 |
INTRODUCTION |
Extracellular pH is a key environmental signal that influences
growth, physiology, and differentiation (1-9). 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+-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+-ATPase (called Ena1p or
Pmr2p (14)) and the multi-subunit vacuolar membrane
H+-ATPase (15). Ena1p hydrolyzes ATP to pump
Na+ out of the cell, and the Na+ 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+-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 (23-25). 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, 32-34). 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.
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EXPERIMENTAL PROCEDURES |
Strains--
Yeast strains (Table
I) were derived from strain SK1 (35)
through genetic crosses and transformation. The RIM101-HA2,
RIM101-531-HA2-TRP1, and rim101
12
alleles have been described (32); the
rim13
::HIS3 and
rim13
10::TRP1 mutations are
described below.
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 ~35
mM HCl) or pH 8 (with ~75 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 µg/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
A600 of 5- and 3.5-µl 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 A600 <0.2 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
ORF1 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
A600 of 0.375. After 2 h, cells were
collected by filtration and frozen at
80° C; at this time the pH 4 culture had reached an A600 of 0.905 and pH 3.6, and the pH 8 culture had reached an A600 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)+ 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 µg of
selected RNA. 0.04 µg of poly(A)+ RNA from the pH 4 culture was used to generate [
-33P]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 ~18 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 µgs 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
[
-32P]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.
lacZ Fusion Genes--
To generate lacZ fusion genes,
1.5 kilobase pairs of DNA sequence upstream of the desired gene was
PCR-amplified from wild-type genomic DNA using the primers designated
with "L" followed by the gene name in Table II. PCR products were
cloned into plasmid pGEM-T Easy (Promega) to yield the plasmids pAED54
(VMA4 5'), pAED50 (YOL154W 5'), pAED51
(ARN4 5'), pAED49 (ENA1 5'), and pAED53 (TIS11 5'). A PstI-EcoRI fragment from
plasmid pAED54 was cloned into PstI- and
EcoRI-cut plasmid YIp356R (38) to yield plasmid pTL112
(vma4-lacZ), which includes 1.5 kilobase pairs of
VMA4 5' sequences and fuses the VMA4 initiation
codon in-frame to lacZ. Also, a
PstI-EcoRI fragment from plasmid pAED53 was
inserted into PstI- and EcoRI-digested plasmid
YIp356R to yield plasmid pTL111 (tis11-lacZ). Similar
fusions were created by inserting SphI-SphI fragments of plasmids pAED49, 50, and 51 into SphI-cut
plasmid YIp356 to yield plasmids pTL107 (ena1-lacZ), pTL105
(yol154W-lacZ), and pTL106 (arn4-lacZ),
respectively. For transformation into yeast, each plasmid was cut with
StuI to direct integration to the ura3 locus.
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 0+:4
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).
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 palB
620 and palB+2526.
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+) from plasmid pFA6aHis3MX6 (39). Primers
pRIM13-5'F and pRIM13-5'R were used to amplify 500 base pairs of
RIM13 5'-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 pRIM13-5'F
and flanking primer pRIM13-3'R. Transformants were also tested for
smooth colony morphology and failure to process Rim101-HA2p.
 |
RESULTS |
Identification of Alkaline Response Genes--
We used a
GeneFilter 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 10-100-fold 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
pH-dependent 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.

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Fig. 1.
Expression of alkaline response genes at
extremes of pH and salt concentration. Strain TLY759 was grown in
pH 4 YPD, pH 8 YPD, YPD, or YPD + 0.8 M NaCl (lanes
1-4, respectively) for preparation of RNA. Transcript levels were
detected by Northern analysis with probes for the genes listed at the
left side of each panel. The numbers under each
lane are the number of cpms in that lane, normalized to the
number of cpms in the ENO1/2 control lane and expressed as a
percentage of the pH 8 signal (lane 2).
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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 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.

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Fig. 2.
Role of Rim101p in expression of alkaline
response genes. Strains TLY758 (rim101 , lanes
1 and 2) and TLY759 (RIM101, lanes
3 and 4) were grown in pH 4 YPD (lanes 1 and
3) and pH 8 YPD (lanes 2 and 4) for
preparation of RNA. Transcript levels were detected by Northern
analysis with probes for the genes listed at the left side
of each panel. The numbers under each lane are the number of
cpms in that lane, normalized to the number of cpms in the
ENO1/2 control lane and expressed as a percentage of the
TLY759 pH 8 signal (lane 4).
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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 determine 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.

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Fig. 3.
Dependence of Rim101p processing on
RIM13. Strain AMP1689
(rim13 10::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 YPD-grown 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.
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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 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.

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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 µg/ml hygromycin B. Growth was
visualized after 2-6 days at 30° C.
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 |
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
Rim101p-dependent 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+-ATPase gene (14,
18). We found here that VMA4 is also induced by alkaline
conditions. VMA4 encodes a subunit of the vacuolar H+-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, sporulation, growth inhibition by 3-aminotriazole or lack of
calcineurin (53-56). 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 Rim101p-dependent. 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 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.