Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway
2007; Springer Nature; Volume: 26; Issue: 15 Linguagem: Inglês
10.1038/sj.emboj.7601796
ISSN1460-2075
AutoresKazuo Tatebayashi, Keiichiro Tanaka, Huiyu Yang, Katsuyoshi Yamamoto, Yusaku Matsushita, Taichiro Tomida, M Imai, Haruo Saito,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoArticle12 July 2007free access Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway Kazuo Tatebayashi Kazuo Tatebayashi Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Keiichiro Tanaka Keiichiro Tanaka Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hui-Yu Yang Hui-Yu Yang Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Yusaku Matsushita Yusaku Matsushita Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Midori Imai Midori Imai Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Kazuo Tatebayashi Kazuo Tatebayashi Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Keiichiro Tanaka Keiichiro Tanaka Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Hui-Yu Yang Hui-Yu Yang Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Katsuyoshi Yamamoto Katsuyoshi Yamamoto Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Yusaku Matsushita Yusaku Matsushita Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Taichiro Tomida Taichiro Tomida Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Midori Imai Midori Imai Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Search for more papers by this author Haruo Saito Corresponding Author Haruo Saito Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan Search for more papers by this author Author Information Kazuo Tatebayashi1,2,‡, Keiichiro Tanaka1,2,‡, Hui-Yu Yang1,2,‡, Katsuyoshi Yamamoto1, Yusaku Matsushita1,2, Taichiro Tomida1,2, Midori Imai1 and Haruo Saito 1,2 1Division of Molecular Cell Signaling, Institute of Medical Sciences, The University of Tokyo, Minato-ku, Tokyo, Japan 2Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan ‡These authors contributed equally to this work. *Corresponding author. Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan. Tel.: +81 3 5449 5505; Fax: +81 3 5449 5701; E-mail: [email protected] The EMBO Journal (2007)26:3521-3533https://doi.org/10.1038/sj.emboj.7601796 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To cope with life-threatening high osmolarity, yeast activates the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase cascade. Activated Hog1 regulates the cell cycle, protein translation, and gene expression. Upstream of the HOG pathway are functionally redundant SLN1 and SHO1 signaling branches. However, neither the osmosensor nor the signal generator of the SHO1 branch has been clearly defined. Here, we show that the mucin-like transmembrane proteins Hkr1 and Msb2 are the potential osmosensors for the SHO1 branch. Hyperactive forms of Hkr1 and Msb2 can activate the HOG pathway only in the presence of Sho1, whereas a hyperactive Sho1 mutant activates the HOG pathway in the absence of both Hkr1 and Msb2, indicating that Hkr1 and Msb2 are the most upstream elements known so far in the SHO1 branch. Hkr1 and Msb2 individually form a complex with Sho1, and, upon high external osmolarity stress, appear to induce Sho1 to generate an intracellular signal. Furthermore, Msb2, but not Hkr1, can also generate an intracellular signal in a Sho1-independent manner. Introduction The budding yeast Saccharomyces cerevisiae survive widely fluctuating osmotic conditions in their natural habitat, such as the surface of ripening grapes. To cope with an increased external osmolarity, yeast synthesize, and intracellularly retain the compatible osmolyte glycerol (Gustin et al, 1998; Hohmann, 2002). There is also a temporary arrest in cell cycle progression and inhibition of protein