Alkaline Response Genes of Saccharomyces cerevisiae and Their Relationship to the RIM101 Pathway*

Environmental pH exerts broad control over growth and differentiation, but the molecular responses to external pH changes are poorly understood. Here we have used open reading frame macroarray hybridization to identify alkaline response genes in Saccharomyces cerevisiae . Northern or lacZ fusion assays confirmed the alkaline induction of two ion pump genes ( ENA1 and VMA4 ), several ion limitation genes ( CTR3 , FRE1 , PHO11/12 , and PHO84 ), a siderophore-iron transporter gene ( ARN4/ENB1 ), two transcription factor genes ( NRG2 and TIS11 ), and two predicted membrane protein genes ( YAR068W/YHR214W and YOL154 W). Unlike ENA1 and SHC1 , these new alkaline response genes are not induced by high salinity. The known pH-responsive genes in other fungi depend on the conserved PacC/ Rim101p transcription factor, but induction of several of these new genes relied upon Rim101p-independent pH signaling mechanisms. Rim101p-dependent genes were also dependent on Rim13p, a protease required for Rim101p processing. The Rim101p-dependent gene VMA4 is required for growth in alkaline conditions, il-lustrating how Rim101p may control adaptation. Because Rim101p activates ion pump genes, we tested the role of RIM101 in ion homeostasis and found that RIM101 promotes resistance to elevated cation concentrations. by transformation into meiotic segregants for analysis of diploids. The mutation caused an invasive growth defect in haploids and a sporulation defect in homozygous diploids, as described for rim13-1 (32). Complementation tests: ymr154C::TRP1 strains failed to complement rim13-1 strains for sporulation ability and for expression of a meiotic ime2-lacZ reporter gene. In addition, a plasmid carrying YMR154C complemented the Rim101p processing defect of a rim13-1 mutant. For the linkage test, a cross of a ymr154C::TRP1 strain to a rim13-1 strain yielded exclusively 0 1 :4 2 meiotic segregation of invasive growth ability in 24 tetrads, as expected from tight linkage of YMR154C and RIM13 . Thus all tests indicate that YMR154C is RIM13 . the of RIM13 described CPL1

Extracellular pH is a key environmental signal that influences growth, physiology, and differentiation (1)(2)(3)(4)(5)(6)(7)(8)(9). Specialized responses to pH changes have been characterized in many organisms, but there is limited information concerning the extent and general features of such a response. Our objective is to use Saccharomyces cerevisiae as a model to understand transcriptional responses to a change in external pH.
Yeast cells grow more rapidly in acidic media than in neutral or alkaline media. They neutralize the cytoplasm through activity of Pma1p, the plasma membrane H ϩ -ATPase, which hydrolyzes ATP to pump protons out of the cell (10). The proton gradient is used for transport of amino acids, nucleotide bases, phosphate, and many other molecules in symport reactions (10). It also provides an electrochemical gradient that favors uptake of cations (11,12). Thus, the plasma membrane proton gradient is required for accumulation of solutes from the medium, and PMA1 is an essential gene (10,13).
In neutral and alkaline conditions, two other ion pumps are vital for growth: the plasma membrane Na ϩ -ATPase (called Ena1p or Pmr2p (14)) and the multi-subunit vacuolar membrane H ϩ -ATPase (15). Ena1p hydrolyzes ATP to pump Na ϩ out of the cell, and the Na ϩ gradient permits uptake of other cations. The vacuolar ATPase is required for vacuolar acidification, which cannot occur through endocytosis in alkaline media (16,17). Null mutants lacking these pumps are hypersensitive to alkaline growth conditions and elevated external cation concentrations but are capable of growth in acidic media.
External pH changes cause a transcriptional response in S. cerevisiae. There are three known alkaline-inducible genes: the Na ϩ -ATPase structural gene ENA1 (14,18), SHC1, and SCY1 (19). ENA1 and SHC1 are also induced in high salt medium; results for SCY1 have not been reported. Thus, it is possible that external pH is sensed by factors that also sense external salt concentration, thereby triggering a common signal transduction cascade and activating a common set of genes. Alternatively, it is possible that a unique regulatory pathway senses and responds to external pH changes.
