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Response of the Saccharomyces cerevisiae Mpk1 Mitogen-Activated Protein Kinase Pathway to Increases in Internal Turgor Pressure Caused by Loss of Ppz Protein Phosphatases

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Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Response of the

Saccharomyces cerevisiae

Mpk1 Mitogen-Activated

Protein Kinase Pathway to Increases in Internal Turgor Pressure

Caused by Loss of Ppz Protein Phosphatases

Stephanie Merchan,

1

Dolores Bernal,

2

Ramo´n Serrano,

1

and Lynne Yenush

1

*

Instituto de Biología Molecular y Celular de Plantas, Universidad Politecnica de Valencia—CSIC, Valencia,1and

Departament de Bioquímica i Biología Molecular, Facultat de Biología, Universitat de Valencia, Burjassot,2Spain

Received 25 September 2003/Accepted 24 October 2003

The Mpk1 pathway ofSaccharomyces cerevisiae is a key determinant of cell wall integrity. A genetic link between the Mpk1 kinase and the Ppz phosphatases has been reported, but the nature of this connection was unclear. Recently, the Ppz phosphatases were shown to be regulators of Kand pH homeostasis. Here, we

demonstrate that Ppz-deficient strains display increased steady-state Klevels and sensitivity to increased

KCl concentrations. Given these observations and the fact that Kis the major determinant of intracellular

turgor pressure, we reasoned that the connection betweenPPZ1and -2andMPK1was due to the combination of increased internal turgor pressure in Ppz-deficient strains and cell wall instability observed in strains lackingMPK1. Accordingly, theMPK1gene was up-regulated, the Mpk1 protein was overexpressed, and the phosphorylated active form was more abundant in Ppz-deficient strains. Moreover, the expression of genes previously identified as targets of the Mpk1 pathway are also up-regulated in strains lackingPPZ1and -2. The transcriptional and posttranslational modifications of Mpk1 were not observed when the internal K

concen-tration (and thus turgor pressure) was lowered by disrupting theTRK1and -2Ktransporter genes or when

the cell wall was stabilized by the addition of sorbitol. Moreover, we present genetic evidence showing that both the Wsc1 and Mid2 branches of the Mpk1 pathway contribute to this response. Finally, despite its role in G1/S

transition, increased levels of activated Mpk1 do not appear to be responsible for the cell cycle phenotype observed in Ppz-deficient strains.

The cell wall is an essential and dynamic organelle of the budding yeast Saccharomyces cerevisiae that is required to maintain cell integrity during vegetative growth, mating, and stress conditions. Several lines of evidence have established an important role for the signal transduction cascade that begins with the Rho1 GTPase, which then activates protein kinase C (Pkc1), and culminates in the activation of a canonical mito-gen-activated protein (MAP) kinase cascade, referred to as the Mpk1 pathway (recently reviewed in references 7 and 8). Phos-phorylation of the Mpk1 MAP kinase (also known as Slt2) has several consequences in the cell, including activation of the serum response factor-like transcription factor Rlm1, respon-sible for regulating the expression of a myriad of genes impli-cated in cell wall biosynthesis (11, 27). Several environmental stimuli are known to induce Mpk1-mediated cell wall integrity signaling, such as elevated temperatures, hypo-osmotic shock, and exposure to mating pheromone (3, 12, 19, 30). Two trans-membrane proteins, Wsc1 and Mid2, have been identified as the major sensors of cell wall stress. Wsc1 (also known as Hcs77 and Slg1) has been proposed to sense cell wall stress at the plasma membrane during vegetative growth (6, 9, 24, 26). Mid2, on the other hand, while also located at the plasma membrane, appears to function primarily during pheromone-induced morphogenesis. Although null mutations of either gene have mild effects on growth in normal media, the

disrup-tion of both genes causes a severe lytic phenotype, suggesting that the functions of these genes are partially redundant during vegetative growth (14, 24).

A genetic link between the Mpk1 pathway and the type 1-like Ppz protein phosphatases has been established. Specif-ically, thePPZ1andPPZ2genes act as multicopy suppressors of the temperature sensitivity ofmpk1orpkc1strains (15). In addition, similar lytic phenotypes and caffeine sensitivity have been observed for strains lacking either components of the Mpk1 pathway or thePPZ1gene (15, 23). Moreover, growth of a mutant strain lackingMPK1, PPZ1, andPPZ2requires the addition of an osmostabilizer, such as 1 M sorbitol (15). Al-though these observations were made some time ago, the con-nection between these genes has remained unclear. Recently, it was reported that the Ppz phosphatases play an important role in the maintenance of K⫹and pH homeostasis and that

regulation of these physiological parameters has important consequences for the determination of membrane potential, cell cycle progression, and cell wall integrity (28).