translation, during which cells readjust to the changed environment (Bilsland-Marchesan et al, 2000; Belli et al, 2001; Teige et al, 2001; Escot et al, 2004). These events are governed by the high-osmolarity glycerol (HOG) signaling pathway, whose core element is the Hog1 MAP kinase (MAPK) cascade. As a result, defects in the HOG pathway cause severe osmosensitivity in cell growth. The upstream part of the HOG pathway is composed of the functionally redundant, but mechanistically distinct, SLN1 and SHO1 branches (Figure 1A). A signal emanating from either branch converges on a common MAPK kinase (MAPKK), Pbs2, which is the specific activator of the Hog1 MAPK (Brewster et al, 1993; Maeda et al, 1994, 1995). For yeast to survive on high-osmolarity media, either the SLN1 or the SHO1 branch alone is sufficient. Figure 1.Hkr1 and Msb2 are redundant in the SHO1 branch of the HOG pathway. (A) A schematic model of the yeast HOG pathway. The gray horizontal bar represents the plasma membrane. Arrows indicate positive signal flow, whereas perpendicular bars represent negative regulation. The crosstalk pathway is indicated by white arrows. (B–D) Phenotypes of hkr1Δ and msb2Δ mutant cells. The following yeast strains were used: TM257 (ssk2/22Δ), QG153 (ssk2/22Δ sho1Δ), KT034 (ssk2/22Δ msb2Δ), KT060 (ssk2/22Δ hkr1Δ), KT063 (ssk2/22Δ hkr1Δ msb2Δ), and KT064 (ssk2/22Δ hkr1Δ msb2Δ sho1Δ). The complete genotypes of these and other strains used in this work are listed in Supplementary Table I. The hkr1Δ msb2Δ double mutant is osmosensitive (B), defective in osmostress-induced Hog1 MAPK phosphorylation (C), and defective in osmostress-induced 8xCRE-lacZ reporter induction (D). Throughout the paper, 8xCRE-lacZ expression is presented as an average of three or more independent samples, and is expressed in Miller units (Miller, 1972). Where indicated, cells were treated with (+) or without (−) 0.4 M NaCl for 5 min (C) or 30 min (D). (E) Schematic models of Hkr1 and Msb2 proteins. Cyto, cytoplasmic domain; HMH, Hkr1-Msb2 Homology domain; SS, signal sequence; ST-rich, serine/threonine-rich; TM, transmembrane segment. Download figure Download PowerPoint For each branch, there must be an osmosensor that generates an intracellular signal in response to extracellular osmolarity variations. It is believed that the osmosensor for the SLN1 branch is Sln1, a transmembrane (TM) histidine kinase (Maeda et al, 1994). Sln1 detects turgor changes and transmits a signal via the Sln1-Ypd1-Ssk1 phospho-relay system (Posas et al, 1996; Reiser et al, 2003). Unphosphorylated Ssk1 binds and activates the functionally redundant Ssk2/Ssk22 MAPKK kinases (MAPKKK) that activate the Pbs2 MAPKK (Posas and Saito, 1998). In contrast, the osmosensor in the SHO1 branch has been elusive. There are three candidates, but none has been convincingly shown to be an osmosensor. The first candidate is the branch's namesake, Sho1, which is, to date, the most upstream known component of the pathway. Sho1 has four TM domains, TM1∼TM4, separated by short loops (Loop-1∼Loop-3) of five to eight amino acids each (Maeda et al, 1995) (see Figure 4A for a schematic structure of Sho1). The arrangement of the tightly packed four TM domains is highly conserved across fungi that possess an Sho1 ortholog, suggesting that it may have a more specific function than simple membrane targetting (Krantz et al, 2006). Sho1 predominantly localizes to the cytoplasmic membrane at areas of polarized growth, such as the emerging bud and the bud neck (Raitt et al, 2000; Reiser et al, 2000). The Sho1 C-terminal cytoplasmic region contains an SH3 domain and binds both the Pbs2 MAPKK and the complex of the Ste11 MAPKKK and the Ste50 adaptor protein (Maeda et al, 1995; Zarrinpar et al, 2004; Tatebayashi et al, 2006). Thus, Sho1 