Copyright1999 by the Genetics Society of America
Activation of the
Saccharomyces cerevisiae
Filamentation/Invasion Pathway by
Osmotic Stress in High-Osmolarity Glycogen Pathway Mutants
K. D. Davenport, K. E. Williams, B. D. Ullmann and M. C. Gustin
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892 Manuscript received January 7, 1999
Accepted for publication July 6, 1999
ABSTRACT
Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the five MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identified: MSG5, CAK1, and TRX1. Msg5p is a dual-specificity phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1D could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a filamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of filamentation/invasion pathway repression contributes to the HOG mutant phenotype.
Y
EAST cells, like many other eukaryotes, utilize mito- rate activating signal and physiological response (for gen-activated protein kinase (MAPK) cascades to review seeGustin et al. 1998), it is becomingincreas-transmit signals from plasma-membrane-associated sen- ingly clear that the MAPK pathways interact with each sory complexes to the nucleus, where transcriptional other. For example, the HOG pathway MAPK Hog1p is responses are elicited (for review see Banuett 1998; rapidly dephosphorylated in response to decreases in Gustin et al. 1998). MAPK cascades are composed of extracellular osmolarity in a Slt2p-dependent manner three conserved families of protein kinases: the MAPK, (Davenportet al. 1995), suggesting that the HOG
path-the MAPK and ERK kinase (MEK), and path-the MEK kinase way is negatively regulated by the cell integrity pathway. (MEKK; for review seeDavis1993). The MEKK receives The phosphorylation of the pheromone response path-a signpath-al from path-an upstrepath-am ppath-athwpath-ay component path-and way MAPK Fus3p in response to hypertonic stress is phosphorylates a conserved threonine and serine resi- enhanced by the deletion of HOG1 or the HOG pathway due within the MEK’s activation domain (Kyriakiset al. MEK gene PBS2 (Hall et al. 1996). The observation
1992;Lange-Carteret al. 1993). Once phosphorylated, that the pheromone response pathway is activated in
the activated MEK phosphorylates a threonine and a response to hypertonic stress in the absence of Hog1p tyrosine residue within the activation domain in the is further supported by evidence that a transcriptional MAPK (CrewsandErikson1992). The phosphorylated target of the pheromone response pathway, FUS1, is also MAPK is then able to phosphorylate the targets of the induced by osmotic stress in the absence of catalytically MAPK cascade, including transcription factors (Gilleet active Hog1p (Hall et al. 1996;O’Rourkeand Hers-al. 1992;Sethet al. 1992) and other regulatory proteins kowitz 1998). It has been suggested that these data
(Cooket al. 1996). indicate that Hog1p prevents the inappropriate
activa-The budding yeast Saccharomyces cerevisiae contains five
tion of the pheromone response pathway by osmotic MAPK cascades, each with its own unique MAPK: Fus3p
stress. in the pheromone response pathway, Kss1p in the
fila-These cross-pathway interactions may provide a mech-mentation/invasion pathway, Hog1p in the
high-osmo-anism for establishing and maintaining signaling speci-larity-growth (HOG) pathway, Slt2p in the cell integrity
ficity. The question is whether these interactions are pathway (Figure 1), and Smk1p in the spore wall
assem-physiologically significant or merely introduced by the bly pathway. Although these pathways each have a
sepa-genetic manipulations of the organism. For example, the activation of Fus3p by high osmolarity in the absence of Hog1p (Hallet al. 1996) may indicate an important
Corresponding author: Mike Gustin, Department of Biochemistry and
role for Hog1p in the maintenance of signal specificity,
Cell Biology, MS140, Rice University, 6100 S. Main, Houston, TX
77005-1892. E-mail: [email protected] or it may be an artifact of the experimental
mutant, pbs2-3. Prevention of Kss1p phosphorylation by the overexpression of the MAPK phosphatase MSG5, the deletion of KSS1, or the deletion of upstream fila-mentation/invasion pathway MAPK cascade genes is suf-ficient to suppress not only the osmosensitivity of pbs2-3, but also one of the morphological phenotypes associ-ated with HOG pathway mutants on high-osmolarity media: the formation of long projections. These data indicate that the filamentation/invasion pathway is in-appropriately activated by hyperosmotic stress in cells lacking a functional HOG pathway.
MATERIALS AND METHODS
Strains, media, and general methods:The yeast strains and plasmids used in this study are listed in Table 1. The bacterial strain DH5awas used for all plasmid amplifications and isola-tions. Growth media (YEPD, supplemented SD, and LB) were prepared as described (Kaiseret al. 1994). Common proce-dures (DNA manipulations, bacterial propagation, etc.) were followed as described (Sambrook et al. 1989). Techniques used for genetic crosses, sporulation, dissection, and propaga-tion of S. cerevisiae are described elsewhere (Kaiseret al. 1994). Yeast were transformed by the one-step method (Chenet al. 1992).
Figure1.—MAPK cascades in yeast.
Isolation, cloning, and sequencing ofpbs2-3:Wild-type strain MG159B was mutagenized with ethyl methanesulfonate (EMS) and screened for colonies that failed to grow on YEPD supple-sion of FUS3. Other examples, such as the activity of
mented with 900 mmNaCl or 1.5msorbitol and that showed Fus3p in the repression of the filamentation/invasion a reduced production of glycerol in high-osmolarity media. pathway and the maintenance of pheromone response Four complementation groups of recessive mutations were identified and designated hog1–hog4 (Brewsteret al. 1993). pathway signal specificity (Madhaniet al. 1997), seem
The mutants in the hog4 complementation group (alleles of to indicate that some interactions are physiologically
PBS2;Brewsteret al. 1993) were then screened for the ability significant. However, in general, the prevalence and
to grow at an intermediate osmolarity of 400 mmNaCl (YEPD physiological significance of these cross-pathway interac- plus 400 mmNaCl). Only one mutant, hog4-3 (renamed pbs2-tions are not yet well known. 3), was able to grow at this osmolarity. Thus, the phenotype The HOG pathway mutants hog1 and pbs2 were first of pbs2-3 was intermediate between a pbs2Dstrain and the wild-type strain. pbs2-3 was backcrossed to W303-1A four times to isolated in a screen for yeast unable to grow or produce
produce strain KDY1. Genomic DNA was extracted (Hoffman glycerol in high-osmolarity media (Brewster et al.