Transcriptional responses to external pH have been characterized in several other fungi. For example, alkaline pH induces transcription of IPNA and other penicillin biosynthetic genes in Aspergillus nidulans and Penicillium chrysogenum (20,21), the protease gene XPR2 in Yarrowia lipolytica (22), and cell wall protein genes in Candida albicans (23)(24)(25). These responses depend upon a conserved signal transduction pathway (20, 22, 26 -29) that is best understood from work on the A. nidulans transcription factor PacC. PacC is required for responses to alkaline pH. It accumulates in an apparently inactive full-length form under acidic growth conditions and is activated by C-terminal proteolytic cleavage under alkaline growth conditions (30). Several alkaline response regulatory genes, palA, palB, palC, palF, palH, and palI, are required for proteolytic cleavage of PacC (30,31). Their sole known function is to promote PacC cleavage, because loss-of-function pal mutations cause similar phenotypes to loss-of-function pacC mutations and because expression of C-terminally truncated PacC derivatives suppresses pal mutant defects (30,31).
S. cerevisiae has a PacC-like pathway. Rim101p is a homolog of PacC, and it is activated by C-terminal proteolytic cleavage (32) that depends upon pal gene homologs (26,(32)(33)(34). However, unlike PacC cleavage, which is completely dependent upon external alkaline pH, Rim101p cleavage occurs under both acidic and alkaline growth conditions (32). Therefore, it is uncertain whether the S. cerevisiae RIM101 pathway also governs pH-responsive gene expression.
Here we have used a gene expression survey to identify pH-responsive S. cerevisiae genes. We have used these genes to determine the relationship between gene induction by alkaline pH and by salt. In addition, we have asked whether the S. cerevisiae RIM101 pathway is required for alkaline gene expression, and how cleavage of Rim101p impinges upon regulation of alkaline genes. Our results provide insight into both the functional conservation of the PacC/RIM101 pathway as a pH response pathway and upon the nature of external pH as a global transcriptional regulatory signal.
Media, Growth Conditions, and ␤-Galactosidase Assays-Yeast growth media (YPD and SC) were of standard composition (36). For growth in liquid YPD medium at a specified pH, YPD containing 0.1 M HEPES was titrated to pH 4 (with ϳ35 mM HCl) or pH 8 (with ϳ75 mM NaOH) and filter sterilized. YPD plates at pH 3 or pH 9 contained 150 mM HEPES and were titrated before autoclaving. Other additions to YPD included 0.4 or 0.8 M NaCl, 1.2 M sorbitol, 50 g/ml hygromycin B, and 20 mM LiCl. For plates, these additions were always made after autoclaving. To assess growth on various media, YPD overnight cultures were adjusted to an A 600 of 5-and 3.5-l samples of serial 5-fold dilutions in YPD were spotted on test plates. All cultures and plates were incubated at 30°C.
For ␤-galactosidase assays, strains were grown to saturation in pH 4 YPD and then inoculated at an A 600 Ͻ0.2 in pH 4 YPD or pH 8 YPD and grown for at least two doublings. ␤-Galactosidase assays were carried out on permeabilized cells as described (36), except that cells were suspended in 0.15 ml of Z buffer, permeabilized, and assayed with 0.7 ml of 1 mg/ml o-nitrophenyl-␤-D-galactopyranoside in Z buffer.
