![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 20, Issue 13, 3101-3114, July 1, 2009
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Biological Sciences, State University of New York at Buffalo, Buffalo, NY 14260-1300
Submitted July 24, 2008;
Revised April 8, 2009;
Accepted May 5, 2009
Monitoring Editor: Charles Boone
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
). For example, mitogen-activated protein kinase (MAPK) pathways utilize overlapping or shared components to coordinate different aspects of cellular behaviors (Bardwell, 2006). The overlap between pathways can be extensive, and it remains unclear how a particular signal transmitted through an interconnected network elicits a specific response. Given that inappropriate cross talk between MAPK pathways is an underlying cause of cancer and other diseases (Santen et al., 2002), understanding how signaling pathways precisely coordinate cellular behaviors is an important question.
In the budding yeast Saccharomyces cerevisiae, MAPK pathways regulate the response to a variety of stimuli (Errede et al., 1995). In response to nutrient limitation, cells undergo filamentous growth (FG; pseudohyphal/invasive growth; Gimeno et al., 1992; Liu et al., 1993; Roberts and Fink, 1994; Cullen and Sprague, 2000), a cellular differentiation characteristic of many fungal species including pathogens (Lo et al., 1997; Whiteway and Bachewich, 2007). FG is regulated by a typical MAPK pathway (Roberts and Fink, 1994; Borneman et al., 2007). At the head of the FG pathway, the signaling mucin Msb2p and adaptor protein Sho1p (O'Rourke and Herskowitz, 1998; Cullen et al., 2004) connect to the polarity establishment Rho (Ras homology) GTPase Cdc42p (Peter et al., 1996; Leberer et al., 1997), a global regulator of cell polarity and signaling (Johnson, 1999). In its activated (GTP-bound) state, Cdc42p associates with the p21-activated kinase (PAK) Ste20p (Peter et al., 1996; Leberer et al., 1997), which results in the activation of a typical MAPK cascade composed of the Ste11p, Ste7p, and Kss1p protein kinases in a phosphorelay circuit (Madhani et al., 1997). Phosphorylation of the transcription factors Ste12p and Tec1p by Kss1p results in the induced expression of FG pathway target genes (Madhani and Fink, 1997; Madhani et al., 1999). Although it is not entirely clear how nutritional information connects to FG pathway signaling, activation of the FG pathway requires processing and release of the extracellular inhibitory domain of Msb2p by the aspartyl protease Yps1p, which occurs preferentially under nutrient-limiting conditions (Vadaie et al., 2008).
Most of the proteins that function in the FG pathway are required for the activation of other MAPK pathways in the same cell (see Figure 1; Schwartz and Madhani, 2004; Qi and Elion, 2005; Murphy and Blenis, 2006). Msb2p, Sho1p, Cdc42p, Ste20p, Ste11p (O'Rourke and Herskowitz, 1998; O'Rourke et al., 2002; Tatebayashi et al., 2006) and the Ste11p-adaptor protein Ste50p (Posas et al., 1998; Jansen et al., 2001; Truckses et al., 2006) function in the FG pathway and the high osmolarity glycerol response (HOG) pathway (Raitt et al., 2000; Reiser et al., 2000; Tatebayashi et al., 2007), which controls the response to osmotic stress (Hohmann, 2002; Chen and Thorner, 2007). The Ste11p branch of the HOG pathway converges with the Sln1p branch at the level of the MAPKK Pbs2p (Maeda et al., 1994). A second mucin, Hkr1p, functions redundantly with Msb2p in the HOG pathway (see Figure 1; Tatebayashi et al., 2007). Whether Hkr1p also functions in the FG pathway is not known. Cdc42p, Ste20p, Ste50p, Ste11p, and Ste7p also function in the pheromone response pathway, which controls mating of complementary haploid cells that sense and respond to secreted peptide pheromones (Bardwell, 2004; Schwartz and Madhani, 2004; Dohlman and Slessareva, 2006). The three MAPK pathways are activated by different stimuli and induce different target genes and morphological responses (Roberts et al., 2000; McClean et al., 2007). Specificity between the FG and HOG pathways is regulated at least in part at the level of Pbs2p, which binds to Sho1p and Ste11p, presumably to recruit these proteins to the HOG pathway (Maeda et al., 1995; Zarrinpar et al., 2004).
|
), but is commonly found in the wild associated with the grape-producing plant Vitis vinifera. Extensive cultivation over thousands of years has reinforced the relationship between the two organisms (Cavalieri et al., 2003). We therefore developed a "grape assay" that among other things uncovered a multimodal FG response mediated by the FG pathway. Communication between the HOG and FG pathways led to modulation of the FG response, and we specifically show that the signaling mucins Msb2p and Hkr1p exert different effects on FG pathway activity. Our findings therefore bring to light a new point of discrimination between MAPK pathways by members of the signaling mucin family of proteins. More generally, our findings suggest a possible rationale for why MAPK pathways share components, to coordinate a cellular response through mutually exclusive activation between pathways. |
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Rose et al., 1990). All experiments were carried out at 30°C unless otherwise indicated. ß-Galactosidase assays were performed as described (Cullen et al., 2000) and represent at least two independent trials. The single cell invasive growth assay (Cullen and Sprague, 2000) and the plate-washing assay (Roberts and Fink, 1994) were performed to evaluate FG. The pGAL-inducible plasmid containing the PBS2DD allele was generously provided by H. Saito (University of Tokyo, Tokyo, Japan) (Wurgler-Murphy et al., 1997). Plasmids containing FG pathway targets fused to lacZ and plasmid V84 were provided by C. Boone (University of Toronto, Toronto, Canada) (Roberts et al., 2000). Reporters included lacZ fusions to a filamentation response element (FRE; Madhani et al., 1997), MSB2, a transcriptional target that encodes an upstream regulator of the pathway (Cullen et al., 2004), KSS1, which encodes the MAPK (Figure 1; Madhani and Fink, 1997); PGU1, which encodes a secreted enzyme that hydrolyzes polygalacturonic acid, present in plant cell walls (Madhani et al., 1999), and YLR042c, which encodes a protein of unknown function (Roberts et al., 2000). Plasmid pIL30-URA3 containing FgTy-lacZ was provided by B. Errede (University of North Carolina, Chapel Hill, NC) (Laloux et al., 1994), and pFRE-lacZ was provided by H. Madhani (University of California, San Francisco, San Francisco, CA) (Madhani et al., 1997). Overexpression constructs were obtained from an ordered collection obtained from Open Biosystems (Huntsville, AL; Gelperin et al., 2005). Gene disruptions and GAL1 promoter fusions were made by PCR-based methods (Baudin et al., 1993; Longtine et al., 1998), including the use of antibiotic resistant markers (Goldstein and McCusker, 1999) and epitope fusions (Schneider et al., 1995). Integrations were confirmed by PCR analysis and phenotype. Internal epitopes were at 298 residues for the Hkr1p protein, and 500 residues for the Msb2p protein. The FUS1-lacZ reporter (typically a mating pathway reporter) was used to evaluate the activity of the FG pathway for some experiments, which in 
1278b strains lacking an intact mating pathway exhibits Msb2p-, Sho1p-, and Ste12-dependence (Cullen et al., 2004). FUS1-HIS3 expression was used to confirm FUS1-lacZ reporter data and was measured by spotting equal amounts of cells onto synthetic medium lacking histidine and containing 4-amino-1,2,4-triazole. Budding pattern was based on established methodology (Chant and Pringle, 1995) and was confirmed for some experiments by visual inspection of connected cells. Endopolygalacturonase activity of strains was assessed using the method adapted from Gainvors et al. (1994), except that equal concentrations of cells were spotted onto synthetic complete (SC) media containing 6.7 g/l of yeast nitrogen base without amino acid, 2% glucose, 1% polygalacturonic acid (Fluka, Ronkonkoma, NY), and 50 mM potassium phosphate buffer (pH 8.0). Plates were incubated for 2 d at 30°C. After incubation plates were flooded with 1% ruthenium red (Sigma, St. Louis, MO) overnight to ensure uniform staining. Plates were washed with a gentle stream of distilled water and photographed. The RCK2 gene was identified in a genetic screen using an inducible plasmid library (
YES library; Ramer et al., 1992). Plasmids were transformed into a wild-type strain (PC538), and >10,000 colonies were examined by replica plating from S-GAL-URA medium to S-GAL-URA-HIS medium to identify those that failed to express the FUS1-HIS3 reporter and failed to grow. A single plasmid-dependent isolate containing RCK2 (PC1460) was identified, which was confirmed by retesting. Plasmid mutagenesis of pSHO1-GFP was performed as described (Vadaie et al., 2008).
