Phosphorelay-Regulated Degradation of the Yeast Ssk1p Response Regulator by the Ubiquitin-Proteasome System

In Saccharomyces cerevisiae, a phosphorelay signal transductionpathway composed of Sln1p, Ypd1p, and Ssk1p, which are homologousto bacterial two-component signal transducers, is involved inthe osmosensing mechanism. In response to high osmolarity, thephosphorelay system is inactivated and Ssk1p remains unphosphorylated.Unphosphorylated Ssk1p binds to and activates the Ssk2p mitogen-activatedprotein (MAP) kinase kinase kinase, which in turn activatesthe downstream components of the high-osmolarity glycerol response(HOG) MAP kinase cascade. Here, we report a novel inactivationmechanism for Ssk1p involving degradation by the ubiquitin-proteasomesystem. Degradation is regulated by the phosphotransfer fromYpd1p to Ssk1p, insofar as unphosphorylated Ssk1p is degradedmore rapidly than phosphorylated Ssk1p. Ubc7p/Qri8p, an endoplasmicreticulum-associated ubiquitin-conjugating enzyme, is involvedin the phosphorelay-regulated degradation of Ssk1p. In ubc7 ${Delta}$ cells in which the degradation is hampered, the dephosphorylationand/or inactivation process of the Hog1p MAP kinase is delayedcompared with wild-type cells after the hyperosmotic treatment.Our results indicate that unphosphorylated Ssk1p is selectivelydegraded by the Ubc7p-dependent ubiquitin-proteasome systemand that this mechanism downregulates the HOG pathway afterthe completion of the osmotic adaptation.

INTRODUCTION

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Introduction

In eukaryotic cells, the mitogen-activated protein kinase (MAPK)cascades are activated by a variety of external stimuli suchas growth factors, cytokines, UV irradiation, and osmotic stress.In the yeast Saccharomyces cerevisiae, the high-osmolarity glycerol(HOG) response MAPK pathway is activated by increased externalosmolarity (Fig. 1) (6, 18, 20, 49, 54). An upstream His-Aspphosphorelay signaling system, which is homologous to bacterialtwo-component systems, regulates the downstream HOG MAPK cascade(38, 45, 50). Under normal osmolarity conditions, Sln1p, a membrane-associatedhistidine kinase, phosphorylates a histidine residue in itself.The phosphate group is in turn transferred to an aspartic acidresidue in its receiver domain. Subsequently, the phosphategroup is transferred to the Ypd1p phosphotransmitter and thento the receiver domain of the Ssk1p response regulator. Highosmolarity inactivates Sln1p and thus lowers the phosphorylationlevel of Ssk1p (26, 27, 38, 44, 50). Unphosphorylated Ssk1pbinds to the Ssk2p, and perhaps the Ssk22p, MAPK kinase kinases(MAPKKKs) through its receiver domain and activates them (48).Activated Ssk2p phosphorylates and activates the Pbs2p MAPKkinase, which in turn phosphorylates and activates the Hog1pMAPK (36, 48, 49). Phosphorylated Hog1p activates the transcriptionof genes required in the response to high osmolarity (1, 12,53). Deletion of SLN1 or YPD1 induces constitutive activationof the HOG pathway as the consequence of failure in phosphorylatingSsk1p (38, 50). In addition to Ssk1p, S. cerevisiae has anotherresponse regulator, Skn7p, that is also phosphorylated via theSln1p-Ypd1p phosphorelay (8, 29, 32). Skn7p has a DNA-bindingdomain homologous to heat shock transcription factors, as wellas a receiver domain, and in fact acts as a transcription factorinvolved in multiple physiological processes, such as maintenanceof cell wall integrity, cell cycle regulation, and oxidativestress response (4, 7, 8, 31, 39, 40). Under hypo-osmotic conditions,the receiver domain of Skn7p remains as phosphorylated as thatof Ssk1p, and induction of the target genes, including OCH1,is dependent on the phosphorylation (7, 32, 33, 63). In contrast,induction of the target genes for oxidative stress responseis independent of the phosphorylation (31). Skn7p is not implicatedin the regulation of the HOG pathway except for some marginalgenetic interaction (29).