andWinston1987) from strain KDY1 and used as a template 1993). Additionally, budding and growth defects have to amplify the PBS2 locus by PCR. PCR products from three also been described for these mutants (Brewsterand independent reactions were cloned using the TA cloning kit Gustin1994). In high-osmolarity media, hog1Dor pbs2D (Invitrogen, San Diego) to produce pKD10, pKD11, and pKD12. One clone, pKD10, was sequenced (Seqwrite) and cells will abandon a small bud and grow a new bud.
compared to the Saccharomyces Genome Database and pre-This double-budded phenotype may indicate a defect
viously published sequences (BoguslawskiandPolazzi1987). in cell cycle regulation in HOG pathway mutants that
Discrepancies between the pbs2-3 sequence and published se-are exposed to high-osmolarity media (Brewsterand quences of PBS2 were verified by sequencing portions of Gustin 1994). A second growth defect observed in pKD11 and pKD12 to ensure that the mutations identified in pKD10 were not introduced by PCR. Two mutations were HOG pathway mutants grown in high-osmolarity media
identified, which are predicted to code for the following substi-is the production of long cellular projections
(Brew-tutions in the polypeptide sequence: proline for serine at sterandGustin1994), indicating stimulated or
unreg-position 168 (S168P) and aspartate for glycine at unreg-position 509 ulated polarized growth in HOG mutants exposed to (G509D). Restriction fragments from pbs2-3 containing either high osmolarity. However, the precise cause of these one or both of the identified mutations were used to replace morphological defects is not known. the corresponding restriction fragments of a plasmid con-taining PBS2 to produce pKD13, pKD14, and pKD15. A strain In this article, we provide evidence that part of the
deleted for PBS2 (KDY9) was transformed with these plasmids HOG pathway mutant phenotype is a consequence of
and assayed for growth on high-osmolarity media (YEPD plus the loss of HOG-pathway-dependent inhibition of a
sec-400 or 900 mmNaCl) to identify which of the mutations were ond MAPK pathway, the filamentation/invasion path- responsible for the pbs2-3 growth phenotype.
TABLE 1
Strains and Plasmids
Genotype Reference or source
Strain
MG159B MATaura3-52 Brewsteret al. (1993)
hog4-3 MATa pbs2-3 (congenic with MG159B) Brewsteret al. (1993) W303-1A MATa ade2-1 his3-11 leu2-3,112 trp1-1 ura3-1 can1-100 Laboratory stock
KDY1 pbs2-3 This work
KDY2 pbs2-3 kss1D::URA3 This work
KDY3 pbs2-3 fus3D::LEU2 This work
KDY4 pbs2-3 slt2D::TRP1 This work
KDY5 pbs2-3 ste12D::URA3 This work
KDY6 pbs2-3 ste7D::URA3 This work
KDY7 pbs2-3 ste11D::URA3 This work
KDY8 pbs2-3 sho1D::URA3 This work
KDY9 pbs2D::LEU2 This work
KDY10 hog1D::TRP1 This work
KDY11 pbs2D::LEU2 kss1D::URA3 This work
KDY12 pbs2D::LEU2 ste7D::URA3 This work
KDY13 hog1D::TRP1 kss1D::URA3 This work
KDY14 hog1D::TRP1ste7D::URA3 This work
KDY15 pbs2-3 kss1D::URA3 fus3D::LEU2 This work
KDY16 pbs2-3 kss1D::URA3 slt2D::TRP This work
KDY17 pbs2-3 fus3D::LEU2 slt2D::TRP This work
KDY18 pbs2-3 kss1D::URA3 fus3D::LEU2 slt2D::TRP This work Plasmid
pRS426 URA3 2m Christiansonet al. (1992)
pRS423 HIS3 2m Christiansonet al. (1992)
pKD1 URA3 2mMSG5 This work
pKD2 URA3 2mTRX2 This work
pKD3 URA3 2mCAK1 This work
pKD4 HIS3 2mMSG5 This work
pKD5 HIS3 2mTRX1 This work
pKD6 HIS3 2mTRX2 This work
pKD7 HIS3 2mCAK1 This work
pKD8 pGEM-7ZF1KSS1 This work
pKD9 pGEM-7ZF1kss1D::URA3 This work
pSL 1077 pBR322 Ste7D::URA3 Fieldset al. (1988)
pSL 1094 pBluescript KS1ste11D::URA3 Fieldset al. (1988)
pSL 1311 pSPGS ste12D::URA3 Fieldset al. (1988)
pYEE98 pUC119 fus3-6D::LEU2 Elionet al. (1990)
YEpT-KSS1 TRP1 2mKSS1 Bardwellet al. (1996)
YEpT-kss1 TRP1 2mkss1 Y-F, T-A Bardwellet al. (1996)
YEpL-FTyZ LEU2 2mTy1::lacZ Cooket al. (1997)
YEpU-FTyZ URA3 2mTy1::lacZ Cooket al. (1997)
All strains used in this study are congenic with W303-1A, with the exception of MG159B and hog4-3.
libraries (M. F. Hoekstra, unpublished results; C. J. Con- Plasmids:pKD2, pKD3, and pKD5–pKD7 were constructed by first amplifying the appropriate open reading frame (TRX2, nellyandP. Hieter,unpublished results). The transformants
(z30,000 per library) were screened for the ability to grow TRX1, or CAK1) and 1-kb flanking DNA from wild-type (W303-1A) genomic DNA using primers designed to add XhoI sites on YEPD supplemented with either 1mKCl or 1msorbitol.
Plasmid DNA was isolated from osmoresistant colonies, ampli- to the ends of the PCR product. The PCR products were digested with XhoI and ligated to SalI-digested pRS426 (2m fied in bacteria, and transformed into KDY1 to test for
suppres-sion of pbs2-3 osmosensitivity. Twelve colonies that demon- URA3) or pRS423 (2mHIS3;Christiansonet al. 1992). Plas-mids pKD1 and pKD4 were constructed by subcloning an strated plasmid-dependent osmotic resistance were isolated.