GeneFilter Analysis-Yeast ORF 1 GeneFilters (Research Genetics, GF100I and II) were probed with labeled cDNA from strain TLY759 as follows. A late exponential pH 4 YPD culture was used to inoculate 250 ml cultures of pH 4 YPD and pH 8 YPD at an A 600 of 0.375. After 2 h, cells were collected by filtration and frozen at Ϫ80°C; at this time the pH 4 culture had reached an A 600 of 0.905 and pH 3.6, and the pH 8 culture had reached an A 600 of 0.625 and pH 7.5. Total RNA was produced as follows. Thawed cell pellets were resuspended in 20 ml AE (50 mM sodium acetate, pH 5.2, 10 mM EDTA) and brought to 1% SDS. After vortexing and incubation at 65°C in the presence of 25 ml of acid phenol, the aqueous phase was separated, and total RNA was precipitated. For each sample, poly(A) ϩ RNA was prepared as follows. 1 mg of total RNA was batch bound to 40 mg of oligo(dT) cellulose (Roche Molecular Biochemicals) in NETS buffer (0.6 M NaCl, 10 mM EDTA, 10 mM Tris-HCl, pH 8.0, 0.2% SDS), transferred to a Poly-Prep column (Bio-Rad), washed four times with NETS, eluted in ETS (10 mM EDTA, 10 mM Tris-HCl pH 8.0, 0.2% SDS) at 65°C, and precipitated, yielding between 15 and 30 g of selected RNA. 0.04 g of poly(A) ϩ RNA from the pH 4 culture was used to generate [␣-33 P]dCTP-labeled cDNAs through reverse transcription, following the Research Genetics protocol. After purification on a BioSpin6 column (Amersham Pharmacia Biotech) and subsequent boiling, the labeled cDNAs were added to GeneFilters which had been prehybridized at 42°C for 3 h in 10 ml of MicroHyb (Research Genetics, HYB250-GF). Filters were hybridized at 42°C for ϳ18 h in roller bottles, washed as recommended, and exposed to a phosphor screen for 26 h. The filters were scanned at a resolution of 50 microns on a Storm2000 Phosphorimager, and the data were imported into Pathways software (Research Genetics) for analysis. Filters were then stripped by washing twice with boiling 0.5% SDS and reprobed with cDNAs prepared from the pH 8 culture. Pathways software was used to normalize each ORF signal to all data points for each probe, and then a ratio of the pH 8 to pH 4 signal was obtained for each ORF. We identified 71 ORFs that had a pH 8 to pH 4 ratio of 2.1-fold or greater. Because of the order of hybridization and incomplete stripping of the pH 4 signal, our experiment may not have reliably identified genes that are up-regulated at pH 4 compared with pH 8.
Northern Blots-For Northern analysis, 15-20 gs of total RNA per lane was run on dentaturing formaldehyde gels, transferred to a nylon membrane and hybridized as described (37). To generate Northern blot probes, genomic DNA was PCR-amplified using the primers designated with "N" followed by the gene name in Table II. The ENA1 and ENO1 PCR fragments were gel purified, labeled with [␣-32 P]dCTP using a High Prime kit (Roche), and QIAquick PCR purified (Qiagen). All other PCR-amplified fragments were cloned into plasmid pGEM-T Easy (Promega), and plasmid inserts were obtained by NotI digestion, gel purified, and labeled as described above.
RIM13 Cloning and Analysis-We identified RIM13(YMR154C) initially through a S. cerevisiae genomic BLAST search for a PalB homolog (60). Our conclusion that YMR154C is RIM13 is based upon tests of phenotype, complementation, and linkage. For phenotypic tests, a ymr154C::TRP1 insertion-deletion allele (rim13⌬10::TRP1) that removes the entire ORF was introduced into haploid strain AMP1604 by transformation and was moved into meiotic segregants for analysis of diploids. The mutation caused an invasive growth defect in haploids and a sporulation defect in homozygous diploids, as described for rim13-1 (32). Complementation tests: ymr154C::TRP1 strains failed to complement rim13-1 strains for sporulation ability and for expression of a meiotic ime2-lacZ reporter gene. In addition, a plasmid carrying YMR154C complemented the Rim101p processing defect of a rim13-1 mutant. For the linkage test, a cross of a ymr154C::TRP1 strain to a rim13-1 strain yielded exclusively 0ϩ:4Ϫ meiotic segregation of invasive growth ability in 24 tetrads, as expected from tight linkage of YMR154C and RIM13. Thus all tests indicate that YMR154C is RIM13. During the course of our studies, RIM13 was independently described as CPL1 (34).
a All strains were of genotype ura3 trp1 leu2 lys2 hoϻLYS2 rme1⌬5ϻLEU2, except as indicated, and carried additional markers listed in the table.
The rim13⌬10::TRP1 mutation was constructed as follows. Primers palB-ATG and palB-TAA were used to amplify a TRP1 cassette, and the PCR product was transformed into haploid strain AMP1604. Homologous integration was verified by PCR with flanking primers palBϪ620 and palBϩ2526.