Both the Mpk1 pathway and the Ppz phosphatases are known to have effects on G1cell cycle progression. Specifically,

Mpk1 is activated not only during cell wall stress but also during the G1/S transition of the cell cycle and in response to

mating pheromone (3, 12, 19, 30). In addition, Mpk1 has been shown to physically associate with and phosphorylate compo-nents of the SBF transcription factor, which plays a vital role in the yeast cell cycle (16). Moreover, the function of the up-stream activator of the Mpk1 pathway, Pkc1, is partially de-pendent on the presence of the yeast cyclin-dede-pendent kinase, Cdc28, and overexpression of thePKC1 gene can rescue the

* Corresponding author. Mailing address: Instituto de Biología Mo-lecular y Celular de Plantas, Universidad Politecnica de Valencia— CSIC, Camino de Vera s/n, 46022 Valencia, Spain. Phone: 34 96 387 7860. Fax: 34 96 387 7859. E-mail: lynne@ibmcp.upv.es.

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bud emergence defect of a mutant lackingswi4, a gene encod-ing a component of the SBF transcription factor (6, 17). There-fore, the Mpk1 pathway is required for normal progression through the G1/S transition of the cell cycle. On the other

hand, the Ppz phosphatases appear to negatively affect the G1

phase of the cell cycle. Overexpression of the PPZ1 gene causes a pronounced slow growth defect and accumulation of cells at G1(2). Conversely, strains lacking Ppz activity recover

faster from␣-factor-induced G1cell cycle arrest (4, 28).

How-ever, the mechanism by which Ppz activity affects cell cycle progression has yet to be identified.

In addition to the observed effects on cell cycle progression, strains deficient in Ppz activity display sensitivity to agents that are thought to destabilize the cell wall, such as caffeine and Calcofluor white (23, 28). The sensitivity of Ppz-deficient strains to these agents is relieved by further disruption of the genes encoding the high affinity K⫹transporters encoded by

theTRK1andTRK2genes. Thus, it appeared that a connection must exist between the alterations in K⫹homeostasis and cell

wall stability observed in these strains. Since K⫹is the major

determinant of turgor pressure in living cells, we reasoned that if strains lacking Ppz activity display a relative accumulation of K⫹ under steady-state conditions, then the internal turgor

pressure of these strains would also be increased. This increase in turgor pressure would represent a constant stress on the cell walls of these strains. Therefore, constitutive reinforcement of the cell wall, presumably mediated by the Mpk1 pathway, would be required in these cells. Here we present several lines of evidence that support the proposed hypothesis.

MATERIALS AND METHODS

Yeast strains and culture conditions.All strains ofS. cerevisiaeused in this work are listed in Table 1. TheWSC1gene was deleted by cloning genomic fragments corresponding to bp⫺200 to⫹100 (relative to ATG) and bp 1 to 424 (after the stop) by PCR, digesting with restriction enzymes specific for sites in the primers, and inserting them into the pJJ252 vector (10), flanking theLEU2open reading frame. For theMID2deletion, an identical approach was followed except that genomic PCR fragments corresponding to bp⫺496 to⫺120 (before ATG)

and bp 29 to 470 (after the stop) of theMID2gene were used. Correct integration of the disruption cassettes was confirmed by genomic PCR using a 5⬘primer upstream of the disruption cassette and a 3⬘primer corresponding to the gene used for selection. Complete media contained 2% glucose, 2% peptone, and 1% yeast extract (YPD). Where indicated, YPD was supplemented with the indi-cated concentrations of sorbitol or KCl. Synthetic medium (SD) contained 2% glucose, 0.7% yeast nitrogen base (Difco) without amino acids, 50 mM MES [2-(N-morpholino)ethanesulfonic acid] adjusted to pH 5.5 with Tris, and the amino acids and purine and pyrimidine bases required by the strains.

Kmeasurements.Internal [K] was determined by high-performance liquid

chromatography (HPLC) analysis as described previously (28).

Cell size measurements.Cell size was analyzed in exponentially growing cells (after brief sonication) in the Particle Count and Size Analyzer, model Z2 (Coulter Inc.).