|
1 x 105 yeast cells were applied to 0.5-cm lateral sections of V. vinifera berries macerated with a wooden applicator. Yeast cells applied to sectioned grapes grew robustly on macerated sections more so than unperturbed sections. Inoculated tissues were incubated at 30°C. Pilot experiments using different inoculation strategies and sources of V. vinifera gave similar outcomes. To confirm that yeast cells in V. vinifera arose from input cells, strains contained plasmids carrying genes that encoded fluorescently tagged proteins (pSHO1-GFP URA3 in pRS316; Sikorski and Hieter, 1989) or pEB0876 pho4NLS-GFP in pRS316 (Strawn et al., 2004). An osmometer (5100B Vapor Pressure Osmometer, Wescor, Logan, UT) was used to determine the osmolarity of V. vinifera extracts, and Glucostix were used to estimate glucose concentrations.
For growth assays, yeast strains were inoculated onto halved and skinned V. vinifera tissues in sterile tubes containing 0.5 ml of water. Cultures were incubated for 24 h at 30°C, the mixture was vortexed briefly, and serial dilutions were plated onto synthetic media. Cell adherence was determined similarly except that nonadherent cells were removed by washing extracts. To examine the colonization of V. vinifera, inoculated extracts were examined at 96 h by embedding in 2% agarose, based on established procedures (Ruzin, 1999). Tissues embedded in agarose were cut into sections and incubated in 5% paraformaldehyde for 16 h. Paraformaldehyde-treated tissues were embedded in tissue-sectioning holders using Optimal Cutting Temperature (OCT) Compound (Tissue-Tek, Product No. 4583, Sakura, Torrance, CA) at –20°C. Approximately 15–20-µm lateral tissue sections were taken using a cryostat (Riechert-Jung, Walldorf, Germany; Cryocut 1800). Tissue sections were immediately placed onto ice-cold microscope slides and observed by microscopy. The FG pathway was not required for growth under laboratory conditions, in filtered V. vinifera extracts (not shown), or in high-glucose medium (Supplemental Table S2), which indicates that the defect does not arise from the high glucose levels found in this environment.
DNA Microarray Analysis
Wild-type (PC538), GAL-MSB2 (PC1083), and GAL-HKR1 (PC2746) strains were induced in YEP-GAL medium for 6 h. At the 6-h time point, cells were examined by microscopy for the characteristic filamentation response. RNA was prepared by hot acid phenol and passage over an RNeasy column (Qiagen, Chatsworth, CA). DNA microarray analysis was performed as described (DeRisi et al., 1997; Lashkari et al., 1997). Microarray construction, target labeling, and hybridization protocols were as described (Fazzio et al., 2001). Sample comparisons were independently replicated at least three times, each of which was derived from a separate induction. Fluoro-reverse experiments were used to identify sequence-specific dye biases. Arrays were scanned using a GenePix 4000 scanner (Axon Instruments, Burlingame, CA). Image analysis was performed using GenePix Pro3.0. Array features (i.e., spots) having low signal intensities or signals compromised by artifacts were removed from further analysis. Background subtracted Cy5/Cy3 ratios were log2 transformed and a loess normalization strategy (f = 0.67) was applied for each array using S-Plus (MathSoft, Cambridge, MA). Each feature where the |log2 (ratio)| 
0.8, the corresponding gene was considered differentially expressed.
Genomic Screening Approach
The MATa haploid deletion collection containing 4800 haploid strains (Giaever et al., 2002) was transferred to YEPD medium and YEPD medium containing 1.4 M glucose, 1 M sorbitol, or 1 M KCl. Plates were incubated for 4 d at 30°C. Deletion strains were manipulated using a 96-fixed pin pinning tool (V&P Scientific, San Diego, CA; VP 408) and a plate replication tool (V&P Scientific, VP 381) in Omnitrays (VWR International, Bridgeport, NJ.). For growth-curve analysis, strains were diluted in 96-well format in 250 µl water and pinned to YEPD, YEPD + 1.4 M glucose, and YEPD + 0.9 M NaCl liquid media. Growth curves were performed at 0, 2, 4, 6, 8, 24, 48, and 72 h using a Spectra MR spectrophotometer (Dynex, Richfield, MN). Microscopic examination of glucose-sensitive mutants showed a range of phenotypes in high-glucose medium including cell lysis, hyperpolarized growth, or no phenotype indicative of a contribution from multiple pathways in the response (data not shown). Process/function and GO annotations were made using Saccharomyces Genome Database (Cherry et al., 1998; Hong et al., 2008).
Microscopy
Differential interference contrast (DIC) and fluorescence microscopy using rhodamine, FITC, and yellow and cyan fluorescent protein (YFP and CFP, respectively) filter sets were performed using an Axioplan 2 fluorescent microscope (Zeiss, Thornwood, NY) with a Plan-Apochromat 100x/1.4 (oil) objective (NA 0.17). Digital images were obtained with the Axiocam MRm camera (Zeiss). Axiovision 4.4 software (Zeiss) was used for image acquisition and analysis and for rendering 3D Z-stack images. Images were further analyzed in Adobe Photoshop (San Jose, CA), where adjustments of brightness and contrast were made.
Protein Analysis
Immunoblots were performed as described (Cullen et al., 2004). Proteins were separated by SDS-PAGE on 10% precast gels (Bio-Rad, Hercules, CA) and transferred to nitrocellulose membranes (protran BA85, VWR International, Bridgeport, NJ). Membranes were incubated in blocking buffer (5% nonfat dry milk, 10 mM Tris-HCl, pH 8, 150 mM NaCl, and 0.05% Tween 20) for 1 h at 25°C. ECL Plus immunoblotting reagents were used to detect secondary antibodies (Amersham Biosciences, Piscataway NJ). Nitrocellulose membranes were incubated for 18 h at 4°C in blocking buffer containing a primary rabbit polyclonal IgG antibody against dually phosphorylated p38 (9211S, Cell Signaling Technology, Beverly, MA) or a mouse monoclonal IgG antibody against green fluorescent protein (GFP; Roche Diagnostics, Mannheim, Germany) to detect Hog1p-GFP. Phosphotyrosine assays were performed based on the following protocol (Reiser et al., 2000). A wild-type strain (PC538) and isogenic strain containing HOG1-GFP (PC2063) were grown to midlog phase in YEPD media at 30°C. Cells were harvested by centrifugation and resuspended in YEPD, YEPD + 1.4 M glucose, YEPD + 1 M sorbitol, or V. vinifera extracts at 30°C for 30 min. Cells were harvested and resuspended in 8 M urea, 5% SDS, and 1% ß-mercaptoethanol, 0.1 mM sodium metavanadate (Sigma), and Complete EDTA-free protease inhibitor cocktail (Roche Diagnostics). After the addition of glass beads, cells were disrupted by vortexing, and protein extracts were separated by SDS-PAGE analysis. Protein alignments were made using ClustalW (European Bioinformatics Institute, Cambridge, United Kingdom), and protein domain determinations were made using the Prosite database (ExPASy; Swiss Institute of Bioinformatics).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
, Figure 2A). Microscopic examination showed a colonization defect in FG pathway mutants (Figure 2B) that was confirmed by quantitation of cell numbers in grapes (Table 2). Yeast cells underwent FG in grapes (Figure 2C, panel 1; Table 2). Filamentous cells were observed 48 h after inoculation, presumably when nutrients had become limiting, which resulted in a mixed population of yeast-form and filamentous-form cells (Figure 2C, panels 1 and 2; and see Supplemental Figure S1C). The FG pathway was required for the cellular differentiation to the filamentous morphology in grapes (Figure 2C, panel 3; Table 2).