The rim13⌬::HIS3 mutation was constructed as follows. Primers Pada-RIM13F and Pada-RIM13R were used to amplify the HIS3-MX6 cassette (Schizosaccharomyces pombe his5 ϩ ) from plasmid pFA6aHis3MX6 (39). Primers pRIM13-5ЈF and pRIM13-5ЈR were used to amplify 500 base pairs of RIM13 5Ј-flanking sequences, and two-way PCRs were performed to fuse this sequence to the amplified HIS3-MX6 cassette. The fused PCR product was transformed into strains WXY169 and WXY170, and homologous integration was verified by PCRs with primer pRIM13-5ЈF and flanking primer pRIM13-3ЈR. Transformants were also tested for smooth colony morphology and failure to process Rim101-HA2p.

RESULTS
Identification of Alkaline Response Genes-We used a Gene-Filter macroarray to identify 71 S. cerevisiae genes that appeared to be expressed at higher levels at pH 8 than at pH 4 (Table III). These genes included ENA1, which is induced at pH 8 (14, 18), but not SHC1 or SCY1, which are induced at higher pH (19). Northern blot hybridization confirmed the expression pattern of genes that represent several major classes in Table  III. Of 9 Northern probes, seven detected transcripts of appropriate size, and all of these transcripts accumulated at 10 -100fold higher levels at pH 8 than at pH 4 ( Fig. 1). These genes included phosphate metabolism genes PHO84 and PHO11/ PHO12 (a duplicated gene pair), metal transport genes CTR3, FRE1, and ARN4(ENB1), putative membrane protein genes YAR068W/YHR214W (a duplicated gene pair), and transcription factor gene NRG2 (Fig. 1). Levels of a control transcript from ENO1/ENO2 (a duplicated gene pair) were unaffected by external pH. Two additional transcripts were not reliably detected on Northern blots but gave alkaline-dependent GeneFilter signals: VMA4, a vacuolar ATPase subunit gene, and YOL154W, a putative cell surface protein gene. To verify pHdependent expression of these genes and of TIS11, a transcription factor gene, we created strains carrying the respective lacZ fusion genes and assayed ␤-galactosidase activity after growth at pH 4 and pH 8 (Table IV, RIM101 strain). Control ena1-lacZ and arn4-lacZ fusions were expressed at 100-fold higher levels at pH 8 than at pH 4, as expected from Northern analysis, and the lacZ fusion vector with no insert was not expressed at either pH. The vma4-lacZ, yol154W-lacZ, and tis11-lacZ fusions were expressed at 100-fold higher levels at pH 8 than at pH 4. These results indicate that expression of several genes increases substantially in response to a shift from acidic to alkaline pH. We refer to these genes below as alkaline response genes.
To determine whether alkaline pH and high salt induce the same genes, we examined expression of alkaline response genes after treatment of cells with 0.8 M NaCl (Fig. 1). ENA1 was induced by NaCl, as expected (40,41), but the other alkaline response genes were not. In addition, we observed no induction of vma4-lacZ, yol154W-lacZ, or tis11-lacZ by 0.8 M NaCl, whereas ena1-lacZ was strongly induced (data not shown). These results indicate that gene expression responses

Control of Yeast Alkaline Response Genes
to alkaline pH and high salt are distinct. Requirement for Rim101p in Alkaline Response Gene Expression-To determine whether Rim101p is required for alkaline response gene expression in S. cerevisiae, we compared alkaline response transcript levels in RIM101 and rim101⌬ strains (Fig. 2). The strains were grown at pH 4 and then shifted to pH 4 or pH 8 for 2 h prior to RNA preparation. Under these conditions, growth of the two strains was comparable. Northern analysis revealed that there are three classes of genes. One class was not induced at pH 8 in the rim101⌬ strain; this class included ARN4 and YAR068W/YHR214W. The second class was partially induced at pH 8 in the rim101⌬ strain; this class included ENA1 and NRG2. The third class was induced at pH 8 in the rim101⌬ strain to levels as great or greater than in the RIM101 strain; this class included PHO11/PHO12, PHO84, FRE1, and CTR3. Control transcripts UBC4 and ENO1/2 were unaffected by the rim101⌬ mutation. These results indicate that Rim101p is required for expression of some, but not all, alkaline response genes.