Western blot analysis.The indicated strains were grown to mid-log phase in YPD supplemented or not with 1 M sorbitol. A total of 107cells were pelleted

by centrifugation and then frozen at⫺70°C. Cell pellets were resuspended in loading buffer, separated on a sodium dodecyl sulfate–8% polyacrylamide gel electrophoresis gel, transferred to a nitrocellulose membrane, and immunoblot-ted with either an anti-Mpk1 antiserum (1:1,000; a kind gift from M. Molina) or an anti-phospho-p44/42 MAP kinase monoclonal antibody (1:5,000; Cell Signal-ing Technology, Inc.). Anti-Mpk1 blots were visualized usSignal-ing the ECL detection system (Amersham), and anti-phospho-p44/42 MAP kinase blots were visualized using alkaline phosphatase.

Northern blot analysis.Total RNA was isolated from yeast cells that were grown to mid-log-phase in a rich medium supplemented or not with 1 M sorbitol. Approximately 20␮g of RNA per lane was separated in formaldehyde gels and transferred to nylon membranes (Hybond-N; Amersham). Radioactively labeled probes were hybridized in PSE buffer (300 mM sodium phosphate [pH 7.2], 7% sodium dodecyl sulfate, 1 mM EDTA). PCR fragments derived from yeast chromosomal DNA representing nucleotides 1 to 990 ofMPK1, 1 to 1016 of

SED1, 1 to 977 ofPIR3, and 77 to 706 ofTBP1were used as probes. Pheromone response and recovery.The indicated strains were grown in YPD medium to mid-log phase, and equal numbers of cells were added to top agar (YPD with 0.7% agar) and spread evenly on YPD plates. Immediately after solidification, sterile cellulose disks (diameter, 0.6 cm; Difco) with 14␮g of synthetic␣-factor (Sigma) were placed on the nascent lawn. YPD medium, top agar, and plates were supplemented with the indicated amount of sorbitol. Images of plates were taken after 48 h of incubation at 28°C.

RESULTS

Strains lackingPPZ1and -2show alterations in steady-state [K], are sensitive to high KCl concentrations, and have

in-creased cell sizes.Various lines of evidence suggested TRK1

and -2-dependent alterations in the K⫹homeostasis of strains

lacking thePPZ1 and PPZ2genes (28). Accordingly, we de-tected increases in the steady-state K⫹ levels of ppz1 ppz2

mutants grown in both rich and minimal media compared to levels in the isogenic wild-type strain (Fig. 1A). In addition, further disruption of theTRK1and -2genes in theppz1 ppz2

background significantly lowered the internal K⫹

concentra-tion to below wild-type levels. The slight differences observed between theppz1 ppz2 trk1 trk2strains and thetrk1 trk2mutant are not statistically significant, suggesting that the effect of the loss of the phosphatases on K⫹homeostasis requires the

pres-ence of the TRK1 and -2 genes (Fig. 1A). One important prediction of the hypothesis that the Ppz phosphatases are the negative regulators of K⫹uptake is that, without the genes, the

cell would be unable to impede the entrance of excess K⫹. We

observed that strains lacking the PPZ1 and -2 genes were sensitive to increased concentrations of KCl (Fig. 1B). This effect was specific for K⫹, as no effect on cell growth was

observed in the presence of equivalent osmotic stress due to the addition of sorbitol or NaCl (Fig. 1B and data not shown). In addition, we observed that strains lackingPPZ1and -2were larger than the control strain, presumably due to increased

TABLE 1. Yeast strains used in this study

Strain Relevant genotype Referenceor source

DBY746 Matura3-52 leu2-3 leu2-112 his3-⌬1 trp1-289 A. Rodriguez-Navarro LY 83 DBY746ppz1::URA3 ppz2::TRP1 22

LY 85 DBY746ena1-4::HIS3 28

LY 102 DBY746ena1-4::HIS3 ppz1::URA3 ppz2::TRP1 28 W303-1A Mataade2-1 can1-100 his3-11,15 leu2-3,112

trp1-1 ura3-1 5

W⌬3 W303-1Atrk1::LEU2 trk2::HIS3 20 LY 140 W303-1Atrk1::LEU2 trk2::HIS3 ppz1::URA3

ppz2::TRP1 28

LY191 W303-1Amid2::LEU2 This study

LY 192 W303-1Awsc1::LEU2 This study

LY190 W303-1Ampk1::LEU2 This study

LY165 W303-1Appz1::TRP1 This study

LY194 W303-1Appz1::TRP1 mid2::LEU2 This study LY195 W303-1Appz1::TRP1 wsc1::LEU2 This study LY193 W303-1Appz1::TRP1 mpk1::LEU2 This study LY139 W303-1Appz1::URA3 ppz2::TRP1 28 LY197 W303-1Appz1::URA3 ppz2::TRP1 mid2::LEU2 This study LY198 W303-1Appz1::URA3 ppz2::TRP1 wsc1::LEU2 This study LY196 W303-1Appz1::URA3 ppz2::TRP1 mpk1::LEU2 This study