|
|
, Figure 2D; Supplemental Movie 2; Table 2), which is a target of the pathway that controls cell–cell and cell-substrate adherence (Guo et al., 2000; Lambrechts et al., 1996; Lo and Dranginis, 1996, 1998; Reynolds and Fink, 2001). Unexpectedly, adherence and enmeshment were mediated predominately by yeast-form cells (Figure 2D). As a result, efficient colonization of specific tissues such as veins, tissues, and cavities by yeast-form cells was observed at early time points after inoculation (16 h), as measured by 3D rendering of inoculated grape discs (Figure 2E). Therefore, yeast undergoes a filamentation response in grapes that involves multiple steps in a temporal sequence coordinated by the FG pathway.
The FG Pathway Induces a Multimodal Response
We reasoned that different aspects of FG might be induced at different nutrient levels. To test this possibility, outputs of the FG pathway were examined at different glucose concentrations, given that glucose depletion is a potent inducer of FG (Cullen and Sprague, 2000). Outputs of the FG pathway include Flo11p-dependent adhesion (Rupp et al., 1999), cell elongation (Kron et al., 1994; Madhani et al., 1999), unipolar budding (Gimeno et al., 1992; Cullen and Sprague, 2002), agar invasion (Roberts and Fink, 1994), and secretion of the plant cell wall degrading pectinase Pgu1p (Madhani et al., 1999). Flo11p- and Ste12p-dependent cell–cell adherence was observed between yeast-form cells at high concentrations of glucose (Figure 3A, 20% GLU) and was more apparent at lower glucose concentrations (Figure 3A, 2%, 0.2% GLU). Stationary phase cultures did not exhibit detectable cell–cell adherence (Figure 3A, Stationary). Pectinase secretion was similarly observed from yeast-form cells (Figure 3B). Pectinase secretion was stimulated at lower glucose concentrations (Figure 3B). Agar invasion followed a similar trend (Figure 3C). The appearance of filamentous cells, which exhibit unipolar budding and elongated cell morphologies, was observed at much lower concentrations of glucose (Figure 3A, 0.2% GLU) consistent with previous observations (Cullen and Sprague, 2000).
|
). Therefore, the FG pathway induces different aspects of FG in a sequence coupled to the nutrient-growth cycle. The response involves a linear increase in some outputs (adherence and pectinase) and stepwise induction of others (cell elongation and unipolar budding). This complex modality is consistent with the observed colonization of grapes by yeast cells.
The HOG Pathway Contributes to the Modality of FG Pathway Signaling
We undertook a genomics approach to identify genes required for growth in high-glucose environments similar to those encountered in grapes (Conde et al., 2006). Approximately 90 mutants were identified that were required for growth in YEPD + 25% glucose that were ranked by strength-of-phenotype using growth-curve analysis (Figure 4A; Supplemental Table S2). Among the mutants that showed the strongest defect were those lacking an intact HOG pathway (pbs2 and hog1; Figure 4A; Supplemental Figure S3A). The gpd1 mutant was also identified (Figure 4A), which is a HOG pathway target required for glycerol production (Albertyn et al., 1994). Genes that function in a nutritional capacity were also identified (Supplemental Table S2). A role for the HOG pathway in this context is not surprising given that high concentrations of dissolved sugars would be expected to create an osmotic imbalance. We confirmed that the HOG pathway is activated at high concentrations of glucose based on the phosphorylation (Supplemental Figure S3B) and nuclear localization (Supplemental Figure S3C) of the MAPK for the HOG pathway Hog1p (Maeda et al., 1995; Ferrigno et al., 1998). The HOG pathway was required for growth on grapes (pbs2, Figure 2, A and B, Table 2) and was activated to 80% its maximal level within minutes of exposure to this environment (Supplemental Figure S3D). High glucose represents a commonly encountered osmotic stress that connects the HOG pathway to the nutrient cycle of this organism.
|
; O'Rourke et al., 2002; Westfall and Thorner, 2006), although the relationship between the pathways has not been thoroughly examined. Deletion of the PBS2 gene caused hyperinvasive growth (Supplemental Figure S4) and hyperpolarized growth that was stimulated by increasing concentrations of glucose (Figure 4B, YEPD + 0.5M GLU). The pbs2 mutant showed elevated pectinase activity (Supplemental Figure S5A) and elevated expression of the FG pathway reporter FRE-lacZ (Supplemental Figure S7B). Likewise, overexpression of PBS2 inhibited invasive growth, cell polarization, pectinase levels, and FRE reporter expression (Figure 4C and Supplemental Figure S5A). Osmotic stresses KCl and sorbitol similarly inhibited FG (Supplemental Figure S4). Inhibition of FG by glucose or osmotic stress was more severe than seen in FG pathway mutants (Supplemental Figure S4, cf. ste12 mutant YEPD 10 d with wild-type cells YEPD + 1 M KCl 10 d), indicating that other mechanisms may contribute to the inhibition of FG that extend beyond the FG pathway. Progressive repression of FG by glucose in a concentration-dependent manner is indicative of the gradually sloping activation of the HOG pathway by increasing osmolite (Hersen et al., 2008; Supplemental Figure S7A).
Inhibition of the FG pathway might occur at the level of the Pbs2p-Sho1p and Pbs2p-Ste11p interactions (Maeda et al., 1995; Zarrinpar et al., 2004). Bypass of the inhibitory effect of GAL-PBS2 (Supplemental Figure S5, B and C) and osmotic stress (Supplemental Figure S6) was observed using hyperactive alleles MSB2100–818 (Vadaie et al., 2008), SHO1P120L (Vadaie et al., 2008), and STE11-4 (Stevenson et al., 1992). Because the alleles do not affect protein levels or presumably interfere with the Pbs2p interaction (Stevenson et al., 1992; Vadaie et al., 2008), the bypass is not likely mediated by altered association of the proteins with Pbs2p. The inhibitory effect of GAL-PBS2 or an activated allele PBS2DD (Wurgler-Murphy et al., 1997) required the MAPK Hog1p (Supplemental Figure S7, C and D, respectively), further suggesting that the HOG pathway exerts its effect through downstream MAPK signaling (i.e., targets). Overexpression of many HOG pathway components dampened FG pathway activity, including Ypd1p, Ssk1p, Ssk2p, Ssk22p, Pbs2p, Hog1p, and the transcription factor Hot1p (Figure 4D). We also identified the calmodulin-like protein kinase Rck2p, which is a target of Hog1p (Bilsland-Marchesan et al., 2000; Teige et al., 2001) in a genetic screen for genes that dampen the FG pathway when overexpressed (Figure 4D). Taken together, our results are consistent with an inhibitory role for the HOG pathway on the FG pathway that is potentially mediated at multiple levels.