serves as an obligatory adaptor between the Ste11 MAPKKK and its substrate Pbs2. It has not, however, been experimentally determined if Sho1 serves an osmosensor function as originally postulated (Maeda et al, 1995). Figure 2.Functional domains and subcellular localization of Hkr1 and Msb2. (A, B) Schematic diagrams of the deletion constructs of Hkr1 and Msb2 used in (C) and (D). Abbreviations are the same as in Figure 1E. WT, wild-type. (C, D) Induction of 8xCRE-lacZ in KT063 (ssk2/22Δ hkr1Δ msb2Δ) that carries a plasmid encoding either WT or one of the deletion constructs of Hkr1 or Msb2, expressed from their native promoter. (E–H) Subcellular localization of Hkr1 and Msb2 in the absence of omsostress. GFP-fusion constructs of full-length Hkr1 (E) and Hkr1 ΔHMH mutant (F) were expressed in KT060 (ssk2/22Δ hkr1Δ) using pRS426 vector, and full-length Msb2 (G) and Msb2 ΔHMH mutant (H) were expressed in KT034 (ssk2/22Δ msb2Δ) using pRS416 vector. (I–N) Osmostress induces similar relocalization of Hkr1, Msb2, and Sho1. Subcellular localization of Hkr1-YFP (I), Msb2-YFP (J), and Sho1-YFP (K), in the absence of osmostress. Osmostress treatment (0.4 M NaCl for ∼10 min) induces a similar punctate redistribution of Hkr1-YFP (L), Msb2-YFP (M), and Sho1-YFP (N). The yeast strain KT064 (ssk2/22Δ sho1Δ hkr1Δ msb2Δ) was transformed with the pRS424 vector expressing the indicated fluorescent fusion protein. Download figure Download PowerPoint A second candidate for the osmosensor in the SHO1 branch is Msb2. The MSB2 gene was originally identified as a multicopy suppressor of the budding defect of cdc24-ts (Bender and Pringle, 1992), and its product is a member of the highly glycosylated mucin family. More recently, it was shown that Msb2 is at the head of the filamentous growth (FG) signal pathway (Cullen et al, 2004). In wild-type yeast cells, hyperosmotic stress activates neither the mating pathway nor the FG pathway. However, when osmotic activation of Hog1 is prevented, for example by a pbs2Δ or a hog1Δ mutation, osmostress induces the mating-specific reporter, Fus1-lacZ (Hall et al, 1996; O'Rourke and Herskowitz, 1998). This physiologically inappropriate crosstalk, however, also has characteristics of the FG pathway, such as independence from Ste4 and Ste5 and a strong dependence on Ste50 (Cullen et al, 2004; O'Rourke and Herskowitz, 1998, 2002). Unlike mating factor, furthermore, osmostress can induce Fus1-lacZ even in diploid (pbs2Δ/pbs2Δ) cells (K. Tatebayashi, unpublished data). Indeed, crosstalk induction of an FG-specific reporter (FRE-lacZ) has been observed in pbs2 mutant cells (Davenport et al, 1999). More important, the crosstalk activation of the mating/FG pathways is completely suppressed by a sho1Δ msb2Δ double mutation, but only partially by sho1Δ or msb2Δ alone (O'Rourke and Herskowitz, 1998, 2002), suggesting that Sho1 and Msb2 have related roles in the FG and HOG pathways. A physiological role for Msb2 in the HOG pathway, however, has been dismissed, because msb2Δ mutants (in a host strain that is defective in the SLN1 branch) are osmoresistant, with robust Hog1 phosphorylation and HOG-dependent gene expression upon osmostress stimulation (O'Rourke and Herskowitz, 2002; Cullen et al, 2004). Finally, a third candidate for the osmosensor in the SHO1 branch is Opy2. Opy2 is a type 1 TM protein, recently shown to have an essential role in the SHO1 branch, as opy2Δ ssk1Δ double mutants are synthetically osmosensitive (Wu et al, 2006). However, there is no evidence that Opy2 participates in an osmosensing process. Thus, despite much speculation, the identity of the osmosensor in the SHO1 branch has been elusive. Here, we report that two mucin-like TM proteins Hkr1 and Msb2 are the most-upstream components in the SHO1 branch