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turgor pressure (Fig. 1C). In agreement with our previous studies, both the K⫹sensitivity of theppz1 ppz2strain and the

increase in cell size were relieved by further disruption of the high-affinity K⫹ transporters encoded by the TRK1 and -2

genes. On the other hand, as would be expected, further dis-ruption of the genes encoding the cation extrusion pumps, Ena1p to Ena4p, exacerbated the K⫹sensitivity of the ppz1

ppz2strain (Fig. 1B). Taken together, these data suggest that the absence of Ppz activity results in an inability to regulate the upper limits of K⫹uptake, causing an increase in turgor

pres-sure.

MPK1mRNA, protein, and phosphorylation levels inppz1 ppz2 mutants. Given the results demonstrating that loss of

PPZ1 and 2results in a marked increase in the internal K⫹

concentration and KCl sensitivity, we reasoned that the addi-tive lytic phenotype of appz1 ppz2 mpk1strain may be due to the combination of turgor pressure (caused by PPZ1 and -2

disruption) and a weakened cell wall (observed inmpk1 mu-tants). If this hypothesis is true, then strains lackingPPZ1and -2should display increased activity of the Mpk1 pathway due to the augmented internal turgor pressure. Therefore, we assayed the expression of the MPK1gene, protein, and phosphoryla-tion levels inppz1 ppz2mutant strains. We observed a marked up-regulation of theMPK1mRNA inppz1 ppz2mutants com-pared to levels in the wild-type strain (sixfold induction [Fig. 2A]). Similar results were also observed in the DBY746 back-ground (data not shown). The increase in MPK1 expression

FIG. 1. Analysis of intracellular accumulation of K⫹, KCl toxicity,

and cell size in strains lackingPPZ1and -2. (A) The indicated strains (W303-1A, LY139, LY140, and W⌬3) were grown to mid-log phase in either rich or minimal medium, as indicated, and processed for HPLC analysis of K⫹content as described previously (28). Data are averages

of three separate measurements; error bars, standard deviations. Sim-ilar results were observed in three different experiments. (B) The in-dicated strains (W303-1A, LY139, LY140, W⌬3, DBY746, LY83, LY85, and LY102) were grown to saturation in selective media, serially dilut-ed in sterile water, and spottdilut-ed onto YPD plates containing the indicat-ed amount of KCl or sorbitol. Images were taken after 2 to 3 days of incubation at 28°C. Identical results were obtained in two separate ex-periments. (C) The indicated strains (as in panel A) were grown to early-log phase, and cell size was determined as described in Materials and Methods. Data are averages from three independent colonies of each genotype. Similar results were observed in two separate experiments.

FIG. 2. Northern and Western blot analyses of Mpk1 expression and phosphorylation. (A) The indicated strains (W303-1A, LY139, LY140, W⌬3, and LY190) were grown to mid-log phase in complete media supplemented where indicated with 1 M sorbitol (SORB). Northern blot analysis was performed as described in Materials and Methods by using the indicated probes.TBP1was used as an internal loading control. (B) The same strains described above were grown to mid-log phase in complete media supplemented with the indicated amount of sorbitol, and immunoblots were prepared as described in Materials and Methods. The upper panel represents an immunoblot using an antiserum raised against Mpk1, and the center panel repre-sents an immunoblot with a monoclonal antibody specific for the dually phosphorylated form of Mpk1 (Mpk1*). Similar results were observed in three separate experiments. The Ponceau S staining of the mem-brane is shown in the bottom panel as a control for protein loading.

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was removed by adding an osmotic stabilizer to the external medium (1 M sorbitol) or by lowering the internal K⫹

concen-tration in theppz1 ppz2 mutant by further disruption of the

TRK1and -2genes.