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FIG. 1. The osmosensing signal transduction pathway in yeast. Hyperosmotic stress inhibits the phosphorelay system initiated from the Sln1p membrane-associated histidine kinase, which functions as a homodimer, to cause reduced levels of phosphorylation of Ypd1p and the response regulator Ssk1p. Unphosphorylated Ssk1p activates the Ssk2p MAPKKK, which in turn activates the downstream HOG MAPK cascade. Skn7p is another response regulator phosphorylated by the phosphorelay system, which is a transcriptional factor. Another sensor system that is dependent on the membrane anchor Sho1p also functions, in which Pbs2p is activated by Ste11p (36, 47, 51, 52). Msb2p is also implicated in osmosensing (not shown) (43). MAPKK, MAPK kinase.

In the absence of osmotic stress, or once the adaptation processto high osmolarity is completed, the HOG pathway is negativelyregulated by protein phosphatases. Upon activation of Hog1p,a tyrosine residue and a threonine residue in the activationloop are phosphorylated by Pbs2p (6, 25, 37, 38, 64, 66). Hog1pis inactivated by dephosphorylation of the tyrosine residueby the tyrosine phosphatases Ptp2p and Ptp3p (25, 37, 66) orof the threonine residue by the type 2C serine-threonine proteinphosphatase Ptc1p (37, 64). The dephosphorylation of constituentkinases by protein phosphatases is a common mechanism for thenegative regulation of MAPK cascades. On the other hand, recentreports have shown that the ubiquitin-proteasome system alsonegatively regulates MAPK cascades. The human extracellularsignal-regulated protein kinase (ERK) pathway is downregulatedby the degradation of the ERK MAPKs (35), and the yeast pheromone-inducedMAPK cascade is downregulated by the degradation of the Ste11pMAPKKK (14).

Here, we report that the yeast osmosensing pathway is also downregulatedby the ubiquitin-proteasome system. In this case, the Ssk1presponse regulator is the target for degradation. UnphosphorylatedSsk1p is selectively degraded, and Ubc7p/Qri8p, a ubiquitin-conjugatingenzyme (E2) implicated in endoplasmic reticulum (ER)-associateddegradation (ERAD) (3, 10, 15, 21), is involved in the degradation.This mechanism downregulates the HOG pathway after the completionof the osmotic adaptation. We propose that phosphorelay-regulateddegradation of response regulators can be a general regulatorymechanism of eukaryotic His-Asp phosphorelay signaling systems.

MATERIALS AND METHODS

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Materials and Methods

Bacterial and yeast methods. Standard Escherichia coli and yeast manipulations were performedas described previously (2). E. coli strain XL10-Gold (Stratagene,San Diego, Calif.) was used for plasmid propagation. SGal andSRaf media are modified synthetic complete media with dextrose(SD media) in which glucose is replaced by galactose and raffinose,respectively.

Yeast strains. The yeast strains used in this study are listed in Table 1.UBC7 was disrupted by transformation with pRH1186 (17), SKN7was disrupted by transformation with pNS396 (see below), andRPN9 was disrupted by transformation with pJUN180 (61).

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TABLE 1. Yeast strains used in this study

Plasmids. The plasmids used in this study are listed in Table 2.

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TABLE 2. Plasmids used in this study

To induce the C-terminally epitope-tagged SSK1 from the galactose-inducibleGAL1 promoter, pNS252, pNS114, or pNS462 was used. A three-hemagglutinin(HA)-six-His composite epitope tag was fused to the C terminusof Ssk1p as follows. A SalI site was introduced just beforethe termination codon of SSK1 by PCR. The 2.2-kb ClaI-SalI fragmentcovering the SSK1 open reading frame (ORF) and the 0.2-kb SalI-AscI(blunted) fragment containing a three-HA-six-His composite epitopetag derived from pFA6a-3HA-6His-His3MX6 (T. Maeda, unpublisheddata), which has the three-HA-six-His tag in the common pFA6a-His3MX6backbone (34), were cloned into the ClaI and HincII sites ofp424GAL1 (41) to produce pNS252. The 0.7-kb SalI-SmaI fragmentcontaining the GAL1 promoter from pGSS1 (38), the 2.2-kb ClaI(blunted)-SalI fragment described above, and the 0.4-kb SalI-SacIfragment containing the three-HA-six-His tag and the alcoholdehydrogenase terminator derived from pFA6a-3HA-6His-His3MX6were cloned into the SalI and SacI sites of pRS414 (58) to producepNS114. pNS462 is essentially the same as pNS114 except thatthe parental vector is pRS416 (58). pNS476 is essentially thesame as pFP57 (50) except that the insert is wild-type YPD1.

