From the Department of Biological Sciences and
§ Department of Biochemistry, University of Iowa,
Iowa City, Iowa 52242
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ABSTRACT |
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The HOG mitogen-activated protein kinase pathway
mediates the osmotic stress response in Saccharomyces
cerevisiae, activating genes like GPD1 (glycerol
phosphate dehydrogenase), required for survival under hyperosmotic
conditions. Activity of this pathway is regulated by Sln1p, a homolog
of the "two-component" histidine kinase family of signal
transduction molecules prominent in bacteria. Sln1p also regulates the
activity of a Hog1p-independent pathway whose transcriptional output
can be monitored using an Mcm1p-dependent lacZ
reporter gene. The relationship between the two Sln1p branches is
unclear, however, the requirement for unphosphorylated pathway intermediates in Hog1p pathway activation and for phosphorylated intermediates in the activation of the Mcm1p reporter suggests that the
two Sln1p branches are reciprocally regulated. To further investigate
the signals and molecules involved in modulating Sln1p activity, we
have screened for new mutations that elevate the activity of the
Mcm1p-dependent lacZ reporter gene. We
find that loss of function mutations in FPS1, a gene
encoding the major glycerol transporter in yeast activates the reporter
in a SLN1-dependent fashion. We propose that
elevated intracellular glycerol levels in the fps1
mutant shift Sln1p to the phosphorylated state and trigger the
Sln1-dependent activity of the Mcm1 reporter. These observations are consistent with a model in which Sln1p
autophosphorylation is triggered by a hypo-osmotic stimulus and
indicate that the Sln1p osmosensor is tied generally to osmotic
balance, and may not specifically sense an external osmolyte.
Yeast cells maintain elaborate mechanisms for adaptation and
growth under a variety of adverse environmental conditions. For example, an increase in the osmolarity of the medium elicits an osmotic
stress response that includes transient cell cycle arrest, restructuring of the actin cytoskeleton, and an elevation in the intracellular glycerol concentration (1-4). Glycerol is produced intracellularly to partially offset the rise in external osmolarity thus preventing water loss that would lead to dehydration and eventual
death (5-9). Although the exact mechanism by which cells sense a
change in osmotic pressure is not known, components of the
intracellular signaling pathway that responds to osmotic stress response have begun to be elucidated (10-12). In Saccharomyces cerevisiae, the response to osmotic stress is mediated, in part, by the Hog1p MAP1
kinase pathway (10). Two membrane-associated osmosensors, Sln1p and
Sho1p, regulate the activity of the Hog1p pathway, although the
mechanism by which they do so differs (11). Sho1p associates with and
activates the Ste11p MEK kinase which phosphorylates the Pbs2p MEK,
which in turn activates the MAP kinase Hog1p (10, 12). Sln1p, on the
other hand, is a yeast homolog of "two-component" regulators
prominent in bacterial signal transduction (13). The two-component
designation refers to the two modules, a sensor/transmitter (histidine
kinase) and a response regulator (receiver plus output) module that
comprise the basic signaling unit. Two-component signal transduction
involves autophosphorylation of the transmitter module on a histidine
residue followed by phosphotransfer to an aspartate residue of the
receiver module (14). Sln1p controls the activity of the Hog1p MAP
kinase pathway via a phosphorelay system involving Ypd1p (a
phosphorelay intermediate) and Ssk1p (a response regulator with an
output domain). The dephosphorylated form of the Ssk1p response
regulator activates the MEK kinases, Ssk2p and Ssk22p which then
activate the Pbs2p MEK and Hog1p MAP kinase (11, 15, 16). Hog1p is
thought to phosphorylate transcription factors responsible for turning
on a family of osmoresponse genes that includes glycerol phosphate
dehydrogenase (GPD1) which is involved in the synthesis of
glycerol (8).
Recently, we have shown that Sln1p also regulates the activity of a
Hog1p-independent pathway whose transcriptional output can be monitored
using a lacZ reporter gene we call P-lacZ, which consists of a CYC1-lacZ fusion in which the UAS
of CYC1 has been replaced by a palindromic-binding site (P
site) for the Mcm1p transcription factor (17, 18). Potential and known
target genes of Mcm1p encode proteins involved in cell type
determination, cell wall and membrane integrity, cellular metabolism,
and cell cycle progression (19). Null mutations in SLN1
decrease P-lacZ reporter gene expression whereas activator
mutations in the gene (sln1*) increase P-lacZ
reporter gene expression. As is the case for the HOG pathway, Sln1p
control of the P-lacZ reporter appears to require the kinase
and phosphotransfer functions of Sln1p (17). The sln1*
mutations that result in increased P-lacZ reporter gene activity map to positions likely to affect phosphorylation. More compelling evidence for the role of phosphorylation and phosphotransfer in sln1* activation of the P-lacZ reporter comes
from the osmosensitive phenotype of these mutants which is a reflection
of reduced HOG pathway activity (17). The demonstration of a second
pathway controlled by Sln1p led us to propose that the Sln1p osmosensor sends two signals in response to changes in osmolarity. Activation of
Hog1p accounts for the increase in glycerol, whereas changes in Mcm1p
activity might account for changes in parameters of cell growth
(e.g. cell cycle progression, cell wall composition, and expansion) that are postulated to occur in response to osmotic stress.