We next examined the steady state levels of the Mpk1 pro-tein in the same strains. In agreement with the results from Northern blot analysis, higher levels of Mpk1 protein were detected in Ppz-deficient strains (an increase of approximately fourfold [Fig. 2B]). As observed at the level of mRNA, this increase in Mpk1 expression was not observed in Ppz-deficient strains lackingTRK1and -2. In the presence of 1 M sorbitol, the expression levels of Mpk1 were decreased in ppz1 ppz2

strains but remained slightly higher than those in the isogenic wild-type strain. Since dual phosphorylation of Mpk1 has been thoroughly shown to correlate with activation of the kinase activity, we used a dual phosphorylation-specific monoclonal antibody to assay the levels of Mpk1 activation (18, 26). We observed that under steady-state conditions, higher levels of phosphorylated Mpk1 were detectable in strains lackingPPZ1

and -2than in the wild type (Fig. 2B, lanes a and c). The Mpk1 phosphorylation was not observed upon addition of an osmotic stabilizer (1 M sorbitol) to the medium to counteract the in-ternal turgor pressure and remove the cell wall stress (Fig. 2B, lanes c and d). As indicated by Fig. 7A below, phosphorylation was not observed in the presence of sorbitol when equal amounts of Mpk1 protein were analyzed. Moreover, in agree-ment with previous results, Mpk1 phosphorylation was not detectable in Ppz-deficient strains lackingTRK1 and -2(Fig. 2B, lane e). These analyses demonstrate that in strains lacking

PPZ1and -2, under steady-state conditions, more Mpk1 pro-tein is present in the cell and increases in the phosphorylated form of Mpk1 are readily detectable, suggesting that this path-way may be more active inppz1 ppz2strains.

Transcriptional regulation of targets of the Mpk1 pathway in strains lacking PPZ1 and -2. Having observed that the protein and phosphorylation levels of Mpk1 are higher in strains lacking Ppz activity, we wanted to use an independent experimental approach to determine whether this pathway is

activated. To this end, we examined the transcriptional regu-lation of some representative targets of the Mpk1 pathway. Based on a previous study that identified many genes regulated by the Mpk1 pathway (11), we chose two representative genes encoding putative cell wall proteins. TheSED1gene encodes a protein of unknown function, but it is predicted to be a glyco-sylphosphatidylinositol (GPI)-modified protein and a compo-nent of the plasma membrane and cell wall (1). ThePIR3gene is a member of the PIR family of cell wall proteins implicated in resistance to the antifungal protein osmotin (29). This class of proteins has been shown to differ from GPI-linked proteins in being attached to the cell wall directly through␤-1,3-glucan, and to date, two members of this family,PIR1andPIR2, have been implicated in tolerance to heat shock (13). We observed increases in the steady-state transcript levels of these genes in strains lackingPPZ1and -2(5- and 3.5-fold induction ofSED1

and PIR3, respectively [Fig. 3]). Moreover, this response was not observed in Ppz-deficient strains lacking TRK1 and -2. These data are in good agreement with those observed at the level of Mpk1 protein expression and phosphorylation, and they provide strong evidence that the Mpk1 pathway is acti-vated in Ppz-deficient strains in a TRK1- and -2-dependent manner.

Exacerbation of lytic phenotypes by exogenous KCl.To es-tablish phenotypic confirmation of the model presented, we examined the growth of various mutants in media supple-mented with KCl. Our hypothesis would contend that the ad-dition of exogenous KCl should exacerbate the lytic phenotype

ofppz1 ppz2 mpk1strains. We clearly observed that the lytic

phenotype of the compound mutant was augmented upon ad-dition of exogenous KCl (Fig. 4). The growth of strains lacking

MPK1is equivalent to that of the wild-type strain in rich media supplemented with 0.8 M KCl. But, as would be predicted, strains lacking MPK1and PPZ1 and -2grow very poorly in media supplemented with KCl. Identical results were observed

inppz1 mpk1strains (data not shown). However, this effect is

not observed when an equivalent osmotic stress, in the form of sorbitol, is added to the medium, demonstrating that the sen-sitivity of these strains is KCl specific.

FIG. 3. Target genes of the Mpk1 pathway are up-regulated in Ppz-deficient strains. The indicated strains (W303-1A, LY139, LY140, W⌬3, and LY190) were grown to mid-log phase in complete media sup-plemented, where indicated, with 1 M sorbitol (SORB). Northern blot analysis was performed as described in Materials and Methods by us-ing the indicated probes.TBP1was used as an internal loading control.

FIG. 4. The KCl sensitivity of theppz1 ppz2strain is exacerbated by the further disruption of MPK1. The indicated strains (W303-1A, LY190, LY139, and LY196) were grown to saturation in YPD plus 1 M sorbitol (SORB.), serially diluted in sterile 1 M sorbitol, and spotted onto YPD plates containing the indicated amount of KCl or sorbitol. Images were taken after 2 to 3 days of incubation at 28°C. Identical results were obtained in two separate experiments. Identical results were also observed in minimal media (data not shown).

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The Wsc1- and Mid2-mediated branches of the Mpk1 path-way contribute to the response to internal turgor pressure.