Plasmids were isolated and divided into five classes by restric- z2.2-kb XhoI-NotI fragment of the library isolate containing MSG5 to XhoI-NotI-digested pRS426 or pRS423, respectively. tion mapping. The largest class (five clones) consisted of
plas-mids containing PBS2. Two plasplas-mids contained HOG1. The Primers designed to add an XhoI site on both ends of the resulting PCR product were used to amplify a fragment con-remaining three classes of high-copy suppressors contained
MSG5 (two clones), CAK1 (two clones), or TRX1 (one clone), taining the KSS1 open reading frame and 500 bp of upstream-and downstream-flanking DNA from wild-type (W303-1A) ge-as determined by the comparison of the plge-asmid DNA
KSS1-coding sequence and flanking DNA was ligated to XhoI- primed labeling kit (Ambion, Austin, TX). The membrane and probe were then incubated overnight at 658, washed, digested pGEM-7ZF(1) (Promega, Madison, WI) to produce
pKD8. pKD8 was used as a template to amplify the 500-bp dried briefly, and exposed to a phosphoimager plate. The phosphoimaging plate was then processed using a Fujix regions flanking the KSS1 open reading frame and the vector,
but not the KSS1-coding sequence, using primers that add a BAS100 phosphoimager.
b-Galactosidase assays:Cells were grown to saturation over-NotI site to the ends of the resulting PCR product. Primers
containing NotI sites were also used to amplify the URA3 gene night, diluted to OD600z0.4 in YEPD, and grown for an
addi-tional 2 hr prior to the addition of solutes for the desired from YDp-U (Berbenet al. 1991). These two PCR products
were digested with NotI and ligated to produce pKD9. pKD9 stress. After the desired time had elapsed, cells were pelleted, transferred in 500 ml of ice-cold Z buffer (16.1 g/liter Na2
was used as a template to amplify the URA3 and flanking DNA
by PCR, and the product was used to replace the genomic HPO4·7H2O, 5.5 g/liter NaH2PO4·H2O, 0.75 g/liter KCl, 0.246
g/liter MgSO4·7H2O, pH 7.0) to a 2-ml tube, pelleted, and
copy of KSS1 in yeast. The deletion of KSS1 was confirmed in
uracil prototrophs by PCR and by sterility in combination with frozen on dry ice. To isolate the protein, 250ml Z buffer and 12.5ml 40 mmPMSF were added to the pellet prior to four fus3D.
The plasmids used in the disruption of STE11, STE7, and freeze/thaw cycles (10-sec incubation in liquid nitrogen and 90-sec incubation in a 378water bath). Cell debris was removed STE12 were a gift from George Sprague. pYEE98 (pUC119
fus3-6::LEU2) was a gift from the Elion lab. Lee Bardwell pro- by centrifugation, and the protein content of the supernatant was determined (Bio-Rad, Richmond, CA). Supernatant (100 vided YEpT-KSS1 and YEpT-kss1. YEp352-CAK1, provided by
Ed Winter, was used to further test whether overexpressed ml) was added to 900 ml Z buffer with 200 ml 4 mg/ml o-nitrophenyl-b-d-galactopyranoside (Sigma). After a 5- to 20-CAK1 can suppress pbs2-3 osmosensitivity.
Growth analysis:Overnight cultures of the desired strains min incubation, 1 ml 1 m Na2CO3 was added to stop the
reaction, andb-galactosidase activity was determined by mea-were diluted to OD600z0.1 in YEPD and grown for 2–3 hr at
308. Cell densities were again adjusted to OD60050.1 in YEPD suring the OD420.
Microscopy:Cells were grown to log phase, stressed with and used to make serial dilutions in a 96-well plate. Equal
volumes of these cultures were transferred to YEPD plates the addition of solute (1mKCl, 1msorbitol, or 900 mmNaCl), and fixed by addition of formaldehyde to a final concentration containing various solutes by use of a 48-prong replicating
tool. Plates were incubated at 308for various lengths of time, of 3.7%. After incubation for at least 1 hr, cells were pelleted, washed once with PBS (8 g/liter NaCl, 0.2 g/liter KCl, 1.44 as indicated in the figure legends.
g/liter Na2HPO4, 0.24 g/liter KH2PO4, pH 7.4), and
resus-Immunoblots:Protein extracts were prepared from treated
pended in PBS for storage. Cells were then diluted, sonicated and untreated cells, as described previously (Brewsteret al.
briefly to disrupt cell clumps, and spotted onto Superfrost 1993). A total of 20mg of each protein sample was separated
Plus slides (Fisher Scientific, Pittsburgh). After allowing the by SDS-PAGE and transferred to Protran nitrocellulose filters
cells to adhere to the slide, the remaining liquid was removed (Schleicher & Schuell, Keene, NH). Molecular weight markers
by aspiration. Five microliters of mounting media (Vecta-(10 kD; GIBCO BRL, Gaithersburg, MD) were visualized by
shield; Vector Laboratories, Burlingame, CA) was added di-staining the membrane with Ponceau S (Sigma, St. Louis) and
rectly to the slide, covered with a coverslip, sealed using nail marking the location with a pencil. The membrane was rinsed
polish, and stored at 48. The yeast cells were visualized using and incubated with blocking buffer [3% BSA in TBS-T (6.056
an Axioskop (Zeiss, Thornwood, NY) set for differential inter-g/liter Tris, 8.766 inter-g/liter NaCl, 0.05% Tween 20, pH 8.0)].
ference contrast (DIC) microscopy. Monoclonal antibodies [antiphosphotyrosine (Upstate
Bio-technology, Lake Placid, NY) or anti-phosphoERK (Sigma)] were diluted 1:1000 in blocking buffer, added to the
mem-brane, and incubated at room temperature for at least 90 RESULTS min. Following incubation, the membranes were washed three
times for 10 min in TBS-T. Secondary antibody (HRP-conju- Isolation of a partially functional allele of PBS2: In gated anti-mouse; Boehringer Mannheim, Indianapolis) was a screen for osmosensitive mutants defective in HOG diluted 1:5000 in blocking buffer and incubated with the mem- pathway function (seematerials and methods), a par-branes for at least 40 min. The mempar-branes were then washed
tially functional allele of PBS2, pbs2-3, was isolated. four times for 10 min in TBS-T prior to the addition of ECL
pbs2-3 exhibits an intermediate osmosensitivity
com-reagent (Amersham, Arlington Heights, IL) and exposure to
Hyperfilm ECL (Amersham). pared to wild-type and pbs2D strains. A pbs2-3 strain, Northern analysis:Cells were grown overnight, diluted to unlike a pbs2Dstrain, can grow on media supplemented OD600z0.4 in YEPD, and grown for an additional 2 hr prior with 400 mmNaCl (Figure 2A). However, a pbs2-3 strain
to the addition of solute (NaCl, KCl, or sorbitol) to produce
cannot grow on media supplemented with 900 mm a hyperosmotic shock. Cells were pelleted and then lysed by
NaCl, though a wild-type strain can grow under these vortexing with glass beads in the presence of phenol and RNA
lysis buffer (0.5mNaCl, 10 mmEDTA, 50 mmTris, pH 8.0). conditions (Figure 2A).