To induce N-terminally three-FLAG-tagged SSK1 from the GAL1promoter, pTB426 was constructed as follows. An EcoRI site wasintroduced just in front of the initiation codon of SSK1 byPCR. The 2.6-kb EcoRI-BstXI segment covering the entire ORFand the 3' flanking region of SSK1, together with a vector-derivedlinker sequence adjacent to the BstXI site, was isolated asan EcoRI fragment. The EcoRI fragment was cloned into the EcoRIsite of pcDNA3.1/N-3xFLAG (T. Maeda, unpublished), which hasa three-FLAG tag in the common pcDNA3.1(+) backbone (InvitrogenCorp., Carlsbad, Calif.), to produce pTB422. The 2.7-kb HindIII-XhoIfragment, containing the N-terminally three-FLAG-tagged ORFand the 3' flanking region of SSK1, was cloned into the HindIIIand XhoI sites of p416GAL1 (41) to produce pTB426.

To express C-terminally FLAG-tagged SSK1, pNS454 was constructedas follows. The 2.2-kb ClaI-SmaI fragment containing the SSK1ORF from pNS252 and the 0.1-kb BamHI (blunted)-ApaI (blunted)fragment containing a single FLAG epitope from pcDNA3-FLAG werecloned into the ClaI and HincII sites of p426TEF (42) to producepNS454. The ubiquitin-like (UBL) domain of Ssk1p was deletedas follows. An EcoRV restriction site was introduced at nucleotides709 to 714 of the SSK1 ORF (mutated from GAA TCA to GAT ATC)by PCR. The 0.7-kb ClaI-EcoRV fragment containing the 5' portionof SSK1, the 1.3-kb EcoRV-SmaI fragment containing the 3' portionof SSK1 from pNS358, and the 0.1-kb BamHI (blunted)-ApaI (blunted)fragment described above were cloned into the ClaI and HincIIsites of p426GPD (42) to produce pNS456 containing SSK1 ${Delta}$ UBL (aminoacids 243 to 290 were deleted).

pNS396, the plasmid used for the deletion of SKN7, was constructedas follows. The 3.5-kb SmaI-XbaI fragment containing the SKN7locus from pRS315-SKN7 (8) was cloned into the HincII and XbaIsites of pBluescript KS(+). The 1.5-kb PstI-HincII fragmentwithin the SKN7 ORF was then replaced with the 1.0-kb PstI-PmeIfragment containing the TRP1 cassette from pFA6a-TRP1 (34).

Detection of epitope-tagged Ssk1p. Cells expressing tagged SSK1 were cultured in SD medium andthen transferred to SRaf medium. Cells were grown to an opticaldensity at 600 nm of $~$ 0.8, and galactose was added to the mediumto a final concentration of 2%. After 3 h, cells were transferredto SD medium containing cycloheximide (0.5 mg/ml). Aliquotswere collected at the indicated times and centrifuged. Cellpellets were suspended in the Laemmli sample buffer and boiledfor 5 min. Proteins were resolved by sodium dodecyl sulfate-7to 10% polyacrylamide gel electrophoresis. Proteins were transferredto a polyvinylidene difluoride membrane, and the blot was incubatedwith the mouse anti-HA monoclonal antibody 12CA5, peroxidase-conjugatedsheep anti-mouse immunoglobulin (Amersham Biosciences, Piscataway,N.J.), and then enhanced chemiluminescence detection reagent(Amersham Biosciences). Actin was detected with the mouse anti-actinmonoclonal antibody C4 (ICN Biomedicals, Costa Mesa, Calif.).In some experiments (see Fig. 3B), the primary antibody wasreplaced with the mouse anti-FLAG monoclonal antibody M2 (Sigma,St. Louis, Mo.).

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FIG. 3. The stability of Ssk1p is dependent on both the phosphotransfer and the proteasome system but not on the SKN7 pathway. (A) The stability of Ssk1p is dependent on the phosphotransfer from Ypd1p and the proteasome system. C-terminally tagged Ssk1p was expressed from the low-copy-number plasmid pNS114 in the YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ RPN9⁺ (NS318), ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ RPN9⁺ (NS320), YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ rpn9 ${Delta}$ (NS337), and ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ rpn9 ${Delta}$ (NS336) strains, and the Ssk1p content was monitored by Western blotting. (B) N-terminally three- FLAG-tagged Ssk1p is also degraded in a phosphorelay-regulated manner. N-terminally three-FLAG-tagged Ssk1p was expressed from the low-copy-number plasmid pTB426 in NS318 and NS320 strains, and the Ssk1p content was monitored. (C) Ypd1p-Ssk1p interaction does not affect the stability of Ssk1p. NS320 strains with pNS114 were transformed with pRS416 (vector), pFP57 (ypd1 [H64Q]), or pNS476 (YPD1⁺), and the Ssk1p content was monitored. (D) Deletion of SKN7 does not reduce the stability of Ssk1p. Tagged Ssk1p was expressed by pNS462 in NS318, NS320, and skn7 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ (NS325) strains, and the Ssk1p content was monitored. +, present; ${Delta}$ , deleted.