Msb2p and Hkr1p Differentially Activate the FG Pathway
The signaling mucin Hkr1p functions redundantly with Msb2p in the HOG pathway (Tatebayashi et al., 2007), but its role in the FG pathway has not been examined. To determine whether Hkr1p functions in the FG pathway, the HKR1 gene was disrupted in a wild-type strain of the 1278b background and assessed for FG and MAPK activity. The hkr1 mutant did not show a defect in agar invasion (Figure 5A), whereas the msb2 mutant was defective for invasive growth (Figure 5A; Cullen et al., 2004). Deletion of HKR1 in an msb2 mutant restored agar invasion (Figure 5A), which indicates that Hkr1p negatively regulates the FG pathway in this context. Deletion of HKR1 in the sho1 mutant failed to restore agar invasion (Figure 5A), indicating that the inhibitory effect of Hkr1p is mediated through Sho1p. The expression of FG pathway reporters corroborated the invasive growth phenotypes (Figure 5B). Overexpression of HKR1 did not induce agar invasion (Figure 5C), unlike overexpression of MSB2, which caused hyperinvasive growth (Figure 5D; Cullen et al., 2004). In fact, overexpression of HKR1 dampened MAPK activity in cells carrying activated alleles of MSB2 and SHO1 (Figure 5D). Together, these results demonstrate that Hkr1p is not a component of the FG pathway and has an inhibitory effect on the pathway in certain genetic contexts.
|
35 genes, 14 of which are targets of the HOG pathway (Figure 6A, HOG; O'Rourke and Herskowitz, 2004; Posas et al., 2000). Overexpression of HKR1 did not induce FG pathway targets. Overexpression of MSB2 induced the expression of 30 genes that include FG pathway targets (Figure 6B, FG; Madhani et al., 1999; Roberts et al., 2000) and genes involved in nutritional scavenging (Figure 6B, S) but not HOG pathway targets. Msb2p-dependent induction of FG pathway targets might be underrepresented because under this condition (YEP-GAL) FG targets are induced in wild-type cells.
|
; Singh and Hollingsworth, 2006). Msb2p and Hkr1p represent the only members of that family in S. cerevisiae (Laura Grell and P. J. Cullen, unpublished observations). Like other signaling mucins, Msb2p and Hkr1p are single-pass transmembrane proteins that contain tandem S/T/P-rich repeats in their extracellular domains (Figure 7A). Despite the similarity in overall topology, the proteins exhibit little sequence similarity in their open reading frames (ORFs) or upstream regulatory domains (Figure 7A).
|
), and an HKR1-lacZ fusion was expressed at lower levels than an MSB2-lacZ fusion (Figure 7B). HKR1-lacZ expression was modestly induced by osmotic stress (Figure 7B), whereas MSB2-lacZ expression was somewhat reduced (Figure 7B). MSB2-lacZ expression was induced under nutrient-limiting conditions that promote FG (Figure 7B). The MSB2 promoter contains upstream regulatory elements for the transcription factors Tec1p and Ste12p (Figure 7A), and its expression is induced by an autofeedback loop (Cullen et al., 2004). Starvation-dependent induction of MSB2-lacZ was dependent on Sho1p and Ste12p (Figure 7B) and was not observed using a construct that contains a point mutation in the Ste12p-consensus binding site (MSB2AG-lacZ; Figure 7, A and B). The upstream regulatory sequence of the HKR1 promoter does not contain FG pathway regulatory elements (Figure 7A), and the HKR1-lacZ reporter did not show Sho1p or Ste12p-dependent induction (Figure 7B) or induction by nutrient limitation (Figure 7B). Published expression profiling data closely agree with these results (O'Rourke and Herskowitz, 2004).
Msb2p is processed as part of its activation mechanism, and the glycosylated extracellular domain of the protein is shed from cells (Vadaie et al., 2008). Colony blot analysis showed that the extracellular domain of an epitope-tagged Hkr1p-hemagglutinin (HA) fusion was also shed (Figure 7C). Processing of Msb2p correlates with its activity and is elevated under glucose-limiting conditions (Figure 7D; Vadaie et al., 2008). Shedding of Hkr1p-HA was not induced by nutrient limitation (Figure 7D). Osmotic shock did not cause a boost in secretion of either mucin (Figure 7D); however, osmotic shock may induce the formation of cell-surface vesicles that trap shed proteins (see below), which complicates the interpretation of this result. The expression and secretion profiles of the two mucins are consistent with the idea that they primarily function in different pathways.
Msb2p Induces Sho1p-Dependent Polarization, Whereas Hkr1p Does Not
Msb2p and Hkr1p function through the adaptor Sho1p (Tatebayashi et al., 2007; Vadaie et al., 2008) and may exert different effects on the Sho1p protein. To test this possibility, we first examined the properties of the Sho1p protein with respect to FG and HOG pathway activation. Sho1p contains four membrane-spanning domains and a cytoplasmic SH3 domain. Biochemical analysis showed that the protein migrates as a dimer (Supplemental Figure S8) as previously reported (Hao et al., 2007) and mutational analysis identified a residue (S149F) adjacent to the tetraspan domain that was required for the Sho1p-Sho1p interaction (Supplemental Figure S8). The Sho1p-Sho1p interaction was not affected by nutrient limitation, osmotic stress, or the activation state of the protein (Supplemental Figure S8), which indicates that the oligomerization of Sho1p may not underlie its specification between pathways.
The localization of Sho1p was examined in response to nutrient limitation and osmotic stress. In nutrient-rich conditions, Sho1p-GFP was localized preferentially to buds as previously reported (Figure 8A; Raitt et al., 2000; Reiser et al., 2000). Under nutrient-limiting conditions, Sho1p-GFP localization to highly polarized sites (Figure 8A) was dependent on the SH3 domain of the protein (Supplemental Figure S10). In response to osmotic shock, Sho1p-GFP localized to punctate sites (Figure 8B) that did not require the SH3 domain or Pbs2p (Figure 8B). The punctate pattern is indicative of global invaginations in the plasma membrane (PM;Slaninova et al., 2000), and Sln1p, Msb2p, and Hkr1p have been reported to localize to similar sites in response to osmotic shock (Reiser et al., 2003; Tatebayashi et al., 2007). Indeed, other cell-surface proteins like Bud8p-GFP showed a similar punctate pattern in response to osmostress (Figure 8B). Structural deconvolution followed by 3D rendering confirmed that Sho1p-GFP was localized to cell-surface sites that were stable with respect to each other and the cell periphery (Supplemental Figure S9; Supplemental Movies 3 and 4). The punctate sites did not overlap with eisosomes, static sites for endocytosis of receptor complexes (Walther et al., 2006), as visualized by colocalization with an eisosome-specific marker (Figure 8C).
|
; Lillie and Brown, 1994; Yuzyuk et al., 2002; Yuzyuk and Amberg, 2003). The localization of Sho1p to punctate sites was independent of the depolarization of the actin cytoskeleton (Figure 8D). The localization of Sho1p to punctate sites and concomitant depolarization of the actin cytoskeleton may contribute to the lack of Sho1p- and MAPK-dependent polarization during osmotic stress.