so far identified, and thus are likely candidates for the osmosensors. We also investigate how Sho1 might function with the Hkr1/Msb2 in transmitting the osmostress signal. Results Mucin-like transmembrane proteins, Msb2 and Hkr1, are functionally redundant in the SHO1 branch To search for an osmosensor in the SHO1 branch, we used the following criteria. First, the osmosensor is likely to be a TM protein. Second, null mutants of the sensor will be unable to respond to osmostress. Third, the osmosensor should be the most upstream element in the SHO1 branch. And fourth, certain mutations of osmosensor may alter the sensor's kinetic properties. According to the first criterion, Msb2 is one of the potential candidates (Figure 1A). It has been dismissed as the osmosensor only because disruption of the MSB2 gene does not have any appreciable effects on the cell's ability to activate the HOG pathway upon osmostress, or on cellular growth on high-osmolarity media (O'Rourke and Herskowitz, 2002). However, because of the high importance of osmostress signaling for yeast, functional redundancy of key molecules is a recurring feature in the HOG pathway. Thus, if there is a gene that is functionally redundant with MSB2 in the SHO1 branch, it would mask the essential involvement of Msb2 in the HOG pathway. To test this possibility, we screened for a mutant that is osmosensitive only in an msb2Δ background. Note that to focus on the SHO1 branch only, all yeast strains used in this work are of the ssk2Δ ssk22Δ (hereinafter abbreviated as ssk2/22Δ) genetic background, unless stated otherwise. Thus, we mutagenized an msb2Δ ssk2/22Δ strain with ethyl methanesulfonate, and screened for mutants that were osmosensitive and unable to express the HOG-specific reporter gene 8xCRE-lacZ (Tatebayashi et al, 2006) upon osmotic stress. Into each of the ∼350 mutants thus selected, a plasmid encoding the wild-type MSB2 gene was introduced, and the mutants were screened for those that became both osmoresistant and capable of reporter gene expression. In this manner, we identified three mutants that were both osmosensitive and incapable of expressing the 8xCRE-lacZ reporter gene, but only in the absence of the MSB2 gene. To identify the mutant gene responsible for this phenotype, we screened for genomic DNA clones that could complement the osmosensitive defect of the mutants. All three mutants were rescued by genomic DNA clones that contain the HKR1 gene. To verify that hkr1 mutations are responsible for the osmosensitive phenotype of the original mutants, we disrupted the HKR1 gene in various host cells. As shown in Figure 1B, hkr1Δ or msb2Δ alone (in the ssk2/22Δ background) conferred no osmosensitivity to yeast cells, whereas the hkr1Δ msb2Δ double-mutant cells were severely osmosensitive. Osmostress-induced phosphorylation of the Hog1 MAPK (which is a measure of Hog1 activation by the Pbs2 MAPKK) was not significantly reduced by hkr1Δ or by msb2Δ alone, but was completely abolished in the hkr1Δ msb2Δ double mutant (Figure 1C). Osmostress-induced expression of the HOG-specific reporter, 8xCRE-lacZ, also followed the same pattern; the hkr1Δ msb2Δ double mutant was defective in reporter expression, whereas neither hkr1Δ nor msb2Δ alone reduced the reporter expression significantly (Figure 1D). Thus, Hkr1 and Msb2 serve critical, although redundant, roles in the SHO1 branch. Hkr1 and Msb2 are single-pass TM proteins of 1802 and 1306 amino acids, respectively (Figure 1E). Their extracellular regions have three notable similarities. First, both have a highly Ser/Thr-rich (STR) domain. Hkr1 residues 51–1200 are 44% Ser/Thr, and Msb2 residues 51–950 are 49% Ser/Thr. Second, within the STR domain, both proteins have tandem Ser/Thr/Pro-rich repeats reminiscent of