The RNA in the aqueous phase was then precipitated, dried Hypertonic-stress-induced changes in Hog1p phos-briefly, and then resuspended in 50ml diethyl pyrocarbonate- phorylation and GPD1 transcription were analyzed to treated water. A total of 2ml 103NBC (0.5mboric acid, 10
determine if the pbs2-3 mutation affected signal trans-mmsodium citrate, 50 mmNaOH, pH 7.5), 3ml formaldehyde,
duction through the HOG pathway. As a MAPK, Hog1p 10ml formamide, 2ml 103loading buffer (15% Ficoll, 0.1m
EDTA, 0.25% bromophenol blue), and 1ml ethidium bromide is activated by phosphorylation on a conserved tyrosine (1 mg/ml) were added to 4mg RNA. Following electrophoretic and threonine residue, allowing detection of activated separation, the RNA was transferred to a Hybond N1mem- Hog1p using commercially available antiphosphotyro-brane (Amersham), UV cross-linked, and incubated for at
sine antibodies. There is increased phosphorylation of least 1 hr at 658with Church buffer (7% SDS, 250 mmNa2PO4,
Hog1p and subsequent induction of GPD1 mRNA accu-pH 7.5;ShifmanandStein1995). Radiolabeled probes were
(Figure 2, B and C), consistent with previous observa-tions (Brewster et al. 1993; Albertyn et al. 1994).
These responses to increases in osmolarity are absent in pbs2Dstrains. In a pbs2-3 strain, the levels of Hog1p phosphorylation and GPD1 mRNA following osmotic stress were intermediate between the wild-type and dele-tion strains. These data show that the partial osmosensi-tivity of the pbs2-3 strain is correlated with a partial loss of signaling through the HOG pathway.
To identify the mutations responsible for the pbs2-3 phenotype, the pbs2-3 locus was cloned and sequenced. Two mutations were identified by comparison of the
pbs2-3 sequence to published PBS2 sequences. The pbs2-3 mutations are predicted to result in a substitution
of proline for serine at position 168 (S168P) and a substitution of aspartate for glycine at position 509 (G509D) in the polypeptide chain of this MEK. Plasmids were constructed in which only one of the two mutations was present in an otherwise wild-type PBS2 gene. When the single mutant plasmids were introduced into pbs2D strains, the plasmid containing the S168P PBS2 muta-tion appeared to complement fully while the plasmid coding for the G509D PBS2 mutation gave a phenotype intermediate between wild type and pbs2D, similar to that seen in a pbs2-3 strain. Thus, the G509D substitution appears to be responsible for the phenotype of a pbs2-3 strain. The G509D substitution occurs near the residues (S514 and T518) predicted to be phosphorylated by the MEKKs of the HOG pathway (Maedaet al. 1995). The
substitution of alanine for either S514 or T518 in Pbs2p prevents signaling through the HOG pathway in
re-sponse to osmotic stress (Maedaet al. 1995). Although Figure 2.—Description of the pbs2-3 phenotype and the three high-copy suppressors of pbs2-3 osmosensitivity. (A) The the exact effect of the G509D substitution on Pbs2p
indicated strains were grown as described inmaterials and activity is unknown, it may be that the perturbation of
methods, spotted on YEPD plates supplemented with the the activation site by the G509D substitution in pbs2-3p indicated osmolytes, and grown for 2 days (control and 400 could interfere with the efficient activation of pbs2-3p. mmNaCl) or 5 days (1msorbitol, 1mKCl, 900 mmNaCl). The G509D substitution is also within the kinase domain (B) Protein was isolated from W303-1A pRS426 (WT 2m), KDY1 pRS426 (pbs2-3 2m), KDY9 pRS426 (pbs2D 2m), and of Pbs2p and, therefore, could also affect the catalytic
KDY1 pKD1 (pbs2-3 2mMSG5) before and after the addition activity of Pbs2p independently of any effect on
activa-of NaCl (400 mm). The protein samples were used to prepare tion of the MEK. antiphosphotyrosine immuoblots, as described inmaterials High-copy suppressor screen:The partially osmosen- and methods.(C) RNA was isolated from the indicated strains sitive pbs2-3 strain (KDY1) was used in a screen to identify grown in YEPD followed by growth in YEPD supplemented with 1msorbitol for 1 or 3 hr. Total RNA (4mg) was used proteins that affect growth on high-osmolarity media.
for each sample in the RNA blot, as described inmaterials
pbs2-3 was transformed with two high-copy yeast
geno-and methods. mic libraries and screened for plasmid-dependent
growth on high-osmolarity media (YEPD plus 900 mm
NaCl). The HOG pathway genes PBS2 and HOG1 both strains (data not shown). Msg5p is a dual-specificity (ty-suppressed the osmosensitivity of the pbs2-3 strain (data rosine and threonine) phosphoprotein phosphatase not shown). Three additional genes were also identified that has been implicated previously in the downregula-as high-copy extragenic suppressors of pbs2-3: CAK1, tion of the pheromone response pathway and cell
integ-TRX1, and MSG5 (Figure 2A). Cak1p is the cyclin-depen- rity pathway MAPKs, Fus3p, and Slt2p, respectively (Doi dent, kinase-activating kinase in yeast (Espinoza et al. et al. 1994;Watanabeet al. 1995). The suppression of
1996;Kaldiset al. 1996; Thuretet al. 1996). TRX1 is a MAPK cascade mutant by the overexpression of a one of the two thioredoxin genes in yeast (Gan1991). MAPK phosphatase was intriguing, so MSG5 was selected After obtaining TRX1 in the suppressor screen, a high- for further study.
HOG pathway: One possible explanation for the sup-pression of pbs2-3 by high-copy MSG5 is that the overex-pression of Msg5p increases the signaling efficiency of the impaired HOG pathway. However, neither Hog1p tyrosine phosphorylation nor GPD1 mRNA levels are altered by MSG5 overexpression in a pbs2-3 strain (Fig-ure 2, B and C). Additionally, high-copy MSG5 also weakly suppresses the osmosensitivity of a pbs2Dstrain (data not shown). One hypothesis to explain these data is that the activity of one or more Msg5p-regulated MAPK pathways is/are deleterious to growth at high osmolarity.