Msb2p interacts with the polarity establishment GTPase Cdc42p (Cullen et al., 2004). In cells overexpressing MSB2, cells were hyperpolarized (Figure 8E). This phenotype was partly independent of the MAPK pathway but required Sho1p (Figure 8E). Overexpression of Hkr1p inhibited the hyperpolarized growth of cells containing GAL-MSB2 (Figure 8E), whereas deletion of HKR1 had the opposite effect (Figure 8E). The two mucins also induced different localization patterns of Sho1p-GFP, likely due to their differential effects on cell polarization (Supplemental Figure S10). The inhibitory effect of Hkr1p on cell polarization may or may not be direct, as activation of the HOG pathway by other means exerts a similar effect (Supplemental Figure S10). Therefore, Msb2p can be functionally distinguished from Hkr1p by its ability to induce cell polarization through Sho1p.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Different Signaling Mucins Function Preferentially in Different MAPK Pathways
We have identified a potentially new point of discrimination between the FG and HOG pathways at the level of the signaling mucins Msb2p and Hkr1p. This discovery represents an important contribution, because Hkr1p is the first HOG pathway component to be identified at the pathway's head that does not also function in the FG pathway (among the Msb2p, Sho1p, Cdc42p, Ste20p, Ste50p, and Ste11p proteins; Figure 1). A straightforward possibility supported by this study is that Msb2p functions primarily in the FG pathway, whereas Hkr1p functions mainly in the HOG pathway. Msb2p appears to function as a dedicated component of the FG pathway, based in its ability to induce cell polarization, its expression and secretion profiles, and its processing, which occurs under nutrient-limiting conditions and is required for activation of the FG pathway (Vadaie et al., 2008). Msb2p might functionally substitute for Hkr1p in the HOG pathway (Tatebayashi et al., 2007) in a manner that is analogous to the MAPKs Fus3p and Kss1p, which function primarily in the mating and FG pathways, respectively, but functionally substitute for each other if one MAPK is absent (Madhani et al., 1997).
Likewise, Hkr1p appears to function as a HOG pathway–specific factor. Hkr1p is not required for FG pathway signaling and when overexpressed does not induce FG pathway targets. Expression of the HKR1 gene is not induced by nutrient limitation and is not under the control of the FG pathway. In some settings, Hkr1p inhibits the FG pathway at the level of Sho1p. Taken together, our results indicate that the two mucins function preferentially in different MAPK pathways. This conclusion may slightly oversimplify the biological roles of the two mucins, as their overexpression also induces target genes (such as nonoverlapping genes involved in cell wall biosynthesis, mitochondrial function, and sporulation) that have not been established as targets of either MAPK pathway.
It is not yet clear how Msb2p and Hkr1p mediate their different outputs. An attractive possibility is they recruit different proteins to the cell surface through their cytoplasmic signaling domains, which bear little similarity to each other. The cytoplasmic tail of Msb2p associates directly with Cdc42p and contributes to cell polarization. Hkr1p contains a distinctly different cytoplasmic domain that includes a leucine zipper motif. Both proteins interact with Sho1p, and the conditional inhibition of Sho1p by Hkr1p in cells lacking Msb2p might be explained if the mucins compete for a binding site in the Sho1p protein. Discrimination between different pathway outputs may extend to other members of the signaling mucin family. The mammalian mucin MUC4 imparts specificity in MAPK pathway outputs through differential polarization of the ErbB2 receptor (Ramsauer et al., 2006).
Multimodality of the Filamentation MAPK Pathway
We have found that the FG pathway induces different aspects of FG at different nutrient levels. This conclusion is supported by the fact that different aspects of FG are genetically separable (Cullen and Sprague, 2002) and resolves an established paradox with respect to nutrition and FG pathway signaling. FG is potently triggered by glucose depletion (Cullen and Sprague, 2000; Kuchin et al., 2003), yet the pathway is active in nutrient-rich environments (Cullen et al., 2004; Madhani et al., 1999), in protein glycosylation mutants (Cullen et al., 2000), in mutants that exhibit cross talk (O'Rourke and Herskowitz, 1998), and during mat-form growth (Reynolds and Fink, 2001). The fact that the FG pathway does not exist in ON and OFF states but is active throughout the nutrient-growth cycle unifies these apparently paradoxical observations.
Other MAPK pathways, like the pheromone response pathway, similarly exhibit multimodality (Hao et al., 2008; Moore, 1983). A unique aspect of multimodality in FG pathway regulation is by its inhibition mediated by the HOG pathway, which shares components with the FG pathway but functions in a mutually exclusive manner. HOG pathway-dependent inhibition of the FG pathway accounts for one aspect of pathway modality at high concentrations of glucose (>20–2%). Regulatory feed ins at other glucose concentrations may occur through nutrient-regulatory pathways such as the RAS pathway (Mosch et al., 1996) and the ATP/AMP kinase Snf1p (Hedbacker and Carlson, 2008). In a broad sense, our findings might provide an explanation for why the FG and HOG MAPK pathways have maintained the sharing of common components over evolutionary time, to create mutually exclusive activation states that function in graded opposition as part of a coordinated behavior. Multimodality resulting from exclusive inhibition may extend to other MAPK pathways that share components.
Yeast Foraging and Fungal Pathogenesis
Fungal pathogens remain a significant threat to many aspects of human health (Sanchez and Larsen, 2007), in part because their behaviors in complex environments like metazoan tissues remains largely unclear (Chandra et al., 2005; Lo et al., 1997). By examining yeast foraging in a complex natural setting, we show that adherence and penetration functions occur before dimorphism. Some pathogenic fungi like Cryptococcus neoformans exhibit invasive properties and virulence in the yeast form (Lin and Heitman, 2006), which underscores the potency of these poorly understood processes in pathogenic efficacy. Pathogens moreover induce target genes in a spatiotemporal hierarchy by the induction of early and late genes (Schlumberger and Hardt, 2006). Likewise, budding yeast induces distinct facets of FG in a spatiotemporal hierarchy coupled to the nutrient-growth cycle. Future studies of yeast behaviors in natural settings will build on the overall understanding of fungal foraging that might aid in our appreciation of fungal pathogenesis.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
* These authors contributed equally to this work. 
Address correspondence to: Paul J. Cullen (pjcullen{at}buffalo.edu).
Abbreviations used: FG, filamentous growth; FRE, filamentation response element; GEF, guanine nucleotide exchange factor; GFP, YFP, and CFP, green, yellow, and cyan fluorescent protein, respectively; HA, hemagglutinin; HOG, high osmolarity glycerol response; MAPK, mitogen-activated protein kinase; ORF, open reading frame; PAK, p21 activated kinase; PM, plasma membrane; SC, synthetic complete.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Bardwell, L. (2004). A walk-through of the yeast mating pheromone response pathway. Peptides 25, 1465–1476.[CrossRef][Medline]
Bardwell, L. (2006). Mechanisms of MAPK signalling specificity. Biochem. Soc. Trans 34, 837–841.[CrossRef][Medline]
Baudin, A., Ozier-Kalogeropoulos, O., Denouel, A., Lacroute, F., and Cullin, C. (1993). A simple and efficient method for direct gene deletion in Saccharomyces cerevisiae. Nucleic Acids Res 21, 3329–3330.
Bhattacharyya, R. P., Remenyi, A., Yeh, B. J., and Lim, W. A. (2006). Domains, motifs, and scaffolds: the role of modular interactions in the evolution and wiring of cell signaling circuits. Annu. Rev. Biochem 75, 655–680.[CrossRef][Medline]
Bilsland-Marchesan, E., Arino, J., Saito, H., Sunnerhagen, P., and Posas, F. (2000). Rck2 kinase is a substrate for the osmotic stress-activated mitogen-activated protein kinase Hog1. Mol. Cell. Biol 20, 3887–3895.
Borneman, A. R., Gianoulis, T. A., Zhang, Z. D., Yu, H., Rozowsky, J., Seringhaus, M. R., Wang, L. Y., Gerstein, M., and Snyder, M. (2007). Divergence of transcription factor binding sites across related yeast species. Science 317, 815–819.