highly glycosylated mucin proteins, hence termed the mucin repeats (Supplementary Figure S1A and B). The sequences of these repeats, however, are different from each other. Third, immediately following the STR domain, there is a highly homologous region (47% identity; Supplementary Figure S1C) between Hkr1 (residues 1210–1427) and Msb2 (residues 961–1117), hence termed the Hkr1-Msb2 Homology (HMH) domain. There is no significant sequence similarity between the cytoplasmic domains of Hkr1 and Msb2. Positive- and negative-regulatory domains in Hkr1 and Msb2 To analyze the contribution of each domain of Hkr1 and Msb2 to HOG pathway activation, we constructed various deletions of the HKR1 and MSB2 genes (Figure 2A and B). These constructs were individually introduced into an ssk2/22Δ hkr1Δ msb2Δ host strain, and osmotic induction of 8xCRE-lacZ was measured (Figure 2C and D). The results were essentially identical for the two proteins. Figure 3.Constitutively active mutants of Hkr1 and Msb2 indicate that they are upstream of any other known element in the SHO1 branch. (A–D) Constitutively active HKR1ΔSTR or MBS2ΔSTR, placed in a single-copy plasmid with the GAL1 promoter, was induced by galactose. TM257 (ssk2/22Δ) was used. Expression of HKR1ΔSTR induces the HOG pathway reporter gene 8xCRE-lacZ (A) and phosphorylation of Hog1 (B). Expression of MSB2ΔSTR also induces the HOG pathway reporter (C) and phosphorylation of the Hog1 MAPK (D). (E, F) Activation of the HOG pathway by constitutively active Hkr1 or Msb2 is dependent on Sho1, Ste20, Ste50, and Opy2. Induction of 8xCRE-lacZ by expression of HKR1ΔSTR (E) or MSB2ΔSTR (F) was assayed in mutant cells of the indicated genotypes. Yeast strains used were TM257 (WT), KT064 (sho1Δ), KT032 (ste20Δ), FP67 (ste50Δ), and KY477 (opy2Δ). (G) Altered sensitivity to osmostress of an Hkr1 STR domain deletion mutant. The yeast strains KT034 (ssk2/22Δ msb2Δ HKR1+) and TA039 (ssk2/22Δ msb2Δ hkr1−Δ(50–830)) carrying an 8xCRE-lacZ reporter plasmid were stimulated with the indicated concentration of NaCl for 30 min. 8xCRE-lacZ expression was normalized as the percentage of the maximum expression that occurs at 0.4 M for both strains. Download figure Download PowerPoint Deletion of the HMH domain (ΔHMH) completely abrogated 8xCRE-lacZ induction. This is not due to instability or mislocalization of mutant proteins, because expression levels and subcellular localization of Hkr1 ΔHMH-GFP and Msb2 ΔHMH-GFP were not significantly different from those of their full-length parental constructs (Figure 2E–H). The Hkr1 HMH domain contains a central insertion (residues 1296–1357) that has no counterpart in the Msb2 HMH domain (Supplementary Figure S1C). Deletion of the insertion sequence from the Hkr1 HMH domain only moderately reduced Hkr1 activity, whereas deletion of the conserved sequences on either side of the insertion completely abolished Hkr1 activity (Supplementary Figure S2A). Using a series of short deletion mutants of the Msb2 HMH domain, we found that the entire HMH domain, except for the first 18 amino acids, was required for activation of the HOG pathway (Supplementary Figure S2B). We also found that the HMH domains of Hkr1 and Msb2 are functionally interchangeable; replacement of the Msb2 HMH domain with that of Hkr1 did not significantly impair Msb2 function (Supplementary Figure S2C). Deletion of the entire STR region (ΔSTR) constitutively induced 8xCRE-lacZ expression, in the absence of any osmostress (Figure 2C and D). A more extensive deletion analysis of the Hkr1 STR region (Supplementary Figure S3) suggested that no specific part of the STR region is required for inhibition, but rather it is the overall length of the STR region that is critical. For example, Hkr1-Δ(50–830) is only moderately