In their natural environment, yeast are exposed not only to increased
osmolarity but also to decreased osmolarity. Cellular adjustment to
decreased osmolarity involves a protein kinase C-like protein (PKC)
encoded by the PKC1 gene (20) which regulates a MAP kinase
cascade consisting of the BCK1 MEK kinase (21, 22),
MKK1 and MKK2 MEKs (23), and the MAP kinase,
MPK1 (24, 25). In contrast to HOG pathway mutants which fail
to grow under high osmolarity conditions, mutants lacking PKC pathway
function exhibit cell lysis at low osmolarity (26). The cell lysis
phenotype and corresponding reduction in Although there is now a considerable body of information about the
pathways that lead to changes in gene expression and adaptation to
osmotic stress environments, the mechanism by which osmotic stress is
sensed remains obscure. To further investigate the signals and
molecules involved in modulating Sln1 activity, we have undertaken a
genetic screen to find new mutations that mimic the effect of sln1* mutations in elevating the activity of the
P-lacZ reporter gene. We report here the isolation of loss
of function mutations in FPS1, a gene encoding a major
glycerol transporter of the cell. Mutations in FPS1 are
known to reduce glycerol efflux resulting in increased intracellular
glycerol levels, thus disturbing normal osmotic balance (29). The
identification of Fps1p as a regulator of a Sln1p pathway activity
suggests that the Sln1 protein may generally detect and respond to
deviations from the normal osmotic gradient and may not specifically
bind or sense external osmolytes.
Yeast Strains and Media--
All yeast strains used in this work
(Table I) are from our strain collection
or were constructed for this study. The fps1 deletion was
introduced by one-step transformation (30) using the 4.9-kb
BamHI-HindIII fragment from pWT1040. The
replacement was confirmed by Southern hybridization analysis of
BamHI-HindIII digested genomic DNA using the
2.5-kb BamHI-HindIII fragment from the
FPS1 gene as a probe. Conversion of the LEU2 gene
disruption marker to kanR in various strains was
accomplished as described previously (31). Kan replacements were
confirmed on the basis of the newly gained Leu
Conversion of the SLN1+ allele to
sln1-22 was accomplished by two-step replacement (32).
Plasmid pWT980 which carries the sln1-22 allele was
linearized within the SLN1 sequences using NruI.
Ura+ transformants in which the plasmid was integrated at
the SLN1 locus were then subject to selection on
5-fluoroorotic acid to isolate recombinants containing only the
sln1-22 allele. The presence of the sln1-22
allele was confirmed by sequence analysis (primer: 5'-AGAATGTTGAACTTGGAGGGC-3') of a 0.5-kb polymerase chain reaction product generated from yeast genomic DNA template (primers: SLN1 3200 (5'-GCCACATCAAGTTGAGAATTCCCCACAGTCAAAGACG-3') and SLN1 4119 (5'-
CGCGCAAGCTTTTGATTTCTC-3')).
Integration of the P-lacZ reporter was accomplished by
linearizing the P-lacZ integrating vector, pGY88 within
URA3 using ApaI, and transforming the strain to
Ura+. Successful integration at the URA3 locus
and the number of plasmids integrated in each strain was examined by
Southern analysis of PstI digested DNA. A 1-kb
lacZ polymerase chain reaction product (primers: lacZ 280F
(5'-CGGTTACGATGCGCCCATCTACACC-3') and lacZ 1291R
(5'-GGATCATCGGTCAGACGATTCATTGG -3')) was used for probe synthesis. The presence of multiple copies of the reporter was revealed
by hybridization of the probe to a novel 7-kb PstI fragment.
The media were prepared as described by Sherman et al. (33)
and included synthetic complete medium (SC) lacking one or more specific amino acids (e.g. SC-uracil) and rich medium (YPD).
Plates for the detection of Plasmids--
The reporter plasmids pGY48 (35) and pBG12 (TEF2
lacZ) (36) were previously described. The integrating
derivative of pGY48, pGY88, was constructed by deleting a 2.2-kb
EcoRI fragment containing the 2-µm origin. The
CYC1-lacZ reporter is pLG669Z (37), and the
pGY107 is a 2-µm plasmid containing an MCM1 gene with a
Myc epitope tag. The Myc epitope was inserted into a new
EcoRV site that was introduced by site-directed mutagenesis
between amino acids 3 and 4 of the cloned MCM1 gene. The Myc
epitope was encoded by complementary 30-base pair oligonucleotides
which were annealed to generate a blunt end fragment for cloning into
the EcoRV site. The presence and orientation of Myc
sequences was confirmed by DNA sequence analysis. The modified
MCM1 gene was then subcloned as a 3.5-kb
XbaI-XhoI fragment into pRS316 (URA3,
CEN) (38). The plasmid complemented the
pWT1037 consists of a 2.6-kb BamHI-HindIII
fragment containing FPS1 cloned into the BamHI
and HindIII sites of the LEU2 CEN vector, pRS315
(38). pWT1039 consists of a 2.8-kb HindIII-EcoRI fragment containing SDH2 and YLL042C cloned into the
HindIII and EcoRI sites of pRS315. pWT1040 is a
derivative of pWT1037 from which an internal 0.9-kb
XhoI-PstI fragment was deleted and replaced with
a 3.3-kb XhoI-PstI LEU2 fragment
excised from plasmid YEp13.
pJF1070 is a LEU2 marked disruption of the GPD1
gene. A GPD1 fragment encompassing the entire open reading
frame was polymerase chain reaction amplified using a primer pair
corresponding to open reading frame YDL022W (Research Genetics) and
genomic DNA as template. A 1.9-kb AvaI-BamHI
restriction fragment was cloned into pUC19 (Promega Biotech) to
generate the intermediate plasmid, pJF1068. A deletion of 268 base
pairs was introduced into the GPD1 open reading frame on
pJF1068 by SalI digestion and ligation. A 2.2-kb
SalI-XhoI LEU2 fragment was
subsequently ligated into the SalI site to create pJF1070.
pWT250 is a derivative of pRS424 (38), a 2-µm TRP1 marked
plasmid into which was cloned a 5-kb EcoRI fragment
containing the GPD1 gene isolated from YEpGPD1 (8). Plasmid
pWT980 used in converting SLN1+ strains to
sln1-22 consists of a 2.1-kb
EagI-XbaI fragment containing the
sln1-22 alteration cloned into pRS406 URA3
integrating vector.