Carraway, K. L., Ramsauer, V. P., Haq, B., and Carothers Carraway, C. A. (2003). Cell signaling through membrane mucins. Bioessays 25, 66–71.[CrossRef][Medline]
Cavalieri, D., McGovern, P. E., Hartl, D. L., Mortimer, R., and Polsinelli, M. (2003). Evidence for S. cerevisiae fermentation in ancient wine. J. Mol. Evol 57, (Suppl 1), S226–S232.[CrossRef][Medline]
Chandra, J., Zhou, G., and Ghannoum, M. A. (2005). Fungal biofilms and antimycotics. Curr. Drug Targets 6, 887–894.[CrossRef][Medline]
Chant, J., and Pringle, J. R. (1995). Patterns of bud-site selection in the yeast Saccharomyces cerevisiae. J. Cell Biol 129, 751–765.
Chen, R. E., and Thorner, J. (2007). Function and regulation in MAPK signaling pathways: lessons learned from the yeast Saccharomyces cerevisiae. Biochim. Biophys. Acta 1773, 1311–1340.[Medline]
Cherry, J. M. et al. (1998). SGD: Saccharomyces Genome Database. Nucleic Acids Res 26, 73–79.
Chowdhury, S., Smith, K. W., and Gustin, M. C. (1992). Osmotic stress and the yeast cytoskeleton: phenotype-specific suppression of an actin mutation. J. Cell Biol 118, 561–571.
Conde, C., Agasse, A., Glissant, D., Tavares, R., Geros, H., and Delrot, S. (2006). Pathways of glucose regulation of monosaccharide transport in grape cells. Plant Physiol 141, 1563–1577.
Cullen, P. J., Sabbagh, W., Jr, Graham, E., Irick, M. M., van Olden, E. K., Neal, C., Delrow, J., Bardwell, L., and Sprague, G. F., Jr. (2004). A signaling mucin at the head of the Cdc42- and MAPK-dependent filamentous growth pathway in yeast. Genes Dev 18, 1695–1708.
Cullen, P. J., Schultz, J., Horecka, J., Stevenson, B. J., Jigami, Y., and Sprague, G. F., Jr. (2000). Defects in protein glycosylation cause SHO1-dependent activation of a STE12 signaling pathway in yeast. Genetics 155, 1005–1018.
Cullen, P. J., and Sprague, G. F., Jr. (2000). Glucose depletion causes haploid invasive growth in yeast. Proc. Natl. Acad. Sci. USA 97, 13619–13624.
Cullen, P. J., and Sprague, G. F., Jr. (2002). The roles of bud-site-selection proteins during haploid invasive growth in yeast. Mol. Biol. Cell 13, 2990–3004.
Davenport, K. D., Williams, K. E., Ullmann, B. D., and Gustin, M. C. (1999). Activation of the Saccharomyces cerevisiae filamentation/invasion pathway by osmotic stress in high-osmolarity glycogen pathway mutants. Genetics 153, 1091–1103.
DeRisi, J. L., Iyer, V. R., and Brown, P. O. (1997). Exploring the metabolic and genetic control of gene expression on a genomic scale. Science 278, 680–686.
Dohlman, H. G., and Slessareva, J. E. (2006). Pheromone signaling pathways in yeast. Sci. STKE 2006, cm6.
Errede, B., Cade, R. M., Yashar, B. M., Kamada, Y., Levin, D. E., Irie, K., and Matsumoto, K. (1995). Dynamics and organization of MAP kinase signal pathways. Reprod. Dev 42, 477–485.[CrossRef]
Ferrigno, P., Posas, F., Koepp, D., Saito, H., and Silver, P. A. (1998). Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1. EMBO J 17, 5606–5614.[CrossRef][Medline]
Gainvors, A., Frezier, V., Lemaresquier, H., Lequart, C., Aigle, M., and Belarbi, A. (1994). Detection of polygalacturonase, pectin-lyase and pectin-esterase activities in a Saccharomyces cerevisiae strain. Yeast 10, 1311–1319.[CrossRef][Medline]
Gelperin, D. M. et al. (2005). Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. Genes Dev 19, 2816–2826.
Giaever, G. et al. (2002). Functional profiling of the Saccharomyces cerevisiae genome. Nature 418, 387–391.[CrossRef][Medline]
Gimeno, C. J., Ljungdahl, P. O., Styles, C. A., and Fink, G. R. (1992). Unipolar cell divisions in the yeast S. cerevisiae lead to filamentous growth: regulation by starvation and RAS. Cell 68, 1077–1090.[CrossRef][Medline]
Goldstein, A. L., and McCusker, J. H. (1999). Three new dominant drug resistance cassettes for gene disruption in Saccharomyces cerevisiae. Yeast 15, 1541–1553.[CrossRef][Medline]
Guo, B., Styles, C. A., Feng, Q., and Fink, G. R. (2000). A Saccharomyces gene family involved in invasive growth, cell-cell adhesion, and mating. Proc. Natl. Acad. Sci. USA 97, 12158–12163.
Hao, N., Behar, M., Parnell, S. C., Torres, M. P., Borchers, C. H., Elston, T. C., and Dohlman, H. G. (2007). A systems-biology analysis of feedback inhibition in the Sho1 osmotic-stress-response pathway. Curr. Biol 17, 659–667.[CrossRef][Medline]
Hao, N., Nayak, S., Behar, M., Shanks, R. H., Nagiec, M. J., Errede, B., Hasty, J., Elston, T. C., and Dohlman, H. G. (2008). Regulation of cell signaling dynamics by the protein kinase-scaffold Ste5. Mol. Cell 30, 649–656.[Medline]
Hedbacker, K., and Carlson, M. (2008). SNF1/AMPK pathways in yeast. Front. Biosci 13, 2408–2420.[CrossRef][Medline]
Hersen, P., McClean, M. N., Mahadevan, L., and Ramanathan, S. (2008). Signal processing by the HOG MAP kinase pathway. Proc. Natl. Acad. Sci. USA 105, 7165–7170.
Hohmann, S. (2002). Osmotic stress signaling and osmoadaptation in yeasts. Microbiol. Mol. Biol. Rev 66, 300–372.
Hong, E. L. et al. (2008). Gene Ontology annotations at SGD: new data sources and annotation methods. Nucleic Acids Res 36, D577–D581.
Jansen, G., Buhring, F., Hollenberg, C. P., and Ramezani Rad, M. (2001). Mutations in the SAM domain of STE50 differentially influence the MAPK-mediated pathways for mating, filamentous growth and osmotolerance in Saccharomyces cerevisiae. Mol. Genet. Genom 265, 102–117.[CrossRef][Medline]
Johnson, D. I. (1999). Cdc 42, An essential Rho-type GTPase controlling eukaryotic cell polarity. Microbiol. Mol. Biol. Rev 63, 54–105.
Kasahara, S., Yamada, H., Mio, T., Shiratori, Y., Miyamoto, C., Yabe, T., Nakajima, T., Ichishima, E., and Furuichi, Y. (1994). Cloning of the Saccharomyces cerevisiae gene whose overexpression overcomes the effects of HM-1 killer toxin, which inhibits beta-glucan synthesis. J. Bacteriol 176, 1488–1499.
Kron, S. J., Styles, C. A., and Fink, G. R. (1994). Symmetric cell division in pseudohyphae of the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 5, 1003–1022.[Abstract]
Kuchin, S., Vyas, V. K., and Carlson, M. (2003). Role of the yeast Snf1 protein kinase in invasive growth. Biochem. Soc. Trans 31, 175–177.[Medline]
Laloux, I., Jacobs, E., and Dubois, E. (1994). Involvement of SRE element of Ty1 transposon in TEC1-dependent transcriptional activation. Nucleic Acids Res 22, 999–1005.