Recent work from several laboratories has demonstrated that Mpk1 can be stimulated through two upstream pathways, one mediated by Wsc1 and the other by Mid2 (6, 9, 14, 24, 26). The Wsc1 (also known as Hsc77 or Slg1) part of the pathway has been shown to sense cell wall stress during vegetative growth, whereas Mid2 functions principally in signaling cell wall stress during pheromone-induced morphogenesis. Both pathways converge on the Rho1 guanine nucleotide exchange factor, Rom2, and lead to the activation of the Mpk1 pathway via Pkc1 (21). Because we were interested in establishing which of these sensors is responsible for the response to increased internal turgor pressure, we constructed the corresponding strains lack-ingWSC1orMID2alone or in combination with disruption of the PPZ1 and -2 genes. Interestingly, disruption of either

WSC1orMID2in theppz1 ppz2background showed no phe-notypes at 28°C. However, both theppz1 ppz2 wsc1strain and

theppz1 ppz2 mid2strain displayed a severe lytic phenotype at

37°C, which could be rescued by the addition of 1 M sorbitol to the medium (Fig. 5). However, since disruption of bothPPZ1

andPPZ2causes a severe cell lysis defect at 37°C, we went on to construct disruptions ofWSC1andMID2in theppz1 single-mutant background, where the lytic phenotype at 37°C is not as severe. In these strains, we observed that disruption of either

MID2orWSC1exacerbated the lytic phenotype observed for theppz1mutant. Thus, both Wsc1 and Mid2 contribute to the sensing of internal turgor pressure.

The temperature sensitivity ofppz1 ppz2strains is rescued by further disruption ofTRK1and -2.The temperature-sensi-tive phenotype of mutants lackingPPZ1and -2was originally cited as one of the observations supporting the model that

these phosphatases participate in the cell wall integrity path-way. According to our present hypothesis, this Ppz-deficient phenotype may also be explained by increased turgor pressure provoking lysis during cell wall stress caused by high temper-atures. To examine this possibility, we assayed the growth of various mutant strains at both 28 and 37°C. We observed that further disruption of the TRK1 and -2 genes, which would decrease the turgor pressure, relieved the lytic phenotype ob-served for ppz1 ppz2 mutants at 37°C (Fig. 6). These data suggest that cell wall stress caused by increased turgor pressure is responsible for the temperature-dependent lytic phenotype of Ppz-deficient strains.

Strains lackingPPZ1and -2continue to show accelerated recovery from-factor in the presence of sorbitol.The over-expression and disruption ofPPZ1have been shown to have opposite effects on G1/S transition. Specifically, the disruption

of thePPZ genes leads to an accelerated G1/S transition, as

indicated by accelerated recovery from␣-factor arrest, where-as overexpression ofPPZ1causes a G1block (2). In addition,

previous reports have demonstrated that Mpk1 is activated during the G1/S transition and that this activation is important

for bud emergence (19, 30). Therefore, we tested whether the accelerated G1/S transition observed after␣-factor treatment

inppz1 ppz2mutant strains was due to the constitutive

activa-tion of Mpk1. First, we confirmed the lack of Mpk1 phosphor-ylation in Ppz-deficient strains grown in the presence of an osmotic stabilizer (1 M sorbitol). Since Mpk1 is overexpressed in strains lackingPPZ1 and -2grown in rich media, we first empirically determined the amount of extracts required for equal Mpk1 loading in samples grown with or without sorbitol (Fig. 7A, top panel). The levels of phosphorylated Mpk1 were then assayed in the same samples (Fig. 7A, bottom panel). The absence of detectable phosphorylated Mpk1 in the presence of 1 M sorbitol presented in Fig. 2B was confirmed under condi-tions of equal loading of Mpk1 protein. In addition, phosphor-ylated Mpk1 can still be detected in a fivefold dilution of the sample not treated with sorbitol, suggesting that, in the pres-ence of sorbitol, phosphorylation of Mpk1 is less than 20% of that observed without sorbitol (data not shown). Under these conditions, where Mpk1 activation is not detectable (1 M sor-bitol),ppz1 ppz2mutant strains continue to recover faster from

␣-factor treatment (Fig. 7B). Although these results do not completely discard a role for Mpk1 in thisppz1- and -2

-depen-FIG. 5. Disruption of eitherWSC1orMID2exacerbates the lytic phenotype of the ppz1 mutant. The indicated strains (W303-1A, LY191, LY192, LY190, LY165, LY194, LY195, LY193, LY139, LY197, LY198, and LY196) were grown to saturation in YPD medium supplemented with 1 M sorbitol (Sorb.), serially diluted in sterile 1 M sorbitol, and spotted onto YPD plates containing the indicated amount of sorbitol. Images were taken after 2 to 3 days of incubation at 28 or 37°C. Identical results were obtained in two separate experiments.