Kss1p is the relevant target of Msg5p in the suppres-sion ofpbs2-3osmosensitivity:Msg5p is a phosphopro-tein phosphatase that catalyzes the dephosphorylation
Figure3.—Suppression of pbs2-3 osmosensitivity by dele-of tyrosine and threonine residues dele-of a subset dele-of
acti-tion of KSS1. The indicated strains were grown and spotted vated MAPKs, shifting them to a catalytically inactive
on YEPD supplemented with the indicated osmolytes, as de-state. Previous work identified Msg5p as a negative
regu-scribed inmaterials and methods. The plates were incu-lator of Fus3p and Slt2p, MAPKs in the pheromone bated at 308for 2 days (YEPD) or 5 days (YEPD1osmolyte). response and cell integrity pathways, respectively, but
not as a negative regulator of the MAPK Hog1p (Doi
et al. 1994; Watanabe et al. 1995). Interestingly, both fus3Dstrains have significantly higher levels of induction of the filamentation/invasion pathway reporter gene Slt2p and Fus3p have a threonine-glutamate-tyrosine
(TEY) activation sequence similar to the mammalian FRE::lacZ, and they show enhanced invasion. Thus, the
enhancement of pbs2-3 osmosensitivity by fus3Dcould Erk1p (Robinson and Cobb 1997), while Hog1p has
a threonine-glycine-tyrosine (TGY) activation sequence be due to either the loss of a pheromone response pathway function or an increased activity of the fila-similar to mammalian stress-activated protein kinases
such as p38 (KyriakisandAvruch1996). Of the two mentation/invasion pathway. This latter possibility is further supported by the following results.
other MAPKs in yeast, Kss1p also has a TEY activation
sequence (Courchesneet al. 1989) and is a possible In contrast to the results with slt2Dand fus3D, deletion of KSS1 suppressed the osmosensitivity of a pbs2-3 strain target of Msg5p. Smk1p, however, has a
threonine-gluta-mine-tyrosine (TNY) activation sequence (Krisaket al. on media containing 1m sorbitol or 1m KCl (Figure 3). Although interaction between Msg5p and Kss1p had 1994), is expressed only during sporulation (Pierceet
al. 1998), and is therefore an unlikely candidate. To not been described previously, the growth of the pbs2-3 strain lacking KSS1 on high-osmolarity media suggests identify which of the MAPKs is the relevant target of
Msg5p with regard to the suppression of pbs2-3 osmosen- that Kss1p is a target of Msg5p. This assertion is sup-ported by the observation that there is no additional sitivity, deletions of FUS3, SLT2, and KSS1 were
con-structed in pbs2-3 and PBS2 backgrounds and tested for suppression of osmosensitivity by overexpressing MSG5 in a pbs2-3 kss1D strain (data not shown). These data growth at high osmolarity.
pbs2-3 strains lacking SLT2 did not grow noticeably indicate that activated Kss1p and, by extension, activated filamentation/invasion pathway may be deleterious for different than pbs2-3 alone on high-osmolarity media
(Figure 3). In addition, the osmosensitivity of a pbs2-3 growth at high-osmolarity media in a strain with a defec-tive HOG pathway.
slt2Dstrain is still strongly suppressed by MSG5
overex-pression (data not shown). These data suggest that Slt2p Double and triple MAPK deletions were also made in pbs2-3 in an effort to unmask additional interactions is not part of the putative MAPK pathway predicted to
be downregulated by Msg5p as part of the suppression between the various pathways analyzed. A consistent pattern of suppression by strains lacking KSS1 and sensi-of pbs2-3 osmosensitivity.
pbs2-3 strains lacking FUS3 grew very poorly on the tivity in strains with a KSS1 was observed regardless of the other deletions in the pbs2-3 strain. These data again high-osmolarity media tested, though growth on YEPD
alone was unaffected. This indicates that Fus3p is not support a model where Kss1p is the only relevant MAPK downregulated by Msg5p in the suppression of pbs2-3 the critical target of Msg5p in the suppression of pbs2-3
osmosensitivity. Surprisingly, the deletion of FUS3 not osmosensitivity.
It has been demonstrated recently that Kss1p in its only failed to suppress the osmosensitivity of a pbs2-3
strain, it enhanced the osmosensitivity of this stain rela- nonphosphorylated (nonactivated) state is an inhibitor of the filamentation/invasion pathway (Cooket al. 1997;
tive to pbs2-3 alone (Figure 3). Fus3p mediates mating
responses (Elionet al. 1991;Gartneret al. 1992), and Madhani et al. 1997; Bardwell et al. 1998). When a
Kss1p mutant lacking the threonine and tyrosine phos-it appears to negatively regulate the
was expressed in a pbs2-3 strain, the suppression of the tion/invasion pathway are activated in response to hy-pertonic stress inpbs2-3mutants: The increased
phos-pbs2-3 osmosensitivity was even stronger than that seen
in a pbs2-3 kss1Dstrain (data not shown). This further phorylation of Kss1p observed in a pbs2-3 strain suggests the possibility that the filamentation/invasion pathway supports the assertion that activated Kss1p contributes
to the growth defect of a pbs2-3 strain on high osmolarity. is activated under hyperosmotic conditions. We further investigated this possibility by measuring the induction Kss1p is phosphorylated in response to osmotic shock
in the absence of a functional HOG pathway:If Kss1p of downstream gene targets of the sion pathway. Normally, when the filamentation/inva-dephosphorylation by overexpressed Msg5p suppresses
the osmosensitivity of a pbs2-3 strain, then the level of sion pathway phosphorylates Kss1p, Kss1p activates the transcription factors Ste12p and Tec1p, which in turn Kss1p phosphorylation may be an important
determi-nant for growth at high osmolarity in HOG pathway activate transcription from the filamentation/invasion response elements (FREs) present in the promoters of mutants. To investigate the phosphorylation state of
Kss1p in pbs2-3 strains exposed to high osmolarity, im- genes required for filamentous growth (Madhaniand Fink1997).