Lambrechts, M. G., Bauer, F. F., Marmur, J., and Pretorius, I. S. (1996). Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast. Proc. Natl. Acad. Sci. USA 93, 8419–8424.
Lashkari, D. A., DeRisi, J. L., McCusker, J. H., Namath, A. F., Gentile, C., Hwang, S. Y., Brown, P. O., and Davis, R. W. (1997). Yeast microarrays for genome wide parallel genetic and gene expression analysis. Proc. Natl. Acad. Sci. USA 94, 13057–13062.
Leberer, E., Wu, C., Leeuw, T., Fourest-Lieuvin, A., Segall, J. E., and Thomas, D. Y. (1997). Functional characterization of the Cdc42p binding domain of yeast Ste20p protein kinase. EMBO J 16, 83–97.[CrossRef][Medline]
Lillie, S. H., and Brown, S. S. (1994). Immunofluorescence localization of the unconventional myosin, Myo2p, and the putative kinesin-related protein, Smy1p, to the same regions of polarized growth in Saccharomyces cerevisiae. J. Cell Biol 125, 825–842.
Lin, X., and Heitman, J. (2006). The biology of the Cryptococcus neoformans species complex. Annu. Rev. Microbiol 60, 69–105.[CrossRef][Medline]
Liu, H., Styles, C. A., and Fink, G. R. (1993). Elements of the yeast pheromone response pathway required for filamentous growth of diploids. Science 262, 1741–1744.
Lo, H. J., Kohler, J. R., DiDomenico, B., Loebenberg, D., Cacciapuoti, A., and Fink, G. R. (1997). Nonfilamentous C. albicans mutants are avirulent. Cell 90, 939–949.[CrossRef][Medline]
Lo, W. S., and Dranginis, A. M. (1996). FLO11, a yeast gene related to the STA genes, encodes a novel cell surface flocculin. J. Bacteriol 178, 7144–7151.
Lo, W. S., and Dranginis, A. M. (1998). The cell surface flocculin Flo11 is required for pseudohyphae formation and invasion by Saccharomyces cerevisiae. Mol. Biol. Cell 9, 161–171.
Longtine, M. S., McKenzie, A., 3rd, Demarini, D. J., Shah, N. G., Wach, A., Brachat, A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961.[CrossRef][Medline]
Madhani, H. D., and Fink, G. R. (1997). Combinatorial control required for the specificity of yeast MAPK signaling. Science 275, 1314–1317.
Madhani, H. D., Galitski, T., Lander, E. S., and Fink, G. R. (1999). Effectors of a developmental mitogen-activated protein kinase cascade revealed by expression signatures of signaling mutants. Proc. Natl. Acad. Sci. USA 96, 12530–12535.
Madhani, H. D., Styles, C. A., and Fink, G. R. (1997). MAP kinases with distinct inhibitory functions impart signaling specificity during yeast differentiation. Cell 91, 673–684.[CrossRef][Medline]
Maeda, T., Takekawa, M., and Saito, H. (1995). Activation of yeast PBS2 MAPKK by MAPKKKs or by binding of an SH3-containing osmosensor. Science 269, 554–558.
Maeda, T., Wurgler-Murphy, S. M., and Saito, H. (1994). A two-component system that regulates an osmosensing MAP kinase cascade in yeast. Nature 369, 242–245.[CrossRef][Medline]
Mas, G., de Nadal, E., Dechant, R., de la Concepcion, M. L., Logie, C., Jimeno-Gonzalez, S., Chavez, S., Ammerer, G., and Posas, F. (2009). Recruitment of a chromatin remodelling complex by the Hog1 MAP kinase to stress genes. EMBO J 28, 326–336.[CrossRef][Medline]
McClean, M. N., Mody, A., Broach, J. R., and Ramanathan, S. (2007). Cross-talk and decision making in MAP kinase pathways. Nat. Genet 39, 409–414.[CrossRef][Medline]
McCusker, J. H., Clemons, K. V., Stevens, D. A., and Davis, R. W. (1994). Genetic characterization of pathogenic Saccharomyces cerevisiae isolates. Genetics 136, 1261–1269.[Abstract]
Moore, S. A. (1983). Comparison of dose-response curves for alpha factor-induced cell division arrest, agglutination, and projection formation of yeast cells. Implication for the mechanism of alpha factor action. J. Biol. Chem 258, 13849–13856.
Mosch, H. U., Roberts, R. L., and Fink, G. R. (1996). Ras2 signals via the Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93, 5352–5356.
Murphy, L. O., and Blenis, J. (2006). MAPK signal specificity: the right place at the right time. Trends Biochem. Sci 31, 268–275.[CrossRef][Medline]
O'Rourke, S. M., and Herskowitz, I. (1998). The Hog1 MAPK prevents cross talk between the HOG and pheromone response MAPK pathways in Saccharomyces cerevisiae. Genes Dev 12, 2874–2886.
O'Rourke, S. M., and Herskowitz, I. (2004). Unique and redundant roles for HOG MAPK pathway components as revealed by whole-genome expression analysis. Mol. Biol. Cell 15, 532–542.
O'Rourke, S. M., Herskowitz, I., and O'Shea, E. K. (2002). Yeast go the whole HOG for the hyperosmotic response. Trends Genet 18, 405–412.[CrossRef][Medline]
Peter, M., Neiman, A. M., Park, H. O., van Lohuizen, M., and Herskowitz, I. (1996). Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J 15, 7046–7059.[Medline]
Posas, F., Chambers, J. R., Heyman, J. A., Hoeffler, J. P., de Nadal, E., and Arino, J. (2000). The transcriptional response of yeast to saline stress. J. Biol. Chem 275, 17249–17255.
Posas, F., Witten, E. A., and Saito, H. (1998). Requirement of STE50 for osmostress-induced activation of the STE11 mitogen-activated protein kinase kinase kinase in the high-osmolarity glycerol response pathway. Mol. Cell. Biol 18, 5788–5796.
Qi, M., and Elion, E. A. (2005). MAP kinase pathways. J. Cell Sci 118, 3569–3572.
Raitt, D. C., Posas, F., and Saito, H. (2000). Yeast Cdc42 GTPase and Ste20 PAK-like kinase regulate Sho1-dependent activation of the Hog1 MAPK pathway. EMBO J 19, 4623–4631.[CrossRef][Medline]
Ramer, S. W., Elledge, S. J., and Davis, R. W. (1992). Dominant genetics using a yeast genomic library under the control of a strong inducible promoter. Proc. Natl. Acad. Sci. USA 89, 11589–11593.
Ramsauer, V. P., Pino, V., Farooq, A., Carothers Carraway, C. A., Salas, P. J., and Carraway, K. L. (2006). Muc4-ErbB2 complex formation and signaling in polarized CACO-2 epithelial cells indicate that Muc4 acts as an unorthodox ligand for ErbB2. Mol. Biol. Cell 17, 2931–2941.
Reiser, V., Raitt, D. C., and Saito, H. (2003). Yeast osmosensor Sln1 and plant cytokinin receptor Cre1 respond to changes in turgor pressure. J. Cell Biol 161, 1035–1040.
Reiser, V., Salah, S. M., and Ammerer, G. (2000). Polarized localization of yeast Pbs2 depends on osmostress, the membrane protein Sho1 and Cdc42. Nat. Cell Biol 2, 620–627.[CrossRef][Medline]
Reynolds, T. B., and Fink, G. R. (2001). Bakers' yeast, a model for fungal biofilm formation. Science 291, 878–881.