FIG. 6. The lytic phenotype of theppz1 ppz2mutant is relieved by further disruption ofTRK1and -2. The indicated strains (W303-1A, LY139, LY140, W⌬3, and LY190) were grown to saturation in rich media, serially diluted in sterile water, and spotted onto YPD plates. Images were taken after 2 to 3 days of incubation at 28 or 37°C. Identical results were obtained in two separate experiments.

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dent accelerated␣-factor recovery, it appears that other cell cycle components are largely responsible for thisPPZ -depen-dent phenotype.

DISCUSSION

Elucidating the connection between signal transduction pathways established using genetic approaches is currently an important task in molecular and cellular biology. Some time ago, two lines of evidence established a genetic interaction between a MAP kinase, Mpk1 (Slt2), and the type 1-related phosphatases, Ppz1 and -2. Specifically, PPZ1 and -2 were identified as multicopy suppressors of the temperature sensi-tivity ofmpk1andpkc1mutant strains (15). In addition, strains lacking eitherPPZ1and -2orMPK1display similar tempera-ture-dependent cell lysis defects, and these effects are additive

when all three genes are disrupted (15). These observations were initially interpreted as evidence that both Mpk1 and Ppz phosphatases contributed to a pathway(s) governing the main-tenance of cell wall stability. Here, we demonstrate that the link between these enzymes is not due to changes in the phos-phorylation state of proteins in a common pathway required for cell wall stability. Rather, these enzymes affect very differ-ent but interrelated cellular properties. On the one hand, the Ppz phosphatases regulate K⫹/pH homeostasis within the cell

in a Trk1- and -2-dependent manner (28; this work). This regulation has important implications for ion homeostasis, cell cycle progression, and determination of the internal turgor pressure. On the other hand, the Mpk1 pathway has been shown to up-regulate a number of genes required for cell wall stability in response to cell stresses and during normal cell growth and differentiation (11). The combined effects on tur-gor pressure and cell wall strength explain all of the previously reported connections between these enzymes.

Several lines of evidence support this hypothesis. First, we show that mutants lackingPPZ1and -2accumulate more in-tracellular KCl under both normal growth conditions and NaCl stress (Fig. 1) (28). Ppz-deficient strains are also specifically sensitive to exogenous KCl. Interestingly, we could observe this sensitivity ofppz1 ppz2strains in minimal medium containing only 0.2 M KCl (data not shown). Additionally, we show that these mutants have an augmented cell size. This increase in cell size is observed in all stages of the cell cycle and does not therefore reflect an accumulation of cells at the G2/M phase of

the cell cycle (data not shown). Moreover, we observed that addition of KCl to the external medium greatly exacerbates the lytic phenotype of mpk1 ppz1 ppz2 strains. Taken together, these results demonstrate that the internal turgor pressure of strains lacking Ppz activity is increased over that of the wild-type strain.

As would be expected for a strain under constant cell wall stress, theMPK1mRNA is up-regulated, the Mpk1 protein is overexpressed, and the phosphorylated form is more abundant in strains lackingPPZ1and -2. Moreover, two representative target mRNAs of the Mpk1 pathway,SED1andPIR3, are also up-regulated inppz1 ppz2mutants. Taken together, these data provide strong evidence that the cell wall integrity pathway is activated in response to the increased turgor pressure caused by disruption of the PPZ1andPPZ2genes. These effects on Mpk1 are removed either by addition of an osmotic stabilizer, presumably to counteract the cell wall stress, or by further disruption of the genes encoding the high-affinity K⫹uptake

system,TRK1and -2.

Removal of Trk1 and -2 greatly decreases the capability of the cell to import K⫹from the external medium and would

therefore relieve the internal turgor pressure in strains lacking

PPZ1and -2. Accordingly, removal of theTRK1and -2genes from the ppz1 ppz2 mutant reduces the overexpression and activation of Mpk1 and relieves the temperature-dependent cell lysis defect observed for theppz1 ppz2mutant. These data add to the list of Ppz-deficient phenotypes that depend on the presence ofTRK1and -2. Biochemical studies are in progress to establish whether the Ppz protein phosphatases directly regulate these potassium transporters. As we have also ob-served that Ppz-deficient mutants that do not express TRK1

and -2display a slight but reproducible relative tolerance to

FIG. 7. Strains lackingPPZ1and -2continue to display accelerated ␣-factor recovery in the presence of sorbitol. (A) Strain LY139 was grown to mid-log phase in YPD medium containing the indicated amounts of sorbitol (SORB), processed as described in Materials and Methods. Nitrocellulose membranes were immunoblotted with the anti-Mpk1 antibody (upper panel) and the phosphospecific anti-Mpk1 antibody (lower panel). Similar results were observed in two different experiments. (B) The indicated strains (W303-1A and LY139) were grown to an optical density of 0.4 at 660 nm in complete medium containing the indicated concentration of sorbitol, and the assay was performed as described in Materials and Methods. Images of the plates were taken after 48 h. Identical results were observed in two separate experiments.