We assayed expression of lacZ fusion genes in RIM101 and rim101⌬ strains to extend this analysis (Table IV). At pH 8, the ena1-lacZ fusion was expressed at slightly lower levels in the rim101⌬ strain than in the RIM101 strain, and the arn4-lacZ fusion was expressed at 30-fold lower levels in the rim101⌬ strain than in the RIM101 strain. These results were consistent with Northern analysis. We found that vma4-lacZ and yol154W-lacZ were expressed at 10 -100-fold lower levels at pH 8 in the rim101⌬ strain, compared with the RIM101 strain. Expression of tis11-lacZ was similar in rim101⌬ and RIM101 strains. Taken together, Northern analysis and lacZ fusion assays indicate that the set of alkaline response genes that are largely dependent upon Rim101p includes ARN4, VMA4, YAR068W/YHR214W, and YOL154W.
Role of Rim101p Processing in Alkaline Signal Transduction-Rim101p, like PacC, is activated by proteolytic removal of a C-terminal segment. We used a rim13⌬ mutant to deter-   a Each lacZ fusion gene was integrated at the URA3 locus of strains TLY759 (RIM101) and TLY758 (rim101⌬). Strains were grown to saturation in YPD pH 4 and then grown for at least two doublings in YPD pH 4 or YPD pH 8, as indicated. ␤-Galactosidase activities (Miller units) are the mean of three or four independent transformants, and standard deviations were within 30% of each mean. mine whether C-terminal proteolysis is required for alkaline response gene expression. The rim13⌬ null mutation, like rim13-1 (32), blocks Rim101p processing, as determined with epitope-tagged Rim101-HA2p expressed from the RIM101 genomic locus: a haploid RIM13 strain yielded mainly the processed 90-kDa Rim101-HA2p form (Fig. 3, lane 1); a rim13⌬ mutant yielded the unprocessed 98-kDa Rim101-HA2p form (Fig. 3, lane 2); and failure to process Rim101-HA2p cosegregated with rim13⌬ in two meiotic tetrads (Fig. 3, lanes 3-10). (Our results differ from those of Futai et al. (34), who detected no Rim101p in a rim13 mutant, perhaps because they used a GAL-RIM101 overexpression plasmid.) We then asked whether the rim13⌬ mutation blocks expression of alkaline response gene-lacZ fusions. The rim13⌬ mutation had little effect on ena1-lacZ expression but caused a severe defect in arn4-lacZ, vma4-lacZ, and yol154W-lacZ expression (Table V). These findings show that Rim13p is required for Rim101p processing and for expression of the Rim101p-dependent alkaline response genes.
If Rim13p promotes gene expression through processing of Rim101p, then the rim13⌬ gene expression defect should be suppressed by expression of Rim101-531p, a C-terminally truncated Rim101p derivative (32). We found that the RIM101-531 allele restored expression of arn4-lacZ, vma4-lacZ, and yol154W-lacZ at pH 8 in a rim13⌬ mutant (Table V). Expression of the fusion genes was slightly elevated in both RIM13 RIM101-531 and rim13⌬ RIM101-531 strains at pH 8, compared with the RIM13 RIM101 strain. These results support the idea that Rim13p promotes alkaline gene expression through processing of Rim101p. In addition, Rim101-531p may be hyperactive compared with wild type processed Rim101p because it causes higher levels of alkaline response gene expression than Rim101p.
To determine whether bypass of the Rim101p processing pathway can relieve pH-dependent regulation of alkaline response genes, we compared expression of this panel of lacZ fusions in rim13⌬ RIM101-531 and RIM13 RIM101 strains at pH 4 ( Table V). The yol154W-lacZ fusion was expressed at 10-fold higher levels in the RIM101-531 strain than in the RIM101 strain. Also, ena1-lacZ was expressed at slightly elevated levels in RIM101-531 strains. However, vma4-lacZ and arn4-lacZ were expressed at similar low levels at pH 4, regardless of the RIM101 allele. These results argue that Rim101p proteolysis is necessary for normal alkaline response gene activation. However, processed Rim101p cannot relieve pH-dependent regulation of many target genes.