Genetic Screen--
A kar1 (JF1593 or JF1595) strain
carrying the plasmid to be introduced was mated to candidate mutants
(which also carry two recessive drug resistance alleles). Cytoductants
were selected on drug plates (selecting for the persistence of the
nrp nucleus and loss of the kar1 nucleus) lacking
leucine (selecting for the presence of the plasmid) and tested for
their X-gal phenotype. The starting strain, JF1567 was subject to ethyl
methanesulfonate mutagenesis (18) sufficient to cause 65% killing.
Approximately 54,600 surviving colonies were screened on X-gal plates
to find 550 mutants with increased blue color. Mutants were crossed by a wild type strain of the opposite mating type and diploids tested for
their X-gal phenotype. Fifty diploids out of 550 exhibited a mutant
(blue phenotype) and were thus put aside on the basis of the dominance
of the mutation. SLN1 and GAL11 plasmids were introduced into each of the 500 remaining mutants by a cytoduction strategy (see "Results"). Nine were complemented by the
SLN1 plasmid, and 22 were complemented by the
GAL11 plasmid. From previous studies we were aware that
mutations in the KEX2 gene also (nonspecifically) increase
the color of reporter bearing strains on X-gal plates. Mutations in the
KEX2 gene were in fact the major class in the preliminary
screen that resulted in three sln1* alleles. Consequently, we also introduced a KEX2 plasmid into each mutant. Three
hundred mutants were complemented by the KEX2 plasmid or by
more than one plasmid. One hundred forty-two mutants could not be
complemented by any of the three plasmids. Each of the remaining
mutants was tested for its P site specificity. The P-lacZ
reporter was first eliminated from each strain by selection on
5-fluoroorotic acid plates and then a TEF2-lacZ
reporter introduced. Each strain was examined on X-gal media and
cultured for quantitation of Sporulation--
Cells were cultured in YPD or SC media
overnight. Approximately 8 × 107 cells were harvested
by centrifugation and washed twice with water. Cells were resuspended
in sporulation medium (1% potassium acetate and 10 µg/ml amino acids
required by the strain). Sporulation cultures were incubated with
aeration at room temperature for 5-8 days.
Isolation and Quantitation of RNA--
Cells were grown to a
concentration of 1 × 107 cells/ml at 30 °C in the
designated media. Sorbitol (Fluka) was added to log phase cultures to a
final concentration of 1.0 M as follows. A 5.0 M sorbitol stock was first added to conditioned YPD to a
final concentration of 2.0 M. YPD cultures grown to 1 × 107 cells/ml were then diluted 1:1 with the 2.0 M conditioned YPD/sorbitol to a final concentration of 1.0 M sorbitol. Preparation of RNA, electrophoresis, blotting,
and hybridization were performed as described previously (18).
32P-Labeled probes were prepared using random primers (39,
40). Quantitation was performed using a PhosphorImager (Molecular Dynamics).
Immunodetection of Mcm1 Protein--
Cells were harvested in
exponential growth. Extracts were prepared using a previously described
trichloroacetic acid extraction (41). Samples were subjected to
SDS-polyacrylamide gel electrophoresis (10%; 4.5% stacking gel),
transferred to nitrocellulose, and the blot was probed with anti-Myc
monoclonal antibody 9E10 (1:1000 dilution). Immune complexes were
visualized by ECL chemiluminescence (Amersham Corp.)
Intracellular Glycerol Measurements--
Intracellular
glycerol was assayed enzymatically with a commercial glycerol
determination kit (Boehringer-Mannheim Biochemicals). Extracts
were prepared essentially as described (42). Cells were grown to log
phase in selective media. Three 10-ml aliquots were separately filtered
onto 0.45-µm cellulose nitrate filters (Whatman), washed with 5 ml of
cold YPD, and resuspended in 2 ml 0.5 M Tris-HCl, pH 7.5. One-ml samples were heated to 95 °C for 10 min and cell debris
pelleted by low-speed centrifugation. Pooled aliquots were used in
determining glycerol levels according to manufacturer's
specifications. Protein extracts were prepared from the remaining
cells. Cells were vortexed four times (30 s each) in the presence of
glass beads and cell debris pelleted by high speed centrifugation.
Protein concentrations were determined using the Bio-Rad protein assay
(Bio-Rad). Glycerol concentrations were normalized to total protein.
Fold increase in glycerol concentration was determined by normalizing
to the glycerol concentration in wild type cells. Data represent the
average of six trials using a minimum of three transformants.