Roberts, C. J. et al. (2000). Signaling and circuitry of multiple MAPK pathways revealed by a matrix of global gene expression profiles. Science 287, 873–880.
Roberts, R. L., and Fink, G. R. (1994). Elements of a single MAP kinase cascade in Saccharomyces cerevisiae mediate two developmental programs in the same cell type: mating and invasive growth. Genes Dev 8, 2974–2985.
Rose, M. D., Winston, F., and Hieter, P. (1990). Methods in Yeast Genetics, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Rupp, S., Summers, E., Lo, H. J., Madhani, H., and Fink, G. (1999). MAP kinase and cAMP filamentation signaling pathways converge on the unusually large promoter of the yeast FLO11 gene. EMBO J 18, 1257–1269.[CrossRef][Medline]
Ruzin, S. E. (1999). Plant Microtechniques and Microscopy, New York: Oxford University Press, 73–87.
Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Sanchez, A., and Larsen, R. (2007). Emerging fungal pathogens in pulmonary disease. Curr. Opin. Pulm. Med 13, 199–204.[Medline]
Santen, R. J., Song, R. X., McPherson, R., Kumar, R., Adam, L., Jeng, M. H., and Yue, W. (2002). The role of mitogen-activated protein (MAP) kinase in breast cancer. J. Steroid. Biochem. Mol. Biol 80, 239–256.[CrossRef][Medline]
Schlumberger, M. C., and Hardt, W. D. (2006). Salmonella type III secretion effectors: pulling the host cell's strings. Curr. Opin. Microbiol 9, 46–54.[CrossRef][Medline]
Schneider, B. L., Seufert, W., Steiner, B., Yang, Q. H., and Futcher, A. B. (1995). Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae. Yeast 11, 1265–1274.[CrossRef][Medline]
Schwartz, M. A., and Madhani, H. D. (2004). Principles of map kinase signaling specificity in Saccharomyces cerevisiae. Annu. Rev. Genet 38, 725–748.[CrossRef][Medline]
Sikorski, R. S., and Hieter, P. (1989). A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122, 19–27.
Singh, P. K., and Hollingsworth, M. A. (2006). Cell surface-associated mucins in signal transduction. Trends Cell Biol 16, 467–476.[CrossRef][Medline]
Slaninova, I., Sestak, S., Svoboda, A., and Farkas, V. (2000). Cell wall and cytoskeleton reorganization as the response to hyperosmotic shock in Saccharomyces cerevisiae. Arch. Microbiol 173, 245–252.[CrossRef][Medline]
Stevenson, B. J., Rhodes, N., Errede, B., and Sprague, G. F., Jr. (1992). Constitutive mutants of the protein kinase STE11 activate the yeast pheromone response pathway in the absence of the G protein. Genes Dev 6, 1293–1304.
Strawn, L. A., Shen, T., Shulga, N., Goldfarb, D. S., and Wente, S. R. (2004). Minimal nuclear pore complexes define FG repeat domains essential for transport. Nat. Cell Biol 6, 197–206.[Medline]
Tatebayashi, K., Tanaka, K., Yang, H. Y., Yamamoto, K., Matsushita, Y., Tomida, T., Imai, M., and Saito, H. (2007). Transmembrane mucins Hkr1 and Msb2 are putative osmosensors in the SHO1 branch of yeast HOG pathway. EMBO J 26, 3521–3533.[CrossRef][Medline]
Tatebayashi, K., Yamamoto, K., Tanaka, K., Tomida, T., Maruoka, T., Kasukawa, E., and Saito, H. (2006). Adaptor functions of Cdc42, Ste50, and Sho1 in the yeast osmoregulatory HOG MAPK pathway. EMBO J 25, 3033–3044.[CrossRef][Medline]
Teige, M., Scheikl, E., Reiser, V., Ruis, H., and Ammerer, G. (2001). Rck2, a member of the calmodulin-protein kinase family, links protein synthesis to high osmolarity MAP kinase signaling in budding yeast. Proc. Natl. Acad. Sci. USA 98, 5625–5630.
Truckses, D. M., Bloomekatz, J. E., and Thorner, J. (2006). The RA domain of Ste50 adaptor protein is required for delivery of Ste11 to the plasma membrane in the filamentous growth signaling pathway of the yeast Saccharomyces cerevisiae. Mol. Cell. Biol 26, 912–928.
Vadaie, N., Dionne, H., Akajagbor, D. S., Nickerson, S. R., Krysan, D. J., and Cullen, P. J. (2008). Cleavage of the signaling mucin Msb2 by the aspartyl protease Yps1 is required for MAPK activation in yeast. J. Cell Biol 181, 1073–1081.
Walther, T. C., Brickner, J. H., Aguilar, P. S., Bernales, S., Pantoja, C., and Walter, P. (2006). Eisosomes mark static sites of endocytosis. Nature 439, 998–1003.[CrossRef][Medline]
Westfall, P. J., and Thorner, J. (2006). Analysis of mitogen-activated protein kinase signaling specificity in response to hyperosmotic stress: use of an analog-sensitive HOG1 allele. Eukaryot. Cell 5, 1215–1228.
Whiteway, M., and Bachewich, C. (2007). Morphogenesis in Candida albicans. Annu. Rev. Microbiol 61, 529–553.[CrossRef][Medline]
Wurgler-Murphy, S. M., Maeda, T., Witten, E. A., and Saito, H. (1997). Regulation of the Saccharomyces cerevisiae HOG1 mitogen-activated protein kinase by the PTP2 and PTP3 protein tyrosine phosphatases. Mol. Cell. Biol 17, 1289–1297.
Yang, H. C., and Pon, L. A. (2002). Actin cable dynamics in budding yeast. Proc. Natl. Acad. Sci. USA 99, 751–756.
Yuzyuk, T., and Amberg, D. C. (2003). Actin recovery and bud emergence in osmotically stressed cells requires the conserved actin interacting mitogen-activated protein kinase kinase kinase Ssk2p/MTK1 and the scaffold protein Spa2p. Mol. Biol. Cell 14, 3013–3026.
Yuzyuk, T., Foehr, M., and Amberg, D. C. (2002). The MEK kinase Ssk2p promotes actin cytoskeleton recovery after osmotic stress. Mol. Biol. Cell 13, 2869–2880.
Zarrinpar, A., Bhattacharyya, R. P., Nittler, M. P., and Lim, W. A. (2004). Sho1 and Pbs2 act as coscaffolds linking components in the yeast high osmolarity MAP kinase pathway. Mol. Cell 14, 825–832.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  U. Abdullah and P. J. Cullen The tRNA Modification Complex Elongator Regulates the Cdc42-Dependent Mitogen-Activated Protein Kinase Pathway That Controls Filamentous Growth in Yeast Eukaryot. Cell, September 1, 2009; 8(9): 1362 - 1372. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Birkaya, A. Maddi, J. Joshi, S. J. Free, and P. J. Cullen Role of the Cell Wall Integrity and Filamentous Growth Mitogen-Activated Protein Kinase Pathways in Cell Wall Remodeling during Filamentous Growth Eukaryot. Cell, August 1, 2009; 8(8): 1118 - 1133. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
G –479 from transcriptional start site). Regions of sequence homology (47% identity by ClustalW analysis) are shown in yellow, which overlaps the cleavage domain of Msb2p (CD) and may demark the potential CD for Hkr1p (CD?). Regions of the proteins that show <20% homology are shown in green for Msb2p and red for Hkr1p. The mucin homology domain (MHD), transmembrane domain (TM), and signal peptide (SP) domains are also labeled. The S/T-rich regions are shown in purple. LZ, leucine zipper. (B) Wild-type (PC2519), sho1