Requirement for Rim101p in Ion Homeostasis-Our observations above indicate that one role of Rim101p is to promote ion pump expression. Reduced ion pump activity may contribute to the growth defect of rim101⌬ mutants in alkaline media but also may cause additional rim101⌬ growth defects that arise from ion homeostasis defects. Therefore, we compared RIM101 and rim101⌬ strains by cell dilution assays for cation sensitivity (Fig. 4). The rim101⌬ strain grew poorly in the presence of NaCl and LiCl, as do ena1 mutants (10,42). This growth defect did not arise from general osmotic sensitivity because RIM101 and rim101⌬ strains grew equally well on medium  TRP1 RIM101-HA2, lane 1) was crossed to strain AMP1800 (RIM13 RIM101-HA2, lane 2) to yield meiotic progeny of two tetrads (lanes 3-6 and 7-10, respectively). Epitope-tagged Rim101-HA2p was visualized on an anti-HA immunoblot of extracts of YPDgrown cells. Migration of full-length Rim101-HA2p at 98 kDa and of processed Rim101-HA2p at 90 kDa (32) was confirmed on this gel with control RIM13 RIM101-HA2 and rim13-1 RIM101-HA2 extracts. a Each lacZ fusion gene was integrated at the URA3 locus of strains TLY814 (RIM13 RIM101-HA2), TLY816 (rim13⌬ϻHIS3 RIM101-HA2), TLY818 (RIM13 RIM101-531-HA2), and TLY820 (rim13⌬ϻHIS3 RIM101-531-HA2). Strains were grown to saturation in YPD pH 4 and then grown for at least two doublings in YPD pH 4 or YPD pH 8, as indicated. ␤-Galactosidase activities (Miller units) are the mean of three or four independent transformants, and standard deviations were within 30% of each mean. containing 1.2 M sorbitol (Fig. 4), a medium that blocks growth of an isogenic osmoregulation-defective hog1⌬ mutant (data not shown). The rim101⌬ mutant also grew poorly on media containing the cationic aminoglycoside hygromycin B. These findings suggest that Rim101p has a role in general ion homeostasis.

DISCUSSION
Environmental pH is a factor that influences growth, differentiation, and viability of all organisms. The gene expression analysis here provides a framework for understanding the molecular basis for pH-dependent biological processes in S. cerevisiae and in other eukaryotes. Fungal Rim101p/PacC homologs are known regulators of nonessential pH response genes; we extend this understanding with the identification of a Rim101pdependent gene that is essential for alkaline adaptation, with identification of Rim101p-independent alkaline response genes, and with the finding that Rim101p is required for general ion homeostasis.
One effect of external alkalinity is the disruption of membrane proton gradients, which normally supply energy for nutrient and ion transport (10). Prior studies indicated that yeast responds by expressing ENA1, a plasma membrane Na ϩ -ATPase gene (14,18). We found here that VMA4 is also induced by alkaline conditions. VMA4 encodes a subunit of the vacuolar H ϩ -ATPase, which is required for growth in alkaline media (43,44). VMA4 is a logical target of regulation, because Vma4p levels are thought to govern vacuolar ATPase assembly (15,45). Our results argue that cells adapt to alkaline conditions through increases in both vacuolar and plasma membrane ion pump activity. In addition, we found that alkaline induction of VMA4 depends upon RIM101. Thus, alkaline conditions may induce an adaptive increase in vacuolar ATPase activity through Rim101p-dependent VMA4 induction.
A secondary effect of external alkalinity may be nutrient and ion limitation that arises from disruption of the plasma membrane proton gradient. Consistent with this idea, several alkaline response genes are transporters, and others are targets of known solute-specific regulatory pathways: CTR3 and FRE1 are targets of the copper-responsive transcription factor Mac1p (46,47); YOR154W is a target of zinc-responsive transcription factor Zap1p (48); PHO11/PHO12 and PHO84 are targets of the phosphate-responsive transcription factor Pho4p (49,50). The fact that Pho11p and Pho12p are acid phosphatases (51) further suggests that their induction by alkaline pH is not a specific response to alkaline conditions. However, alkaline response genes do not correspond entirely to other sets of starvation-or stress-induced genes. ENA1, HXT2, and SHC1 are induced by high salt (19,40,41,52), but other alkaline response genes are not. In addition, alkaline response genes are not uniformly induced by stationary phase, heat shock, sporu-lation, growth inhibition by 3-aminotriazole or lack of calcineurin (53)(54)(55)(56). Thus, the response to alkaline pH may be in part a stress and starvation response, but it has unique attributes that distinguish it from other growth limitation conditions.