Coimmunoprecipitation of Ssk1p with the proteasome. NS318 and NS320 strains expressing SSK1 (wild type or ${Delta}$ UBL)-FLAGwere grown to an optical density at 600 nm of $~$ 1.0. Cells werecollected by centrifugation, suspended in lysis buffer (25 mMTris-HCl [pH 7.5], 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100,3 mM phenylmethylsulfonyl fluoride, 40 µg of aprotinin/ml,10 µg of pepstatin A/ml, 20 µg of leupeptin/ml,0.1 mM MG-132), and mixed with an equal volume of glass beads(Sigma). After being vortexed 10 times with 30-s pulses, sampleswere centrifuged at 17,000 x g for 15 min at 4°C. The supernatantwas then incubated with anti-FLAG M2-agarose beads (Sigma) for4 h at 4°C. Beads were collected and washed twice with lysisbuffer and then suspended in Laemmli sample buffer and boiledfor 5 min. Western blotting was performed essentially as describedabove, with 12CA5, M2, and rabbit anti-20S proteasome core subunitpolyclonal antibody (62).

Detection of phosphorylated Hog1p. TM141 and NS327 strains expressing multicopy HOG1 from pHG11(38) were grown overnight in SD medium without sorbitol. Cellswere transferred to yeast extract-peptone-dextrose medium withoutsorbitol and grown for 2.5 h to an optical density at 600 nmof $~$ 0.8. Cells were then resuspended in yeast extract-peptone-dextrosemedium containing 1.5 M sorbitol and collected at the indicatedtimes. Cell pellets were suspended in Laemmli sample buffercontaining 1 mM sodium vanadate and boiled for 5 min. Cell extractwas prepared with glass beads as described above. Western blottingwas performed essentially as described above, with rabbit polyclonalanti-phospho-p38 antibody (New England Biolabs, Beverly, Mass.)and the goat polyclonal anti-Hog1p antibody yC-20 (Santa CruzBiotechnology, Santa Cruz, Calif.).

RESULTS

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Results

Ssk1p is stabilized in proteasome mutants. A recent paper on the high-throughput mass spectrometric proteincomplex identification applied to S. cerevisiae (22) reportedthat Ssk1p binds to the Ssk2p MAPKKK, Ubi4p (ubiquitin), andUbc7p. Ubc7p is a ubiquitin-conjugating enzyme (E2) responsiblefor ERAD, which involves the ubiquitination of misfolded proteinsretrotranslocated from the ER to the cytoplasm (15, 21, 46).Ubc7p also functions in the regulatory degradation of the followingproteins: Hmg2p, a yeast isozyme of the cholesterol-biosynthesisenzyme HMG-coenzyme A reductase (3, 17, 19); Mat ${alpha}$ 2p, a transcriptionalrepressor (10, 60); and Ole1p, an ER-bound ${Delta}$ -9 fatty-acid desaturase(5).

This discovery prompted us to investigate the relationship betweenSsk1p and the ubiquitin-proteasome system. We examined the stabilityof Ssk1p in rpn9 ${Delta}$ (61) and rpn12-1 (30) mutants, which are defectivein a 19S regulatory subunit of the proteasome. C-terminallyepitope-tagged Ssk1p, which had been shown to complement thedeletion of SSK1 (data not shown), was expressed under the GAL1promoter in wild-type (W303-1A), rpn9 ${Delta}$ , and rpn12-1 strains culturedin SGal medium at 30°C, the permissive temperature. Thenthe GAL1 promoter was shut off in SD medium, and novel proteinsynthesis was inhibited by the addition of cycloheximide tothe medium. Simultaneously, cells were shifted to 37°C,the nonpermissive temperature, and sampled every hour. The Ssk1pcontent was monitored by Western blotting. In wild-type cells,Ssk1p content constantly decreased over time, whereas Ssk1pwas more stable in rpn9 ${Delta}$ and in rpn12-1 cells than in wild-typecells (Fig. 2). These results indicate that Ssk1p is degradedby the activity of the proteasome system.