Isolation of New nrp Mutants--
A strain carrying the
MCM1-dependent reporter, P-lacZ
exhibits a pale blue phenotype on plates containing the chromogenic substrate, X-gal (50 mg/liter). Previously, we have described recessive
alleles of SLN1 (nrp2-1, nrp2-2, and
nrp2-3; renamed sln1-21, 22, and 23)
and GAL11 that exhibit increased P-lacZ reporter activity (18, 35). To identify additional NRP
(negative regulators of P-lacZ)
genes, we repeated the screen using a strategy designed to rapidly
detect (and discard) additional SLN1 and GAL11
alleles. SLN1 and GAL11 plasmids (LEU2
selectable marker) were introduced separately into each individual
mutant using a cytoduction strategy based on the dominant nuclear
fusion defect of the kar1-1 mutant (43) (see "Experimental
Procedures"). Using this procedure, it was possible to simultaneously
screen hundreds of candidates on a small number of Petri plates for
mutants whose phenotype failed to be complemented by the
SLN1 or GAL11 genes. A similar tactic was used to
determine which mutants had a specific effect on the
P-lacZ reporter. In this case mutants were first cured of
the P-lacZ plasmid and subsequently mated to a
kar1 strain containing the TEF2-lacZ
reporter, a reporter whose transcription is independent of Mcm1p.
Of the 12 mutants exhibiting a 2-fold or greater elevation in
P-lacZ activity, four showed clear 2:2 segregation of the
X-gal phenotype suggesting that the mutation was due to a change at a
single locus. Each of these four was examined more thoroughly for the
specificity of the reporter gene activation phenotype by transformation
with Mcm1-independent reporter plasmids, TEF2-lacZ and
CYC1-lacZ, as well as a reporter lacking a UAS insert (Table II). Two mutants, nrp0831 and
nrp0932, continued to exhibit a specific increase in
Mcm1-dependent reporter gene expression, whereas
nrp0427 and nrp1002 were eliminated from further
consideration on the basis that they were not P-lacZ
specific. Subsequent analysis revealed that the increased
P-lacZ activity in the nrp0932 mutation was due
to an effect of the mutation on the copy number of the reporter plasmid
(data not shown) and we therefore eliminated it from further study. The
specificity of the reporter gene activation phenotype in
nrp0831 was confirmed by comparing the effect of the
nrp0831 mutation on a reporter carrying 4 copies of the P site or 4 copies of a mutated version of the P site. The activating effect of the nrp0831 mutation was eliminated in the mutated
P site reporter (nrp0831/wt = 1.1).
Cloning of NRP0831 by Complementation--
The X-gal phenotype of
the nrp0831 mutation is recessive (Fig.
1B, bottom) so it was possible
to clone the gene by complementation. A CEN based genomic
library was introduced by transformation into the nrp0831
strain, JF1709, a product of a primary backcross. Sixteen thousand
transformants were screened on X-gal plates for restoration of the pale
blue (wild type) phenotype. Plasmids were rescued from each of the
seven transformants that were white on X-gal plates and each could be
shown to confer upon the starting strain a pale blue phenotype after
retransformation. Partial sequence analysis of each of four potential
NRP0831 candidates revealed that the four plasmids covered a
common region of chromosome XII encompassing the open reading frames of
two known genes, FPS1 and SDH2, as well as one
additional complete open reading frame (YLL042C) and part of the
VPS13 gene (Fig. 1A). Subclones containing a
2.6-kb BamHI to HindIII fragment that includes
the entire FPS1 coding region and 300-base pair upstream
sequence complemented the nrp0831 X-gal phenotype, whereas a
2.8-kb EcoRI to HindIII fragment including
SDH2 and YLL042C failed to complement the mutant phenotype
(Fig. 1B, top).
The nrp0831 Mutation Is an Allele of FPS1--
To verify the
identification of the FPS1 gene as a regulator of
P-lacZ reporter gene activity, P-lacZ activity
was measured in strains containing a deletion of the FPS1
gene. The fps1
The increased P-lacZ reporter activity in fps1
To examine whether the increase in P-lacZ activity in the
fps1 The fps1
Tests of the requirement for SLN1 were done in an
ssk1
Sln1p regulates the activity of two separate pathways in a reciprocal
fashion. The Hog1p-dependent osmotic response pathway is
kept inactive by phosphorylated Sln1 pathway intermediates (15), while
a second (Hog1-independent) pathway monitored using the
P-lacZ reporter is activated in response to phosphorylation of Sln1p (17). Likewise accumulation of unphosphorylated Sln1p increases HOG pathway activity (15) and decreases activity of the
second branch.2 We have
recently shown that Sln1p activation of the P-lacZ reporter is mediated by the Skn7 receiver
protein.3 To determine the
involvement of Skn7p in fps1 Effect of FPS1 Mutation Is Due to Osmotic Imbalance Caused by
Accumulation of Intracellular Glycerol--
Since FPS1
encodes a glycerol facilitator and the Hog1p pathway regulates the
production of glycerol in response to osmotic stress, it seemed likely
that the fps1 effect on P-lacZ activity would
also require the Hog1p-dependent production of glycerol. GPD1 encodes the major NADH-dependent glycerol
phosphate dehydrogenase activity in yeast and has been shown to be
required for the increase in intracellular glycerol in fps1
mutants (42). We constructed an fps1
FPS1 is required for normal efflux of glycerol (29). Under
hyper-osmotic stress conditions, the channel closes to allow accumulation of glycerol in the cell. Under normal growth conditions the channel is open. In fps1 deletion mutants, glycerol
efflux is prevented and the normal intracellular glycerol concentration increases by a factor of two (29). We used two approaches to determine
whether the fps1 mutation activates the P-lacZ
reporter due to a direct effect of increased glycerol levels, or,
alternatively, whether elevated internal osmolarity might provide an
osmotic imbalance signal that affects downstream target genes, perhaps including the P-lacZ reporter.