hyperactive, whereas Hkr1-Δ(101–1080) is strongly hyperactive. These results indicate, for both Hkr1 and Msb2, that the STR domain inhibits the signaling function of the essential HMH domain. Finally, for both proteins, their C-terminal cytoplasmic domain is not essential for HOG pathway activation (Figure 2C and D). Hkr1 and Msb2 localize to similar membrane sites as Sho1 Sho1 predominantly localizes to the cytoplasmic membrane at areas of polarized growth, such as the emerging bud and the bud neck (Raitt et al, 2000; Reiser et al, 2000). We thus determined, by confocal fluorescent microscopy, whether Hkr1 and Msb2 localized in the same subcellular regions as Sho1. The localization of Hkr1 and Msb2 is similar to that of Sho1, although Hkr1 and Msb2 are distributed on the cell surface more uniformly than is Sho1 (Figure 2I–K). Furthermore, osmostress induces a similar punctate redistribution of Hkr1, Msb2, and Sho1 (Figure 2L–N). However, this redistribution occurs in a mutually independent manner—Sho1 redistribution occurs in hkr1Δ msb2Δ host cells, and Hkr1 and Msb2 redistribution occurs in sho1Δ host cells. Hkr1 and Msb2 are the most-upstream elements in the SHO1 branch known to date Next, we studied the functional relationship between Hkr1/Msb2 and Sho1 by epistasis analyses. For this purpose, we first analyzed constitutively active Hkr1-ΔSTR and Msb2-ΔSTR constructs. When these proteins were overexpressed in SHO1+ cells, using an inducible GAL1 promoter, the HOG-specific 8xCRE-lacZ reporter was strongly induced (Figure 3A and C), and so was the activation-associated phosphorylation of the Hog1 MAPK (Figure 3B and D), indicating that the HOG pathway was activated. Overexpression of full-length Hkr1 or Msb2 only very weakly activated the HOG pathway. More important, HOG pathway activation by either Hkr1-ΔSTR or Msb2-ΔSTR was completely inhibited in host cells that are defective in any one of the SHO1, STE20, STE50, OPY2, STE11, PBS2, and HOG1 genes (Figure 3E and F and data not shown). Figure 4.Sho1 functions downstream of Hkr1/Msb2. (A) A schematic model of Sho1. The horizontal bar represents the plasma membrane. Approximate positions of Pro-120 (P120) and Trp-338 (W338) are indicated. (B–C) Expression of constitutively active Sho1-P120L induces the HOG pathway reporter gene 8xCRE-lacZ (B) and phosphorylation of Hog1 (C). (D) Activation of the HOG pathway by Sho1-P120L is dependent on Ste20, Ste50, and Opy2. Constitutively active SHO1-P120L, placed in a single-copy plasmid with the GAL1 promoter, was induced by galactose for 2 h, following which cell extracts were prepared for reporter assays. Yeast strains used were TM257 (wild-type (WT)), KT034 (msb2Δ), KT032 (ste20Δ), FP67 (ste50Δ), and KY477 (opy2Δ). (E) Sho1-P120L can activate the HOG pathway in the absence of both Hkr1 and Msb2. WT, or the indicated SHO1 mutant, was expressed from the GAL1 promoter for 2 h before reporter activity was measured (without osmostress stimulation). Yeast strains used were QG153 (sho1Δ), KT053 (sho1Δ msb2Δ), KT061 (sho1Δ hkr1Δ), and KT064 (sho1Δ hkr1Δ msb2Δ). (F) Opy2 is essential in the SHO1 branch signaling. Induction of the 8xCRE-lacZ reporter gene by osmostress was assayed in host cells of the indicated genotypes. Cells were treated with (+) or without (−) 0.4 M NaCl for 30 min before reporter assay. Yeast strains used were KY475 (opy2Δ), KY476 (opy2Δ ste11Δ), and KY477 (opy2Δ ssk2/22Δ). (G) Membrane targeting of Ste50. WT STE50 or STE50-Δ1-Cpr (Tatebayashi et al, 2006) was expressed in KY477 from the GAL1 promoter for 1.5 h, and cells were treated with (+) or without (−) 0.4 M NaCl for 30 min before 8xCRE-lacZ reporter assay. Download figure Download PowerPoint These data place Hkr1 and Msb2 upstream of any other known element in