munoblots of proteins extracted from various strains
before and after hyperosmotic shock were probed with Two methods have been used to analyze the transcrip-tion of filamentatranscrip-tion/invasion-pathway-responsive genes a commercially available monoclonal antibody raised
against a phosphorylated ERK1 activation loop. ERK1 under a variety of conditions. The promoter for TEC1 has a FRE, providing a positive feedback loop for the is a mammalian MAPK with the same TEY activation
sequence as the yeast MAPKs Kss1p, Fus3p, and Slt2p. filamentation/invasion pathway and making the accu-mulation of TEC1 mRNA a sensitive, endogenous indica-Due to the sequence similarity in this region, the
anti-phosphoERK antibody also recognizes the phosphory- tor of pathway activation (Madhani and Fink 1997). However, a recent publication has indicated that TEC1 is lated forms of Kss1p, Fus3p, and Slt2p, albeit with
differ-ent sensitivities (data not shown). not under the sole control of the filamentation/invasion pathway, as it is also activated by the pheromone re-An antiphosphoERK immunoreactive band at the
ex-pected mobility for Kss1p (z43 kD) appears in the lane sponse pathway (Oehlen1998), a result independently confirmed by our lab (data not shown). An alternative corresponding to pbs2-3 cells 1 hr following hypertonic
shock, but not in wild-type cells nor in cells overexpress- assay for filamentous pathway activation is the use of a
FRE::lacZ construct (Madhani and Fink 1997). This ing MSG5 under the same conditions (Figure 4A). This
43-kD band is absent in extracts from cells lacking a KSS1 construct contains ab-galactosidase gene under the con-trol of the filamentous pathway responsive elements. gene and is much darker in the lane corresponding to
cells overexpressing KSS1 than in control cells (Figure This construct appears to be very specific for filamenta-tion/invasion pathway signals (Madhani and Fink 4B). In contrast, there was no evidence of increased
phosphorylation of Fus3p or Mpk1p following osmotic 1997). We have analyzed both TEC1 and FRE::lacZ ex-pression to assay the activation of the filamentation/ stress (Figure 4, A and B). This evidence strongly
sug-gests that Kss1p at its normal physiological concentration invasion pathway transcriptional targets.
Wild-type cells had a low basal level of TEC1 mRNA within the cell is phosphorylated in pbs2-3 cells following
osmotic stress. that did not increase appreciably following hyperos-motic shock (Figure 5A). In a pbs2-3 strain, there was a Downstream transcriptional targets of the
high basal level of TEC1 expression (z1.6-fold over wild pathway mutants following osmotic shock, deletions of the upstream components of the filamentation/inva-type). Exposure of the pbs2-3 strain to hyperosmotic
stress induced a two- to threefold increase in the TEC1 sion pathway were constructed in the pbs2-3 back-ground. These strains were assayed for growth (Figure mRNA expression over the elevated basal levels and
remained high. In contrast, pbs2-3 strains overexpress- 6A), FRE::lacZ-dependent b-galactosidase activity (Fig-ure 6B), and expression of TEC1 (data not shown) fol-ing MSG5 had a slightly elevated basal level of TEC1
mRNA (z1.3-fold over wild type) that increased moder- lowing osmotic stress.
The deletion of filamentation/invasion pathway MAPK ately (1.4-fold over basal levels) 1 hr after hyperosmotic
shock and decreased steadily thereafter. cascade components (STE11, STE7, or KSS1) suppresses the osmosensitivity of a pbs2-3 strain (Figure 6A). The Similar results were obtained using an FRE::lacZ
re-porter construct to assay filamentation/invasion path- extent of suppression by these is roughly equivalent to that seen in a pbs2-3 strain overexpressing MSG5 (Figure way activity (Figure 5B). There is an increased basal
level of FRE::lacZ expression in a pbs2-3 strain (1.6-fold) 6A). Interestingly, the deletion of STE7 has a greater suppressive effect on a pbs2-3 strain than the deletion compared to a wild-type strain. In the pbs2-3 strain, a
two-to threefold increase in FRE::lacZ was observed following of either KSS1 or STE11.
The deletions of filamentation/invasion pathway osmotic stress, while there is a modest 1.6-fold increase
in the wild-type strain with the same treatment. These MAPK signaling cascade components also inhibit the expression of FRE::lacZ activity in pbs2-3 cells (Figure data suggest that the
filamentation/invasion-pathway-responsive genes are activated following osmotic stress 6B). The basal levels of FRE::lacZ expression are consid-erably diminished in pbs2-3 strains lacking the upstream in a pbs3-2 strain.
The requirement for KSS1 in the activation of kinases of the filamentation/invasion pathway. Also, strains lacking a functional filamentation/invasion
FRE::lacZ was investigated. Interestingly, the deletion
of KSS1 results in a reduced basal level of FRE::lacZ pathway MAPK cascade no longer have an increase in
FRE::lacZ-dependentb-galactosidase expression follow-expression in the pbs2-3 strain back to the level observed
in wild type. This low basal level does not increase follow- ing osmotic stress. Similar results were obtained when assaying for TEC1 expression following hypertonic stress ing osmotic stress, consistent with the requirement for
Kss1p in the osmotic induction of the filamentation/ in pbs2-3 strains deleted for filamentation/invasion pathway MAPK cascade components (data not shown). invasion pathway in strains lacking a functional HOG
pathway. These data suggest that the transcriptional tar- The loss of filamentous-pathway-responsive gene induc-tion following osmotic stress in strains with deleinduc-tions in gets of the filamentation/invasion pathway are activated
in response to osmotic stress in strains lacking a fully STE11, STE7, or KSS1 indicates that the entire MAPK
module must be present for efficient activation of the functional HOG pathway in a Kss1p-dependent manner.
Ste7p and Ste11p are required for growth inhibition filamentation/invasion pathway in strains lacking a fully functional HOG pathway.
and transcriptional activation of
filamentation/invasion-pathway-responsive genes:To determine if other com- Deletions of filamentation/invasion pathway compo-nents suppress the morphological defects ofpbs2-3on ponents of the filamentation/invasion pathway are
re-quired for the inappropriate activation of Kss1p in HOG high osmolarity:HOG pathway mutants exhibit severe
Figure6.—Upstream components of the filamentation/invasion pathway are required for the inhibition of growth on high-osmolarity and FRE::lacZ expression by high high-osmolarity in a pbs2-3 strain. (A) Strains deleted for various components of the filamentation/invasion pathway were grown in YEPD and spotted to plates supplemented with various solutes, as described in materials and methods.(B) Protein was isolated from the indicated strains containing YEpL-FTyZ (Cooket al. 1997) before (0) or 3 hr following (3) exposure to 1msorbitol. The protein was then assayed for b-galactosidase activity, as described in materials and methods.