To determine if P-lacZ reporter activity is a reflection of
intracellular glycerol concentrations, we compared
If the fps1 phenotype were due to osmotic imbalance due to
accumulation of intracellular glycerol, increasing the osmotic strength
of the medium would be predicted to counteract the signal and suppress
the phenotype. Since the stability of the Our genetic screen for modulators of Sln1 activation of
P-lacZ reporter gene has led to the identification of the
FPS1 gene. FPS1 encodes a MIP family channel
protein which functions in glycerol transport (29). The permeability of
the yeast plasma membrane for glycerol has two components, an
FPS1-independent component attributable to passive
diffusion, and an FPS1-dependent component representing facilitated diffusion (29). The Fps1p channel is closed
under hyperosmotic stress and opens rapidly when hyperosmotic conditions are reversed (29). The Fps1 protein may also play a distinct
role in controlling glycerol production, perhaps by associating with
enzymes involved in glycerol metabolism, since overexpression of
FPS1 unexpectedly leads to increased glycerol levels (29).
Fps1p appears to play a central role in modulating intracellular
glycerol concentrations under conditions of osmotic stress.
Sln1p has been shown to be a yeast osmosensor, however, the mechanism
by which Sln1p senses a change in osmolarity is not known. Cell
fractionation studies indicate that Sln1p is a membrane-associated protein.4 Assuming a plasma
membrane localization, the role of the putative extracellular or
periplasmic domain could be to bind or sense an osmolyte. However, the
nature of the cellular response to osmolarity in Escherichia
coli and in yeast suggest that there is wide array of salts,
sugars, and other small molecules that constitute the osmotic
environment and that the response to osmolarity is independent of the
identity of any of these molecules. Thus, it is unlikely that the
stimulus will take the form of any single osmolyte that binds to an
extracellular domain. More general models of osmosensing have been
suggested. For example, the existence of mechanosensitive ion channels
in the yeast plasma membrane that are activated by stretching of the
membrane has led to the inference that yeasts are able to sense and
respond to physical forces like osmotic pressure (47). Another possible
form of stimulation of the osmotic sensors is suggested by the changes
in cell shape and structure that occur in response to hyper-osmotic
stress. For example, partial dehydration may alter the interaction
between the plasma membrane and the cell wall. It has been previously
proposed that a cell-wall component may serve as a ligand for a plasma
membrane receptor (10). Changes in turgor pressure might cause an
alteration in the interaction between ligand and receptor to create a signal.
Whether the stimulus is sensed extracellularly or as a perturbation in
normal wall-membrane interactions, subsequent steps in signal
transmission are likely to involve structural changes in the Sln1
protein that stimulate or repress the autokinase activity of the
cytoplasmic histidine kinase domain. The discovery that a Sln1
regulated pathway can be activated by a signal generated in an
fps1 mutant may provide some insight into the osmosensing mechanism. Given the known characteristics of the fps1
mutant, two distinct but related stimuli can be postulated. Analysis of membrane composition in fps1 mutants revealed alterations in
phospholipid and glycolipid fractions in the fps1 mutants
(48), suggesting that membrane composition itself may mimic the effect
of osmotic stress on membrane composition, fluidity, or relationship to
the wall. Alternatively, the increase in intracellular glycerol levels in an fps1 mutant might provide the signal. Our data showing
a reduction in levels of P-lacZ activity in the gpd1
fps1 double mutant indicates that the increased level of
intracellular glycerol in the fps1 mutant does, in fact,
contribute to the P-lacZ phenotype in fps1 mutants.
How might an elevation in intracellular glycerol concentration lead to
an increase in P-lacZ activity? One possibility is that
increased levels of glycerol might stimulate P-lacZ
expression by directly affecting the activity of a transcription factor
involved in P-lacZ expression. However, this is unlikely
because we have shown that Sln1p and the downstream receiver, Skn7p are
required for the fps1 phenotype. In testing the requirement
for Sln1p in the fps1 phenotype, we made use of a
sln1 The decrease in reporter activity attributable to the ssk1
mutation may be due to reduced glycerol production in that genetic background. Although glycerol levels have never been directly measured in a strain retaining the Sho1p osmosensor but lacking the
Sln1p contribution to Hog1p activation, both the kinetics and the
responsiveness of Hog1p tyrosine phosphorylation to various osmotic
stress conditions are altered in strains in which Sln1p pathway-specific molecules are defective (11).
The dependence of the fps1 phenotype on SLN1 and
SKN7 indicates that fps1 activation of the
P-lacZ reporter is mediated by the Sln1-Skn7 pathway. The
additivity of the sln1* and fps1 mutations is
likely to reflect the sum of the effects of the individual mutations on
levels of phosphorylated Sln1p in the cell. Our previous studies
suggest that the sln1* mutation shifts the normal
Sln1
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
REFERENCES
-glucan content in the cell
walls of pkc1 mutants (27) indicates that the PKC pathway is
required, in part, for cell wall integrity. Consistent with the idea
that the PKC kinase cascade is an osmosensory system required for
growth under low osmolarity, Davenport et al. (28) have
shown that Mpk1p is rapidly tyrosine phosphorylated in response to
hypo-osmotic conditions. Interestingly, the Hog1 and Mpk1 pathways
appear to cross-talk; tyrosine phosphorylation of Mpk1 was inhibited in cells exposed to an elevated osmotic environment.
EXPERIMENTAL PROCEDURES
and G418
resistance phenotypes. Disruption of the GPD1 locus was
accomplished by transformation of the appropriate yeast strains with a
3.8-kb gpd1
::LEU2 disruption fragment isolated
from plasmid pJF1070.
Yeast strains used in this study
-galactosidase contained 50 mg of
5-bromo-4-chlor-3-indolyl
-D-galactopyranoside
(X-gal)/ml and were prepared as described by Larson et al.