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FIG. 2. Ssk1p is stabilized in proteasome mutants. HA-tagged Ssk1p was expressed from the high-copy-number plasmid pNS252 in the wild-type (WT) (W303-1A), rpn9 ${Delta}$ (J43), and rpn12-1 (YK109) strains cultured in SGal medium at 30°C. Transcription and translation were shut off in SD medium containing cycloheximide at 37°C. Cells were collected at the indicated times, the proteins were extracted, and the Ssk1p content was monitored by Western blotting with anti-HA antibody. Actin was detected as the loading control.

The stability of Ssk1p is dependent on both the phosphotransfer from Ypd1p and the proteasome system. In the absence of hyperosmotic stress, Ypd1p phosphorylatesand inactivates Ssk1p. Deletion of YPD1 keeps Ssk1p constitutivelyunphosphorylated (50). To determine whether the phosphorylationstate of Ssk1p affects its stability, we examined the stabilityof Ssk1p in strains in which YPD1 was intact or deleted. Tocircumvent the lethality of ypd1 ${Delta}$ , the experiments were performedon a hog1 ${Delta}$ background. C-terminally tagged Ssk1p was expressedin both YPD1⁺ and ypd1 ${Delta}$ cells, and Ssk1p content was monitoredby Western blotting as described above, except that SSK1 wasexpressed from a low-copy-number plasmid and the cells werekept at 30°C throughout the experiment. In ypd1 ${Delta}$ cells, inwhich Ssk1p was unphosphorylated, the Ssk1p content decreasedmore rapidly than in YPD1⁺ cells, in which Ssk1p was kept phosphorylated(Fig. 3A). These results indicate that unphosphorylated Ssk1pis less stable than phosphorylated Ssk1p. The instability ofSsk1p in ypd1 ${Delta}$ cells was almost negated by the deletion of RPN9(Fig. 3A). This observation suggests that the instability ofunphosphorylated Ssk1p is caused by its preferred degradationby the proteasome system. Alternatively tagged Ssk1p, with adifferent epitope tag at the other terminus, was also degradedin a phosphorelay-regulated manner as was the C-terminally taggedSsk1p used in the experiments thus far, which indicates thatthe degradation was unlikely to be affected by the epitope tagging(Fig. 3B). Thus, we used the C-terminally tagged version inthe following experiments.

The possibility was tested that the instability of Ssk1p inypd1 ${Delta}$ cells was caused by phosphorelay-independent effects ofYpd1p deficiency, such as the loss of a physical and stabilizingcomplex between Ypd1p and Ssk1p. In ypd1 ${Delta}$ cells, expression ofmutant Ypd1^H64Qp, which is incapable of phosphotransfer butexpected to be able to interact with Ssk1p (26, 50), did notsignificantly stabilize Ssk1p, while wild-type Ypd1p did (Fig.3C). These results suggest that the phosphorylation state ofSsk1p itself is the determinant of the stability.

Instability of unphosphorylated Ssk1p is independent of the SKN7 pathway. In ypd1 ${Delta}$ cells, not only is Ssk1p unphosphorylated and/or activatedbut Skn7p is also unphosphorylated and/or inactivated (8, 29,32) (Fig. 1). To test the possible involvement of the SKN7 pathwayin Ssk1p stability, we examined the stability of Ssk1p in strainswith intact or deleted SKN7. The deletion of SKN7 did not decreasethe stability of Ssk1p in YPD1⁺ cells (Fig. 3D), indicatingthat Ssk1p instability in ypd1 ${Delta}$ cells is not caused by the inactivationof the SKN7 pathway. Conversely, Ssk1p was marginally, but reproducibly,more stable in skn7 ${Delta}$ cells than in SKN7⁺ cells. This may be dueto increased phosphorylation of Ssk1p caused by the absenceof Skn7p, which potentially competes with Ssk1p for phosphotransferfrom Ypd1p.

Ubc7p is involved in the degradation of unphosphorylated Ssk1p. In light of the results of high-throughput mass spectrometricprotein complex identification applied to S. cerevisiae as mentionedabove (22), Ubc7p was expected to be involved in the degradationof Ssk1p. To test the possibility, we examined the stabilityof Ssk1p in ubc7 ${Delta}$ cells. In YPD1⁺ cells, deletion of UBC7 didnot significantly affect the stability of Ssk1p. In ypd1 ${Delta}$ cells,on the other hand, disruption of UBC7 stabilized Ssk1p cellsin comparison to UBC7⁺ cells (Fig. 4). These results indicatethat unphosphorylated Ssk1p is selectively degraded by the Ubc7p-dependentdegradation pathway.

