced by alkaline stress are involved in a large number of cellular functions. It is remarkable, however, that a significant number of these genes, such as UGA2, GRX1, HSP12, TRX2, MCR1, GAD1, GRE2, or PRX1, are related to the response to oxidative stress. A recent screen carried out in our laboratory, using a systematic deletion mutant library, for mutations that result in sensitivity to alkaline pH revealed that mutation of SOD1, SOD2, or CCS1 (LYS7), encoding key enzymes in tolerance to oxidative stress, results in a dramatic alkaline phenotype (28). A possible explanation would be that exposure to an alkaline environment could involve, to some degree, oxidative stress. Here we tested this hypothesis by using two different approaches. We found that a significant percentage of cells exposed to high pH stress produced positive (fluorescent) signal upon preincubation with dihydrorhodamine 123, a widely used probe for detection of reactive oxygen species, although the response was not as intense as that obtained by using 2 mM hydrogen peroxide as the oxidative agent. Furthermore, the alkaline response of the PRX1 promoter (a gene known to respond to oxidative stress) was fully abolished in cells lacking the YAP1 gene, which codes for a bZip transcription factor required for oxidative stress tolerance (for a recent review see Ref. 50). This is not a general effect, because the induction of the alkaline-responsive gene PHO84 was unaltered in yap1 cells. In fact, PRX1 contains a TTAGTGA sequence 705 nucleotides upstream of the start codon, which represent a potential binding site for Yap1. Most interestingly, PRX1 expression seems to be regulated by multiple pathways in response to different stress conditions, and one of these involves the Msn2/ Msn4 activators (51). However, we have observed that the pH-induced response of PRX1 is not altered in msn2 or msn4 cells (not shown). In any case, our data support the notion that part of the phenotypic effects derived from exposure to high pH (including part of the transcriptional response) might be the result of an oxidative stress situation.
Two recent reports (52,53) have addressed the transcriptional response to cell wall damage as a result of specific mutations or chemical or enzymatic treatment. These authors identify 178 genes whose expression is increased at least 1.9fold upon exposure to Congo Red or zymolyase. Comparison of these results with our microarray data reveals a striking overlap of 28 genes, well above the expected overlap (8 genes) if both events were fully unrelated. These common genes include relevant elements of the cell wall, such as GSC2 (component of ␤-1,3-glucan synthase), CRH1 (a cell wall glycosidase), SED1 (a cell surface glycoprotein that contributes to cell wall integrity and stress resistance), or its structural homolog SPI1 (a glycosylphosphatidylinositol-anchored cell wall protein). It is remarkable that all four genes are also induced by mutation of certain cell wall-related genes (52) and that SED1 has been also reported as required for oxidative stress tolerance (54). These results suggest that exposure to alkaline pH might lead (at least up to some extent) to a situation of cell wall damage. This hypothesis is reinforced by the observation that mutation of key elements of the cell wall maintenance of the mitogenactivated protein kinase pathway (BCK1 and SLT2) or several cell wall components, such as SED1, KRE1, GAS1, and others, results in a severe phenotype of alkaline pH sensitivity (21,28). Most interestingly, 12 of the 48 genes (25%) whose deletion results in sensitivity to FK506 (Table II) correspond to genes related to cell polarity and cell wall organization and biogenesis. This may reflect the link between alkaline stress and cell wall damage and the requirement for a functional calcineurin pathway under both stress situations. It is worth noting that it has been reported that exposure to low external pH promotes changes in the organization of the cell wall, although in this case the Slt2/Mpk1 mitogen-activated protein kinase does not seem to be involved (55).
Our data show that the response of 27 genes (10%) that are induced by alkaline stress involves, at least in part, the calcineurin pathway, indicating that this pathway contributes significantly to the alkaline transcriptional response. It is worth noting that, almost without exception, the calcineurinsensitive genes can be classified as early genes (even if they are classed as intermediate, they show already a response after 10 min of stress). This rapid transcriptional response is in agreement with the very rapid kinetics of the calcium burst observed after exposure to alkali. Exposure to external high calcium results in a transcriptional response, recently characterized at the genomic level, which involves increased expression of 153 genes (13). We have compared our data with that of Yoshimoto et al. (13) and found that 22 genes that are presented in Table   III are also induced by high calcium in the medium. Seven of these genes (YBR287W, PHO89, PRB1, PUT1, YNL208W, YOR220W, and SUR1) have also been defined as sensitive to the absence of calcineurin in this work.
Comparison of the transcriptional response to alkaline stress in cnb1 and crz1 mutants indicates that in most cases the absence of CRZ1 results in a decrease in the response quite similar to that observed in a cnb1 strain. This would be in agreement with the observation by Yoshimoto et al. (13) that most of the calcineurin-dependent transcriptional response to high levels of external calcium is lost in a crz1 strain and supports the notion that this transcription factor is the main mediator for the calcineurin-dependent transcriptional response. It should be noted, however, that computer analysis of the upstream regions of calcineurin-and crz1-sensitive genes in Table IV, using consensus sequences reported for Crz1 binding (13,52), gave positive results only in ϳ50% of the cases. This could mean that further work is required to refine the consensus sequence for Crz1 binding. On the other hand, the observation that certain genes are sensitive to the absence of Crz1 but not to that of calcineurin is rather intriguing. A possible explanation would be that this transcription factor could be also regulated in a calcineurin-independent way.