(34).
UAS-lacZ reporter is pLG670Z, a derivative of pLG669Z which lacks
the XhoI fragment encompassing the CYC1 UAS.
Additional P site-dependent reporters included pJF947
(1xP), pCM771 (4xP), and pCM772 (4xmutP). The P site and mut P site
oligonucleotides used in construction of these reporters were as
follows: 5'-TCGAGTTTCCTAATTAGGAAAC-3' (P) and
5'-TCGAGTTTCCTAATTAATAAAC-3' plus 5'-TCGAGTTTATTAATTAGGAAAC-3' (mutP).
LEU2 CEN plasmids used in the genetic screen for new nrp mutants included SLN1 (pGY111) (18), GAL11
(pJF765) (35), and KEX2 (pSBKX, R. Fuller).
-halo defect of
an mcm1-1 mutant.
-galactosidase levels in liquid assays.
Twelve mutants exhibited a 2-fold or greater increase in
P-lacZ activity but less than a 2-fold increase in
TEF2-lacZ reporter activity.
RESULTS
Reporter specificity of the nrp mutants
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Fig. 1.
Identification of nrp0831
complementing sequences on chromosome XII. Diagram of the
transcriptional orientation and boundaries of the open reading frames
included on the genomic library plasmid complementing the
nrp0831 mutant. Complementation by the original library
clone and two subclones (shown below the map) is shown in the
upper part of the table. FPS1 plasmid, pWT1037;
SDH2 plus YLL042 plasmid, pWT1039; vector, pRS315.
Recessiveness of the nrp0831 mutation is shown in the
lower part of the table. -Galactosidase values are given
in Miller units and are the averages of n assays.
mutation caused an increase in
P-lacZ activity similar in magnitude to the one in the
nrp0831 mutant (Fig. 1B, middle). These results suggest that mutant nrp0831 contains a loss of function
mutation in the FPS1 gene. The
nrp0831/fps1
diploid (Fig. 1B,
bottom) was sporulated and 52 tetrads dissected. 158 out of 158 of
the viable spores were blue on X-gal media, showing 100% cosegregation of FPS1 with the nrp0831 locus, consistent with
the hypothesis that the nrp0831 mutation is an allele of
FPS1 (data not shown).
mutants was also reflected at the RNA level. RNA was prepared from wild
type and mutant strains carrying a single integrated copy of the the P-lacZ reporter plasmid, and lacZ levels
quantitated following Northern (RNA) hybridization analysis. After
normalization to URA3 transcript levels at 5, 10, and 20 µg of RNA concentrations lacZ RNA levels were shown to be
approximately 2-fold higher (fps1
/wt 5 µg,
457.2/213.9 = 2.1; 10 µg, 488.7/218.3 = 2.2; 20 µg,
650.8/310.1 = 2.1) in the fps1
mutant than in the
wild type strain (Fig. 2).
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Fig. 2.
Effect of mutation in the FPS1
gene on transcript levels from the P-lacZ
reporter. An equal amount of RNA (5 µg, lanes 1 and 2; 10 µg, lanes 3 and 4; 20 µg, lanes 5 and 6) from wild type (JF1566,
lanes 1, 3, and 5) and fps1
(JF1825, lanes 2, 4, and 6) strains was subjected
to Northern (RNA) hybridization using lacZ and
URA3 probes sequentially. Transcript levels were quantitated
using PhosphorImage analysis (Molecular Dynamics). lacZ
transcript levels were normalized to URA3 levels, to control
for loading differences between lanes.
mutant was due to a change in the activity of Mcm1p
rather than to alterations in Mcm1 protein levels (18), strains bearing a Myc epitope-tagged MCM1 gene were evaluated using anti-Myc
immunoblot analysis. As observed previously for the sln1*
mutants (29) (Fig. 3), Mcm1 protein
levels were not significantly affected in the nrp0831
background, nor in strains deleted for FPS1 (Fig. 3).
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Fig. 3.
Mcm1 protein levels are unaffected by
mutations in FPS1. Protein extracts prepared from wild
type (JF1567, lanes 1-3), nrp0831 (JF1709,
lane 4), fps1 (JF1732, lane 5),
sln1-22 (JF1567, lane 6), and sln1-22
fps1
(JF1733, lane 7) strains carrying pGY107, the
2-µm MCM1-Myc plasmid (lanes 1-7) and the wild type
strain lacking pGY107 (JF1709, lane 8) were subjected to
SDS-polyacrylamide gel electrophoresis in duplicate. One gel was
treated with Coomassie Brilliant Blue (not shown) to examine loading
and an identical gel was treated with anti-Myc antibody for Western
analysis as described in detail under "Experimental Procedures." A
2-s exposure following chemiluminescence detection is shown.
Lanes 1, 2, and 3 were loaded with 0.5, 1, and
2 × protein, respectively; while lanes 4-8 each
contained 1 × protein.
-dependent Increase in P-lacZ Reporter
Activity Requires Sln1 and Skn7--
The elevation in
P-lacZ activity in an fps1 mutant is similar to
the effect of sln1* activating mutations (17, 18). To investigate the relationship between SLN1 and
FPS1, we measured P-lacZ activity in the double
mutant. The results of this analysis indicate that the effect of the
sln1* mutation is enhanced by deletion of FPS1
(Table III). While the additivity of the
sln1* and fps1 phenotypes is consistent with the
existence of two independent pathways leading to activation of the
P-lacZ reporter, an alternate explanation may be that the
sln1* mutation and the fps1 mutation each only
partially shift the pool of Sln1 protein to the phosphorylated form,
and that the combined effects of the two mutations increases the
signaling through a single pathway leading to P-lacZ
activation. To distinguish between these possibilities we examined
whether the fps1 effect was dependent on Sln1p and on Sln1p
pathway intermediates.