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FIG. 4. Ubc7p-dependent degradation of unphosphorylated Ssk1p. Ubc7p is involved in the degradation of Ssk1p. Tagged Ssk1p was expressed from pNS114 in the YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ UBC7⁺ (NS318), YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ ubc7 ${Delta}$ (NS329), ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ UBC7⁺ (NS320), and ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ ubc7 ${Delta}$ (NS333) strains, and the Ssk1p content was monitored as described in the legend to Fig. 3. +, present; ${Delta}$ , deleted.

Ssk2p-independent degradation of Ssk1p. To examine the possible involvement of the Ssk1p-Ssk2p interactionin the regulation of the Ssk1p stability, the level of Ssk1pwas monitored in ssk2 ${Delta}$ cells. Both in YPD1⁺ and ypd1 ${Delta}$ cells, deletionof SSK2 did not significantly affect the stability of Ssk1p(Fig. 5). These results indicate that the Ssk1p-Ssk2p interactionis not required for the degradation of Ssk1p.

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FIG. 5. Ssk2p-independent degradation of Ssk1p. Tagged Ssk1p was expressed from pNS462 in the YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ SSK2⁺ (NS318), ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ SSK2⁺ (NS320), YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ ssk2 ${Delta}$ (NS354), and ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ ssk2 ${Delta}$ (NS355) strains, and the Ssk1p content was monitored. +, present; ${Delta}$ , deleted.

The UBL domain of Ssk1p is not required for the interaction with the proteasome. We found a UBL domain in the N-terminal region (amino acids243 to 290) of Ssk1p (Fig. 6A). Recent reports showed that theUBL domains of Rad23p and Dsk2p bind to Rpn1p, a component ofthe base complex of the 26S proteasome (13, 16, 56, 65). Thus,we examined the interaction between the UBL domain of Ssk1pand the proteasome. Wild-type or UBL domain-deleted Ssk1p (Ssk1^UBLp)fused to a FLAG tag was expressed in both YPD1⁺ and ypd1 ${Delta}$ cells.When expressed from SSK1's own promoter, the expression levelof Ssk1^UBLp was much lower than that of wild-type Ssk1p, probablybecause of the gross structural perturbation of this mutantprotein (data not shown). To minimize the difference betweenthe expression levels in this experiment, Ssk1^UBLp was overproducedfrom the strong GPD promoter while wild-type Ssk1p was overproducedfrom the weaker TEF promoter. Ssk1p was immunoprecipitated withanti-FLAG antibody, and proteasomes bound to Ssk1p were detectedby Western blotting with anti-20S proteasome antibody. Evenwhen the UBL domain was deleted, Ssk1p coprecipitated with theproteasome (Fig. 6B). These results indicate that the UBL domainof Ssk1p is atypical in that it is not required for interactionwith the proteasome.

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FIG. 6. The UBL domain of Ssk1p is not required for the interaction with the proteasome. (A) UBL domain of Ssk1p. In the middle of the molecule (amino acids 243 to 290), Ssk1p contains a small domain homologous to ubiquitin and the UBL domains of human and yeast Rad23p. The UBL domain of Ssk1p shares amino acid identities of 42.6% with human Rad23p, 25.5% with yeast Rad23p, and 25.5% with ubiquitin. (B) Coimmunoprecipitation of Ssk1p with the proteasome. SSK1 (wild type [WT] or ${Delta}$ UBL)-FLAG was expressed from pNS454 or pNS456, respectively, in the YPD1⁺ ssk1 ${Delta}$ hog1 ${Delta}$ (NS318) and ypd1 ${Delta}$ ssk1 ${Delta}$ hog1 ${Delta}$ (NS320) strains. Ssk1p (WT or ${Delta}$ UBL)-FLAG was immunoprecipitated with anti-FLAG M2 beads, and proteasomes bound to Ssk1p were detected by Western blotting. +, present; ${Delta}$ , deleted; -, not present.