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LiCl and NaCl, it is very likely that these protein phosphatases have more than one target in the cell (28). Further studies are aimed at identifying these proteins, but one recent study points to a possible role for the calcineurin pathway (25).

We also present evidence demonstrating that both the Wsc1 and Mid2 branches of the Mpk1 pathway contribute to the sensing of internal turgor pressure. This result is not surprising, considering the previously reported functions of Wsc1 and Mid2 as sensors of cell wall stress. However, our results add to the definition of the function of these sensors by demonstrating that the response to cell wall stress is not limited in its dura-tion, since it is a chronic response in the case of theppz1 ppz2

mutant, and that changes in the external environment are not required. These observations suggest that the cell has a size limit and that when cells are presented with chronically in-creased internal K⫹concentrations, they respond by activating

the Mpk1 pathway to reinforce the cell wall in order to coun-teract an increase in cell volume and avoid lysis.

The genes encoding Mpk1 and Ppz1 and -2 have been im-plicated in cell cycle progression (2, 30). Owing to its important role in stimulating the expression of several cell wall compo-nents, Mpk1 is thought to contribute mainly to the dynamic reorganization of the cell wall required for bud emergence. Accordingly, Mpk1 is activated upon addition of␣-factor, pre-sumably to facilitate cell wall changes necessary for mating (30). However, Mpk1 has also been reported to bind to and phosphorylate components of the SBF transcription factor, which is a key regulator in the yeast G1/S transition (16),

suggesting perhaps a more general role for Mpk1 in cell cycle progression. Indeed, not only has the upstream activator of Mpk1, Pkc1, been identified as a multicopy suppressor of a mutant of one of the components of the SBF transcription factor, Swi4, but this suppression requires the G1cyclins, Cln1

and Cln2 (6). Thus, one plausible explanation for the acceler-ated G1/S transition observed in ppz1 ppz2 mutants treated

with ␣-factor was the relative increase in levels of activated Mpk1. However, under conditions where no Mpk1 phosphor-ylation is detected,ppz1 ppz2mutant strains continue to dis-play the same␣-factor recovery phenotype. Although it is clear that recovery from an ␣-factor-induced cell cycle block does not necessarily indicate changes in the G1/S transition during

vegetative growth, our results provide evidence that Mpk1 activation is not likely to fully account for this phenotype and that other cell cycle components may respond to increases in in-ternal K⫹/pH to drive G

1progression in Ppz-deficient strains.

ACKNOWLEDGMENTS

L.Y. was supported by a postdoctoral fellowship from the European Molecular Biology Organization and is presently an investigator spon-sored by the “Ramo´n y Cajal” Program (Spanish Ministerio de Ciencia y Tecnología, Madrid, Spain). S.M. is supported by a predoctoral grant from the Ministerio de Ciencia y Tecnología (Madrid, Spain). This work was supported by grants PB98-0565-C04-01, BMC2000-1129, and PB98-0565-C04-02 from the Spanish Ministerio de Ciencia y Tec-nología.

We thank Jose´ Ramo´n Murguía for helpful discussions and Maria Molina for anti-Mpk1 antibodies.

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Figure

TABLE 1. Yeast strains used in this study
FIG. 1. Analysis of intracellular accumulation of K�and cell size in strains lacking(W303-1A, LY139, LY140, and Weither rich or minimal medium, as indicated, and processed for HPLCanalysis of Kof three separate measurements; error bars, standard deviations
FIG. 3. Target genes of the Mpk1 pathway are up-regulated in Ppz-deficient strains. The indicated strains (W303-1A, LY139, LY140,
FIG. 5. Disruption of either WSC1phenotype of theLY191, LY192, LY190, LY165, LY194, LY195, LY193, LY139,LY197, LY198, and LY196) were grown to saturation in YPD mediumsupplemented with 1 M sorbitol (Sorb.), serially diluted in sterile 1 Msorbitol, and spot
+2

References

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