Effect of the FPS1 mutation on sln1* activation of the
Mcm1-dependent reporter
background to suppress the otherwise lethal
sln1
mutation (15). Although, in this experiment, the
ssk1 mutation depressed P-lacZ levels by 50%,
the effect of the fps1 mutation is still apparent in the
SLN1+ ssk1
strain (Table
IV, rows 3 and 4). In the
sln1
mutant background, in contrast, elevation in
P-lacZ levels due to fps1
was eliminated (Table IV, rows 5 and 6). The data in Table IV indicates that the Sln1
protein is required for the fps1 phenotype.
Role of SLN1 in the fps1 phenotype
-mediated activation of the
P-lacZ reporter, a skn7 fps1 double mutant was
constructed. The data in Table V show
that Skn7p is required for the fps1
phenotype. Deletion
of the SKN7 gene suppressed the fps1
-mediated enhancement of P-lacZ reporter activity.
Effect of skn7 mutation on the fps1 phenotype
gpd1
double
mutant to test whether the 2-fold increase in intracellular glycerol
levels seen in fps1-
mutants is required for the
fps1 phenotype. We found, as expected, that the elevated P-lacZ phenotype in fps1 mutants was reversed by
the gpd1 mutation (Table
VI).
Effect of GPD1 dosage on glycerol levels and the fps1 phenotype
-galactosidase levels in wild type and fps1-
strains plus and minus the
high copy GPD1 plasmid, pWT250. In an FPS1
strain, the GPD1 plasmid is expected to elevate both
intracellular and extracellular glycerol concentrations (29, 42).
However, in the fps1-
mutant, efflux is blocked and only
intracellular glycerol increases (29, 42). As expected, intracellular
glycerol concentrations were elevated in both the wild type (2.6x) and
fps1-
(4.5x) strains carrying the GPD1 plasmid
(Table VI). Despite the 2.6-fold increase in intracellular glycerol
concentration in the wild type strain, P-lacZ activity was
increased only in the fps1-
strain carrying the
GPD1 plasmid and not at all in the wild type strain (Table VI). These data suggest that the P-lacZ reporter activity is
not tied to intracellular glycerol levels, but may instead reflect the
differential between intracellular and extracellular osmotic strength.
-galactosidase protein
(t1/2 > 20 h) (44) would have precluded
detecting a decline in
-galactosidase activity after transient
exposure of cells to sorbitol, we measured lacZ mRNA
(t1/2 ~ 30 min) (45, 46) levels in this
experiment. An fps1
strain carrying an integrated P-lacZ reporter was cultured in YPD media and harvested at
indicated times following the addition of 1.0 M sorbitol.
lacZ message levels were measured by Northern (RNA)
hybridization analysis (Fig. 4). At short
times after addition of sorbitol there was a striking and
reproducible decrease in lacZ expression in the
fps1
mutant. The reduction in reporter gene expression by
sorbitol addition in the fps1 mutant indicates that the
fps1
effect is likely the consequence of osmotic
imbalance, rather than of the accumulation of glycerol per se.
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Fig. 4.
Effect of sorbitol on P-lacZ
transcript levels in the fps1 mutant. RNA was
harvested from early log phase cells treated with 1.0 M
sorbitol or untreated for the times (min) indicated and subjected to
Northern hybridization analysis. Levels of lacZ
hybridization were quantitated by PhosphorImage analysis (Molecular
Dynamics) of the blot. Hybridization of the same blot to the
DED1 probe was used for normalization. Values shown below
the image were calculated as the ratio of
lacZ/DED1 hybridization at a given time divided
by the ratio of lacZ/DED1 hybridization at time 0 for a given growth condition.
DISCUSSION
ssk1
double mutant. The ssk1 deletion
was necessary to suppress the lethality of the sln1
mutation. However, the use of the ssk1 mutation complicated the interpretation of the results of this test since, in this experiment, the ssk1 mutation alone reduced
P-lacZ activity by 50%. Comparison of fps1
ssk1
and the FPS1 ssk1
strains shows that the
fps1 mutation caused a 3.5-fold increase in reporter activity despite the absence of Ssk1p, but did not cause an elevation in reporter activity in the absence of Sln1p. Our conclusion that Sln1p
is required for the fps1 phenotype is further substantiated by analysis of the skn7 mutant strain (Table V). Like
sln1, the skn7 mutation eliminated the
P-lacZ activating effect due to fps1 deletion and
is consistent with the requirement for Sln1 and Skn7 in the
fps1 phenotype.
0/Sln1-P equilibrium to favor Sln1-P. Sln1-P levels may be
further boosted due to an additional effect on the Sln1 equilibrium by
accumulation of intracellular glycerol in fps1 mutants as
depicted in the model in Fig. 5.
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Fig. 5.
Model illustrating the effect of FPS1
deletion on the activities of Sln1 regulated pathways. In
the absence of Fps1p, intracellular glycerol levels rise relative to
extracellular levels. This differential in osmotic strength may be
sensed by the cell as a hypo-osmotic environment. This activates Sln1p
kinase activity and the Sln1p phosphorelay pathway and leads to the
Ypd1p-dependent phosphorylation of the two receiver
molecules, Ssk1p and Skn7p. Phosphorylation inactivates Ssk1, whereas
phosphorylation of Skn7 leads to its activation. The phosphorylated
form of Skn7 acts either directly or indirectly to activate
transcription of the P-lacZ reporter.