the SHO1 branch of the HOG pathway, although the epistatic relationship between Sho1 and Hkr1/Msb2 needs further analyses (see the next section). This raises the possibility that Hkr1/Msb2 are the osmosensors. If so, appropriate mutations in their genes could conceivably modulate the sensitivity of the cellular response to external osmostress. Indeed, over a range of NaCl concentrations (0.1–0.3 M), Hkr1-Δ(50–830)-expressing cells responded significantly more strongly than Hkr1-WT-expressing cells, whereas their maximal responses at ∼0.4 M NaCl were similar (Figure 3G). In effect, the sensitivity of Hkr1-Δ(50–830) was shifted by ∼50 mM compared to that of wild-type Hkr1. Constitutively active mutations in the Sho1 extracellular domain activate the HOG pathway in the absence of both Hkr1 and Msb2 The epistasis test in the previous section was incomplete in the sense that it might have only proved that the adaptor function of Sho1 is downstream of Hkr1/Msb2. The Sho1 SH3 domain binds to a Pro-rich motif in Pbs2, and it also interacts with the Ste50 and Ste11 proteins, serving as an adaptor between the Ste50/Ste11 complex and the Pbs2 MAPKK (Maeda et al, 1995; Tatebayashi et al, 2006). Without this adaptor function, no activation of the HOG pathway occurs. We thus conducted additional epistasis analyses in the reverse direction using a constitutively active mutant that appears to affect a more upstream function of Sho1. We previously reported several constitutively active Sho1 mutants (e.g., Sho1-R342G) that have mutations in the cytoplasmic domain and have enhanced adaptor function. Those mutants could activate the HOG pathway only in the presence of a constitutively activated Ste11 (Tatebayashi et al, 2006). Using a similar screening strategy, we found an additional Sho1 mutant that can activate the HOG pathway, and can do so in the presence of only wild-type Ste11. This mutant, Sho1-P120L, has Pro-120 in the extracellular Loop-3 mutated to Leu (Figure 4A). Expression of Sho1-P120L induced the HOG pathway reporter 8xCRE-lacZ (Figure 4B) and phosphorylation of the Hog1 MAPK (Figure 4C) in the wild-type cells, in the absence of any osmostress. To determine whether any other mutation at Pro-120 constitutively activates the HOG pathway better than P120L, we changed Pro-120 to several other nonpolar or neutral amino acids. Of those amino acids tested, P120V, P120C, and P120T could, to varying degrees, induce 8xCRE-lacZ reporter expression, although none was more effective than the original P120L mutant (Figure 4B and data not shown). As expected, HOG activation by Sho1-P120L was completely abrogated by deletion of downstream elements in the SHO1 branch, such as ste20Δ, ste50Δ, and opy2Δ in the host strain (Figure 4D), or by the W338F mutation in the Sho1 SH3 domain that blocks interaction with the downstream Pbs2 (Zarrinpar et al, 2003) (Figure 4E). In clear contrast, Sho1-P120L can activate the HOG pathway in hkr1Δ, msb2Δ, or even in hkr1Δ msb2Δ double-mutant host cells (Figure 4D and E), arguing strongly that Sho1-P120L functions downstream of both Hkr1 and Msb2, but upstream of all other known elements in the SHO1 branch. It should be noted, however, that hkr1Δ, and to a lesser extent msb2Δ, moderately reduces the reporter expression by Sho1-P120L. Therefore, it is possible that Hkr1 and Msb2, although not essential, might still interact with Sho1-P120L and modulate its activity. Taken together, these results place Hkr1 and Msb2 upstream of all other known elements in the SHO1 branch. Membrane-anchorage of Ste50 suppresses the opy2 defect Recently, Wu et al (2006) implicated Opy2 in the SHO1 branch. Using the HOG-specific reporter gene 8xCRE-lacZ, we confirmed their conclusion
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