morphological defects following prolonged exposure tion of the filamentation/invasion pathway by hyperos-motic shock. One high-copy suppressor of the HOG to high-osmolarity conditions (BrewsterandGustin
1994). One such defect is the presence of multiple buds, pathway MEK mutant pbs2-3 identified in this study was
MSG5 (Figure 2A), a MAPK phosphatase. The critical
a possible indication of a cell cycle defect in HOG
path-way mutants on high osmolarity. A second form of aber- target of Msg5p in the suppression of pbs2-3 osmosensi-tivity, Kss1p, was identified on the basis of suppression rant morphology is the production of long projections
reminiscent of the hyphae of pathogenic yeast during of pbs2-3 by kss1D (Figure 3) and the lack of effect of high-copy MSG5 in a pbs2-3 kss1D strain (data not virulent growth phase (Loet al. 1997) and the
pseudo-hyphae of S. cerevisiae (Gimeno et al. 1992). To deter- shown). In strains lacking a functional HOG pathway, Kss1p is phosphorylated (Figure 4) and filamentation/ mine if these morphological defects are dependent on
the filamentation/invasion pathway, wild-type, pbs2-3, invasion pathway gene targets are transcriptionally acti-vated (Figure 5) following osmotic stress. The deletion and pbs2-3 strains deleted for STE7 and KSS1 were grown
in YEPD or YEPD supplemented with 1mKCl and ana- of the filamentation/invasion pathway MAPK module components STE7 or STE11 inhibited hyperosmolarity-lyzed by DIC microscopy (Figure 7).
pbs2-3 strains exposed to high osmolarity produce induced filamentation/invasion pathway activation (Figure 6B) and restored high-osmolarity growth in long projections (Figure 7) similar to those observed in
hog1Dand pbs2Dstrains on high osmolarity (Brewster pbs2-3 strains (Figure 6A). Finally, one of the
morpho-logical defects of HOG pathway mutants on high
osmo-et al. 1993). The projections frequently have striations
that are strikingly similar to those produced by the in- larity, the presence of long projections, was demon-strated to be dependent on the presence of an intact complete septation of pseudohyphae (Gimeno et al.
1992). The long projections were not seen in pbs2-3 filamentation/invasion pathway (Figure 7).
The HOG pathway negatively regulates the filamenta-strains lacking KSS1 or STE7. However, several cells with
multiple buds were observed in pbs2-3 strains lacking tion/invasion pathway: Our data demonstrate that the filamentation/invasion pathway is activated in response
KSS1 or STE7 on high osmolarity (white arrows in Figure
7). These data indicate that the aberrant polarized to osmotic stress in strains with a compromised HOG pathway, but that it is not activated in wild-type strains. growth morphology of HOG pathway mutants requires
the filamentation/invasion pathway even though the Kss1p phosphorylation (Figure 4), TEC1 mRNA levels (Figure 5A), and FRE::lacZ expression (Figure 5B) are multiple-bud phenotype does not.
all increased following osmotic stress in strains lacking a fully functional HOG pathway. In strains with an intact DISCUSSION
HOG pathway, there is no detectable change in Kss1p phosphorylation (Figure 4) or TEC1 mRNA (Figure 5A) In this article, we describe a previously unknown
Other results are consistent with a model in which the HOG pathway negatively regulates the filamentation/ invasion pathway. Diploid hog1D/hog1D yeast of theR
strain background show more active pseudohyphal de-velopment on low-nitrogen media than does a wild-type
Rstrain, indicating an increased filamentation/invasion pathway activity (Madhaniet al. 1997;O’Rourkeand Herskowitz1998). Our data may provide a biochemi-cal explanation for the hyperpseudohyphal phenotype of hog1D cells, as we have demonstrated that the basal filamentation/invasion pathway activity, as measured by
TEC1 mRNA and FRE::lacZ activity, is increased in HOG
pathway mutants. We observed that the elevated basal levels of TEC1 and FRE::lacZ could be eliminated by the deletion of the components of the filamentation/ invasion pathway MAPK cascade. Together, these data suggest that it is the loss of filamentation/invasion path-way regulation by the HOG pathpath-way that causes the hyperpseudohyphal phenotype of HOG pathway mu-tants in theRbackground.
The activation of the filamentation/invasion pathway is correlated with growth inhibition on high-osmolarity media: The activation of the filamentation/invasion pathway has a physiological effect on yeast—growth inhi-bition on high osmolarity. The level of FRE-dependent transcription and the amount of pbs2-3 strain growth on high-osmolarity media appear to be inversely correlated with each other. pbs2-3 strains have increased FRE::lacZ and TEC1 expression, and they have a growth defect on high-osmolarity media (Figures 5, B and A, and 2A, respectively). A pbs2-3 ste7D strain has a much lower level of FRE::lacZ expression than does a pbs2-3 strain, and it grows well on high-osmolarity media (Figures 6, B and A, respectively). The growth inhibition of a pbs2-3
kss1D strain is intermediate between that of a pbs2-3 Figure 7.—The formation of long projections by pbs2-3 strain and a pbs2-3 ste7Dstrain (Figure 6A), correlated strains on high osmolarity is prevented by deletion of fila- with the intermediate levels of TEC1 mRNA accumula-mentation/invasion pathway genes. The indicated strains were tion (data not shown) and FRE::lacZ expression (Figure grown to log phase, and samples were removed before
(con-6B). These data suggest that an activated filamentation/ trol) or after continued growth in 1mKCl for 12 hr. Cells were
invasion pathway contributes to the decreased growth fixed in 3.7% formaldehyde and mounted for microscopic
analysis, as described inmaterials and methods.Cells were of a pbs2-3 strain on high-osmolarity media.
visualized by DIC microscopy using a Zeiss Axioskop fitted A pbs2-3 strain with a deletion in STE11 does not seem with a digital camera. White arrows indicate the presence of to fit such a model, though the complex roles of Ste11p a second bud.
may explain the discrepancy. A pbs2-3 ste11Dstrain and a pbs2-3 ste7Dstrain have equally low levels of TEC1 (data not shown) and FRE::lacZ expression following osmotic small increase in FRE::lacZ expression could be detected
in wild-type strains following osmotic stress, the magni- stress (Figure 6B). However, the deletion of STE11 does not suppress the osmosensitivity of pbs2-3 as well as the tude of the increase was lower than in HOG mutants
(Figure 5B). These data are consistent with either the deletion of STE7 does (Figure 6A). The loss of Ste11p results in both a loss of filamentation/invasion pathway inappropriate activation or the loss of repression of the
filamentation/invasion pathway in HOG mutants dur- activation (increased osmotolerance) and the loss of one upstream branch of the HOG pathway (possibly ing osmotic stress. However, the increased basal levels
of TEC1 mRNA (Figure 5A) and FRE::lacZ expression decreased osmotolerance). Though the loss of the Sho1p branch of the HOG pathway did not affect high-(Figure 5B) in pbs2-3, hog1D, or pbs2Dstrains are more
consistent with the latter model. Together, these data osmolarity growth in an otherwise wild-type strain (PosasandSaito1997), the increased sensitivity of the support a role for the HOG pathway in negatively
for the reduced growth on high-osmolarity in a ste11D example, if cells encounter both increased osmolarity and nutrient loss, it may be more advantageous for the strain relative to a ste7Dstrain.