The Hog1p MAPK is ectopically activated in ubc7 ${Delta}$ cells. In ubc7 ${Delta}$ cells, the delay of the Ssk1p degradation was expectedto cause ectopic activation of the Hog1p MAPK. To examine thepossibility, we overexpressed SSK1 under the strong GAL1 promoterin wild-type, ubc7 ${Delta}$ , ptp2 ${Delta}$ , and ubc7 ${Delta}$ ptp2 ${Delta}$ cells on SGal medium.Overexpression of SSK1 was slightly toxic to ubc7 ${Delta}$ cells comparedto wild-type cells, as observed by poorer growth of ubc7 ${Delta}$ cellsthan of wild-type cells. More remarkably, in ptp2 ${Delta}$ cells, inwhich the inactivation process of Hog1p is partially defective(66), the overexpression of SSK1 was lethal when UBC7 was deleted(Fig. 7A). This lethality was suppressed by deletion of HOG1(Fig. 7B). These results suggest that the Ssk1p degradationby the Ubc7p-dependent mechanism represses the activity of theHOG pathway.

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FIG.7. Ectopic activation of the HOG pathway in ubc7 ${Delta}$ cells. (A) Overexpression of SSK1 is toxic to ubc7 ${Delta}$ cells. Wild-type (TM141), ubc7 ${Delta}$ (NS327), ptp2 ${Delta}$ (TM107), and ubc7 ${Delta}$ ptp2 ${Delta}$ (NS358) strains were transformed with pNS473. Transformants were grown on SD and SGal plates. SSK1 was overexpressed under the GAL1 promoter on an SGal plate. Each plate was incubated at 30°C for 4 days. (B) Deletion of HOG1 suppresses the lethality caused by SSK1 overexpression. SSK1 was overexpressed from pNS473 in the NS358 and ubc7 ${Delta}$ ptp2 ${Delta}$ hog1 ${Delta}$ (NS362) strains. Each plate was incubated at 30°C for 5 days. (C) Prolonged phosphorylation of the Hog1p MAPK in ubc7 ${Delta}$ cells after the hyperosmotic treatment. TM141 and NS327 strains expressing HOG1 from pHG11 were treated with or without 1.5 M sorbitol, and phospho-Hog1p was detected by Western blotting.

Next, we monitored the phosphorylation level of Hog1p in wild-typeand ubc7 ${Delta}$ cells. To improve the sensitivity of our assay, HOG1was expressed from a high-copy-number plasmid. Cells were treatedwith or without 1.5 M sorbitol then sampled at the indicatedtimes, and phosphorylated Hog1p was detected by Western blottingwith anti-phospho-p38 antibody. After the hyperosmotic treatment,the dephosphorylation and/or inactivation process of Hog1p wasdelayed in ubc7 ${Delta}$ cells compared with wild-type cells. On theother hand, in the absence of osmotic stress, no significantincrease in the basal activity of Hog1p was observed in ubc7 ${Delta}$ cells compared with wild-type cells (Fig. 7C). These resultsindicate that the Ubc7p-dependent degradation mechanism, bydegrading an unphosphorylated pool of Ssk1p, downregulates theHOG pathway after the completion of the osmotic adaptation,in cooperation with the protein phosphatases that inactivatethe pathway (25, 37, 64, 66).

DISCUSSION

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Discussion

Activation of the yeast HOG MAPK cascade is essential in theorganism's adaptation to high extracellular osmolarity (6, 36,38, 48). On the other hand, proper inactivation of the HOG cascadeis also essential for growth because hyperactivation of thiscascade is toxic to cells (36, 37, 38, 66). Therefore, the HOGcascade must remain inactive unless cells are exposed to highosmolarity and must be inactivated promptly once the adaptationprocess to high osmolarity is completed. Dephosphorylation ofa phosphotyrosine or a phosphothreonine residue in active Hog1pby protein phosphatases is a well-characterized inactivationmechanism (25, 37, 38, 64, 66). Our results demonstrate an additionaldownregulation mechanism in the pathway: unphosphorylated Ssk1presponse regulator is degraded by the ubiquitin-proteasome system.

Two distinct physiological roles are conceivable for this downregulationmechanism. One is to maintain the low basal activity of theHOG pathway in the absence of osmotic stress. This aids cellsin avoiding ectopic induction of osmotic responses under normalosmotic conditions, which is not only unnecessary but also deleterious.The other is to downregulate the HOG pathway after a sufficientsignal is transmitted to complete the adaptation process. Thisensures that adequate adaptation is achieved and that the immediateresumption of growth occurs once the adaptation is complete.Here, we show that the latter is the main role for the degradationmechanism because, as shown in Fig. 7C, the deletion of UBC7caused the prolonged activation of the HOG pathway after thehyperosmotic treatment but did not significantly increase thebasal activity of Hog1p in the absence of osmotic stress.