How intracellular glycerol changes the activity of the Sln1 protein is not clear. One possibility is that glycerol, which is produced by the product of a Hog1p-dependent gene, could play a direct role in modulating the activity of Sln1p. Glycerol, itself, might stimulate Sln1p kinase activity, leading to an increase in the relative levels of Sln1-P in the cell and a corresponding decrease in Hog1 pathway activity. This would in effect lead to a negative feedback loop allowing the cell to precisely modulate the levels of intracellular glycerol that accumulate in response to osmotic stress. Alternatively, increased levels of intracellular glycerol might increase the normal osmotic gradient beyond a certain threshold causing the cell to perceive a hypo-osmotic environment. Although a slight osmotic gradient is required both to drive the entry of water important for cell growth (6, 49) and to create the turgor pressure needed to force wall expansion (50, 51), the magnitude of the "normal" gradient may be constrained. Accumulation of excess intracellular glycerol in the fps1 mutant may exceed this constraint, thus triggering a hypo-osmotic response.
Two experiments addressed this possibility. First, we used a high copy plasmid expressing the GPD1 gene to increase glycerol levels in wild type and fps1 mutants. The presence of the GPD1 plasmid caused intracellular glycerol levels to increase 2.6-fold in the wild type strain and 4.5-fold in the fps1 strain (Table VI). If P-lacZ activity were simply responding to glycerol levels, we would expect increased reporter activity in both strains. However, the activity of the P-lacZ reporter was increased only in the fps1 mutant. Since the fps1 mutant is defective in glycerol efflux, the extra intracellular glycerol contributed by GPD1 overexpression might exacerbate the osmotic imbalance already created by the fps1 mutation, whereas the increased intracellular glycerol concentration in the wild type strain would be rapidly dissipated. Hence, the elevation in P-lacZ activation in the fps1 mutant but not in the wild type strain carrying the GPD1 plasmid is consistent with a model in which the P-lacZ reporter responds to osmotic imbalance.
Results of experiments in which the presumptive gradient caused by the fps1 mutant was collapsed by addition of sorbitol to the medium also favor the interpretation that it is not the increased glycerol levels per se, but rather the osmotic imbalance that is important for P-lacZ activation in the fps1 mutant. The 2-fold elevation in P-lacZ levels in the fps1 mutant (Fig. 1) is eliminated within 15 min of sorbitol addition (Fig. 4). P-lacZ transcript levels continue to decrease, reaching a minimum at 30 min at 25% of fps1 levels (50% of wild type levels). lacZ levels could be seen to increase following sorbitol treatment beginning at 60 min. By 180 min, levels were comparable to those measured at the beginning of the experiment prior to addition of sorbitol, presumably because the normal osmotic balance has been restored.
Taken together, our results are consistent with a model (Fig. 5) in
which the fps1 signal is interpreted by Sln1 (presumably triggering Sln1 phosphorylation) and transmitted through Skn7 to
undefined downstream genes, ultimately stimulating expression of the
P-lacZ reporter. The signal generated by the fps1
mutation appears to be osmotic imbalance due to accumulation of
intracellular glycerol. Elevated intracellular glycerol levels may
mimic a hypo-osmotic stimulus. Saito and colleagues (15, 16) have shown
that hyper-osmotic conditions shift the equilibrium between
phosphorylated and unphosphorylated Sln1 to favor the unphosphorylated
form. The sln1* activating mutations we previously described
appear to shift the equilibrium to favor the phosphorylated form of
Sln1, counteracting the normal hyper-osmotic response (17). In the
present study we show that loss of the Fps1p glycerol facilitator
protein mimics the effect of activating mutations in SLN1,
suggesting that the Sln1p signal need not be extracellular. The model
outlined in Fig. 5 proposes that hyper-osmotic conditions trigger Sln1p
dephosphorylation and Hog1 pathway activation, whereas hypo-osmotic
conditions trigger Sln1 phosphorylation and Mcm1p pathway activation.
Further studies will be required to determine how the Sln1p
phosphorylation status is modulated by hyper- and hypo-osmotic conditions.
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ACKNOWLEDGEMENTS |
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We acknowledge the assistance of Guoying Yu in constructing and characterizing the MCM1-Myc plasmid; Greg Gingerich and Cherie Malone for strain construction and technical assistance; Stefan Hohmann for the GPD1 plasmid, YEpGPD1, and Robert Fuller for the KEX2 plasmid; Ira Herskowitz and members of the Fassler and Deschenes laboratories for helpful discussions; and Robert Malone and Lois Weisman for critical review of the manuscript.
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FOOTNOTES |
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* This work was supported by American Cancer Society Grant VM-148.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. Tel.: 319-335-1542; Fax: 319-335-1069; E-mail: jan-fassler{at}uiowa.edu.
2 S. Dean and J. Fassler, unpublished observations.
3 Li, S., Ault, A., Malone, C. L., Raitt, D., Dean, S., Johnston, L. H., Deschenes, R. J., and Fassler, J. S. (1998) EMBO J. 17, 6952-6962.
4 H. Lin, C. Malone, and R. Deschenes, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
MAP kinase, mitogen-activated protein kinase;
MEK, MAP kinase kinase;
MEK kinase, MAP kinase kinase kinase;
P site, Mcm1-binding site;
P-lacZ, Mcm1-dependent reporter;
UAS, upstream activation sequence;
kb, kilobase pairs;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside;
MIP, major intrinsic protein of
lens fiber gap junctions in mammals;
PKC, protein kinase C..
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REFERENCES |
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