The correlation of filamentation/invasion pathway yeast cell to move the yeast (or its progeny) to an envi-ronment more conducive to growth via filamentation, activity and decreased growth on high osmolarity is
found not only in pbs2-3 cells, but also in other HOG e.g., the inside of a grape vs. the skin. This response
could be selected at the level of intracellular signaling pathway mutants. On high-osmolarity media, a pbs2D
strain has higher levels of TEC1 mRNA and FRE::lacZ if the filamentation/invasion pathway activation by both osmolarity and nutrient deprivation overcomes the expression than does a pbs2-3 strain under the same
conditions (data not shown). The higher activity of the HOG-pathway-mediated repression. However, if the cell is simply being dehydrated but has a nutrient-rich envi-filamentation/invasion pathway in pbs2D relative to
pbs2-3 is correlated with greater growth inhibition of ronment, the HOG pathway repression of the fila-mentation pathway will prevent the expenditure of
cel-pbs2Drelative to pbs2-3 (Figure 2A). Likewise, a hog1D
strain has a greater accumulation of TEC1 mRNA follow- lular energy on filamentation and focus its energy on overcoming the osmotic challenge.
ing hyperosmotic stress than does a pbs2-3 strain, and
it is also less osmotolerant than a pbs2-3 strain (data not Cross-talk between MAPK cascades in yeast:One pos-sible mechanism for cross-pathway regulation is the acti-shown).
The deletion of filamentation/invasion pathway vation by one MAPK pathway of a phosphatase with a specificity for components of a second MAPK pathway. genes failed to fully complement a pbs2-3 strain (Figure
6A) or cells with a deletion of either HOG1 or PBS2 (data In HeLa cells, the MAPK ERK2 induces the expression of the tyrosine/threonine phosphatase MKP1-1, which not shown). These data indicate that the deactivation
of the filamentation/invasion pathway is not the sole is more active against the SAPK and p38 than ERK2 itself (FranklinandKraft1997). Thus, a mechanism determinant of growth on high-osmolarity media. This
is not unexpected, as previous work demonstrated the exists for the downregulation of p38 and SAPK by ERK2 through the transcription of a phosphatase. MAPKs may importance of GPD1 expression in the growth of yeast
on high-osmolarity media (Albertynet al. 1994). The also directly activate MAPK phosphatases, as is the case with ERK2 and the phosphatase MPK3-1 (Camps et al.
expression of GPD1 in a pbs2-3 strain is not altered by
the overexpression of MSG5 (Figure 2C) nor by the 1998). However, it is not yet known if the direct activa-tion of phosphatases by MAPKs is part of the cross-deletion of filamentation/invasion pathway genes (data
not shown). These data indicate that the HOG pathway pathway regulation mechanisms.
There are a number of phosphatases in yeast that coordinates responses to osmotic stress that affect
growth on high-osmolarity media independently of the could act in an analogous manner to the mammalian phosphatases described above. Ptp2p is transcriptionally repression of the filamentation/invasion pathway.
Mechanism of filamentation/invasion pathway activa- regulated by the HOG pathway (Jacoby et al. 1997;
Wurgler-Murphyet al. 1997), but is more specific for
tion after osmotic stress:The research presented here
demonstrates that the filamentation/invasion pathway Hog1p than for the ERK-like MAPKs (Zhanet al. 1997).
Ptp3p is perhaps a more likely candidate, as it is activated is activated following hypertonic stress in HOG pathway
mutants. It is perhaps significant that two pathways share by the HOG pathway (Jacoby et al. 1997; Wurgler-Murphyet al. 1997) and has a higher specificity for the
a MEKK, Ste11p. It is therefore possible that the
activa-tion of the filamentaactiva-tion/invasion pathway following ERK-like MAPKs than for Hog1p (Zhan et al. 1997).
However, neither the overexpression of Ptp2p nor hypertonic stress occurs because of a leakage of signal
through this common component. It is unclear whether Ptp3p suppressed the osmosensitivity of a pbs2-3 strain (data not shown), though a dependence on a wild-type Ste11p freely diffuses between pathways or is bound
tightly in a signaling complex within the individual path- HOG pathway for full phosphatase activity could ac-count for the lack of suppression. Msg5p is specific for ways. In the case of the pheromone response pathway,
it appears that Ste11p is tightly bound to the scaffold the ERK family of MAPKs, including Slt2p (Watanabe
et al. 1995), Fus3p (Doi et al. 1994), and Kss1p (this
protein Ste5p. Though a role for Pbs2p as a similar
scaffold has been suggested (Posas andSaito 1997), study) in yeast, and suppresses pbs2-3 osmosensitivity when overexpressed. However, the transcription of it is unclear how tightly the signaling components of
the HOG pathway are bound. Thus, the activation of the Msg5p is not Hog1p dependent (data not shown), though post-transcriptional activation of Msg5p by the filamentation/invasion pathway may be an unfortunate
consequence of the sharing of Ste11p between the two HOG pathway is still possible.
MAPK phosphatase specificity: MSG5 had been iden-pathways.
Alternatively, the simultaneous activation of the fila- tified previously as a high-copy suppressor of constitu-tively active mutants in pheromone response (Doiet al.
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pro-and division after osmotic stress requires a MAP kinase pathway.
vided evidence that is consistent with the hypothesis
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1994 Msg5p, a novel protein phosphatase, promotes adaptation Gibson for their critique of this manuscript. Research was supported by
to pheromone response in S. cerevisiae. EMBO J. 13: 61–70. the National Science Foundation (grant MCB 9506987), the American
Elion, E. A., P. L. Grisafi andG. R. Fink,1990 FUS3 encodes
Cancer Society (grant BE-224), and the National Aeronautics and
a cdc21/CDC28-related kinase required for the transition from Space Administration (grant NAGS-4072). K.D.D. was supported by
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