The reaction steps in the ubiquitination of substrates to bedegraded require three kinds of enzymes: ubiquitin-activatingenzyme (E1), E2, and ubiquitin ligase (E3). E1 activates ubiquitinby forming a thiol-ester bond between ubiquitin and E1. Activatedubiquitin is then transferred to E2. E3 interacts with E2 andthe substrate and facilitates ubiquitination. The E3 involvedin Ssk1p ubiquitination has yet to be identified. In the caseof the human ERK pathway, MEKK1 (MAPKKK), which contains a RINGfinger-like PHD domain, functions as E3 for the ERK1/2 MAPKsand downregulates the pathway (35). However, our observationthat deletion of SSK2 does not affect the stability of Ssk1pexcludes the possibility that the Ssk2p MAPKKK acts as E3 forSsk1p. Consistently, Ssk2p does not appear to have any of themotifs frequently found in E3s, such as a RING, HECT, or U-boxdomain. The yeast Ste11p MAPKKK is activated by both matingpheromone and osmotic stress (18, 20, 47, 49). When activatedby pheromone, Ste11p is degraded by the ubiquitin-dependentsystem (14). However, E3 for Ste11p has also yet to be identified.In the case of ERAD, Hrd1p (3, 19) and Doa10p (60) have beendefined as ER-associated E3s that act together with Ubc7p, whereasdegradation of Ole1p is independent of these E3s (5). WhetherHrd1p and/or Doa10p is also an E3 in the ubiquitination of Ssk1pawaits further investigation.

The domains in Ssk1p required for its interaction with E2, E3,or proteasomes, if any, have yet to be determined. We founda UBL domain in the N-terminal region (amino acids 243 to 290)of Ssk1p (Fig. 6A). Recent reports showed that the UBL domainsof Rad23p and Dsk2p bind to Rpn1p, a component of the base complexof the 26S proteasome (13, 16, 56, 65). In contrast, the UBLdomain of Ssk1p is dispensable for its interaction to proteasomes.In accord with the difference in function, there are some structuraldifferences between the UBL domains of Ssk1p and Rad23p/Dsk2p.The UBL domain of Ssk1p is shorter than those of Rad23p andDsk2p, and its location is in the center of the molecule, whereasthose of Rad23p and Dsk2p are near the N termini. The UBL domainof Ssk1p may bind to degradation-related proteins other thancomponents of the 26S proteasome, such as E2 and/or E3.

A model for the functional connection between Ssk1p and theubiquitin-proteasome system is shown in Fig. 8. Once the osmoticadaptation is completed, unphosphorylated Ssk1p, which potentiallyactivates Ssk2p, is ubiquitinated by the Ubc7p E2 and an unidentifiedE3 on the surface of the ER. Then unphosphorylated and ubiquitinatedSsk1p is targeted to the 26S proteasome and degraded, whichensures the timely inactivation of the HOG pathway after thecompletion of the osmotic adaptation.

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FIG. 8. Model for the functional connection between Ssk1p and the ubiquitin-proteasome system. See Discussion for explanation.

Phosphorelay-regulated degradation of response regulators bythe ubiquitin-proteasome system may be a general regulatorymechanism in eukaryotic His-Asp phosphorelay signaling systems.Higher plants make use of His-Asp phosphorelay signaling systemsin the signal transduction of plant hormones ethylene and cytokinin(9, 23, 24, 28, 55, 57, 67). Recently, a mutant of an Rpn12phomolog in Arabidopsis was reported to show altered responsesto cytokinin (59). Although response regulators for cytokininsignaling are still to be identified, impaired degradation ofthem may cause the altered responses to the hormone in thisproteasome mutant.

ACKNOWLEDGMENTS

We thank Randolph Hampton, Mark Longtine, Haruo Saito, HowardBussey, and Masahiro Kawabata for plasmids. We also thank EugeneFutai, Michio Hayashi, Hiroyuki Sorimachi, Koichi Suzuki, andall the members of the Maeda laboratory for help, advice, anddiscussion.

This work was supported by a grant-in-aid for scientific researchon priority areas (KAKENHI 14086203) from the Ministry of Education,Culture, Sports, Science, and Technology (MEXT), a grant (no.0253) from the Salt Science Research Foundation, and a grantfrom the Japan Foundation for Applied Enzymology (to T.M.).

FOOTNOTES

* Corresponding author. Mailing address: Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. Phone: 81-3-5841-7820. Fax: 81-3-5841-7899. E-mail: maeda{at}ims.u-tokyo.ac.jp.

REFERENCES

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