Because alkaline conditions inhibit yeast growth, some alkaline response genes may facilitate competition with bacteria. In S. cerevisiae, the alkaline-induced transporter Arn4p imports a bacterial siderophore-iron complex (57), thus capitalizing on a bacterial iron acquisition mechanism. In A. nidulans and P. chrysogenum, penicillin synthesis is stimulated at alkaline pH (20,21). ARN4 and the penicillin biosynthesis genes depend upon Rim101p or PacC for expression. Therefore, the functions of some Rim101p/PacC-dependent alkaline response genes may only be apparent in competitive growth or survival tests.
Despite limited information on alkaline response genes in other eukaryotes, there is an example of a conserved alkaline response gene. YOL154W is the closest S. cerevisiae homolog of the alkaline inducible C. albicans gene PRA1, which encodes a cell surface protein with similarity to Zn metalloproteases (25). YOL154W is known to be a zinc limitation gene (48), as are its homologs in Aspergillus species (58). However, expression of YOL154W/PRA1 in alkaline media cannot solely be due to impaired zinc uptake, because other zinc limitation genes (48) are not alkaline-induced. We found that expression of S. cerevisiae YOL154W, like C. albicans PRA1 (26), depends upon Rim101p. These observations suggest that the relationships among Rim101p, zinc metabolism, and YOL154W/PRA1 may be conserved in diverse fungi. In S. cerevisiae Rim101p interacts with the zinc response transcription factor Zap1p (59). We suggest that Rim101p-Zap1p interaction may be the basis for conserved pH-and zinc-responsive control of YOL154W/PRA1. We anticipate that many other S. cerevisiae alkaline response genes will have alkaline-regulated homologs in other eukaryotes and that other Rim101p/PacC target genes will be conserved.
There are several classes of alkaline response genes, defined by their relationships to the RIM101 pathway. One class of genes is independent of the RIM101 pathway and includes PHO11/12, PHO84, CTR3, FRE1, and TIS11. In contrast to the previously known alkaline response genes in C. albicans, Y. lypolytica, and Aspergillus species, which are all dependent on Rim101p/PacC, this new class may account for Rim101p-independent biological responses to pH (26). A second class is only weakly dependent on Rim101p and includes ENA1 and NRG2. A third class is dependent on Rim101p but is not expressed in acidic medium in the presence of truncated Rim101-531p and includes ARN4 and VMA4. For this class, Rim101p signaling is clearly necessary but not sufficient to promote alkaline expression. A fourth class is also dependent on Rim101p but is expressed in acidic medium in the presence of Rim101-531p and includes YOL154W. The response of this class parallels the PacC/Rim101p target genes of other fungi in that Rim101p signaling is both necessary and sufficient for expression. This finding provides strong genetic evidence that the S. cerevisiae RIM101 pathway is a pH-sensing pathway. We have yet to determine the molecular basis for control of alkaline response genes by Rim101p, but the presence of a PacC recognition sequence (GCCARG) is not sufficient to predict alkaline inducibility or RIM101 dependence. For example, the UBC4 gene has three GCCARG sites within 500 base pairs upstream of its start, but its expression is neither alkaline-induced nor Rim101pdependent. We expect that detailed analyses of these different classes of S. cerevisiae alkaline response genes will reveal novel mechanisms of pH-dependent gene regulation.
The fact that ion pump genes are among Rim101p targets led FIG. 4. Ion and pH sensitivity of RIM101 and rim101⌬ strains. Serial dilutions of strains TLY759 (RIM101) and TLY758 (rim101⌬) were spotted on a control YPD plate and on YPD with these additions or modifications: titrated to pH 3, containing 1.2 M sorbitol, titrated to pH 9, containing 0.4 M NaCl, containing 20 mM LiCl, and containing 50 g/ml hygromycin B. Growth was visualized after 2-6 days at 30°C. us to infer that Rim101p has a role in general ion homeostasis. This idea is supported by the sensitivity of rim101 mutants to elevated cation concentrations. Thus we can now ascribe functional importance to the activating Rim101p cleavage that occurs even in acidic media in S. cerevisiae (32). In addition, these observations may explain why C. albicans rim101/ rim101 strains grow in the liver, but not in the kidney, in infected mice (8): replication in the kidney probably requires greater salt tolerance. Whether Rim101p/PacC pathways in other fungi have similar roles in ion homeostasis remains to be determined. However, the discovery of a new role for S. cerevisiae Rim101p illustrates one way that comprehensive gene expression surveys can provide new functional information.