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Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, California 94143-0448
Received 20 December 2001/ Returned for modification 1 February 2002/ Accepted 29 March 2002
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ABSTRACT |
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INTRODUCTION |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In Saccharomyces cerevisiae, two structurally unrelated membrane-spanning proteins function during conditions of high environmental osmolarity. Each regulates a unique branch of a pathway which converges on the MAPK kinase (MAPKK) Pbs2 (Fig. 1). The osmosensor Sln1 is a protein with two membrane-spanning domains and a cytoplasmic histidine kinase domain, which is homologous to sensor histidine kinases of bacteria, fungi, and plants. Sln1 is constitutively active in media of constant osmotic pressure, where it inhibits the activity of Ssk1 (31). Under conditions of high external osmolarity, Sln1 is inhibited, resulting in the activation of Ssk1 and hence of two redundant MAPKK kinases, Ssk2 and Ssk22 (24, 29), which then activate Pbs2. The second putative high-osmolarity sensor, Sho1, contains four transmembrane domains and an intracellular Src homology 3 (SH3) domain, which binds a proline-rich region of Pbs2 (23). Under conditions of high osmolarity, Sho1 utilizes Ste20 (a p21-activated protein kinase [PAK] homolog) and Ste50 to activate the MAPKK kinase Ste11 (10, 23, 27, 30), which then activates Pbs2. Pbs2 then phosphorylates the MAPK Hog1, resulting in the translocation of Hog1 into the nucleus and the transcriptional induction of a large number of genes, some of which are responsible for the production of glycerol, the compatible solute in yeast cells (1, 4, 12, 33).
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Here we describe our studies seeking to identify an additional osmosensor that feeds into Ste11. We have carried out a hunt for mutants defective in this osmosensor, which has led to the identification of MSB2 as the presumptive osmosensor. MSB2 was discovered as a multicopy suppressor of a temperature-sensitive mutation in the CDC24 gene (2), but its function has been completely obscure. We have found that Msb2 and Sho1 both provide inputs to the Ste11 branch of the HOG pathway in hog1 mutants and perhaps for the normal osmoresponse in HOG1 cells as well. Our data indicate that MSB2 encodes a weak but physiologically relevant osmosensing component in S. cerevisiae which functions in parallel to the Sho1 osmosensing branch.
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MATERIALS AND METHODS |
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For cloning the gene(s) defective in these mutants, strains were transformed with a yeast genomic library (see below) and plated on SC-ura at a density of approximately 500 to 2,000 transformants/plate. After growth for 3 days, colonies were replica plated to a no. 3 Whatman filter on YEPD medium-1 M sorbitol and incubated overnight at 30°C. Filter ß-galactosidase assays were performed to identify lacZ-expressing strains. Such blue colonies were picked directly from the filters, streaked to SC-ura plates, and retested.
Construction of a yeast genomic library. Yeast genomic DNA was prepared from strain SO329 as described previously (18). This DNA was then treated with Sau3A for various times, and 8- to 12-kb DNA fragments were selected on 0.8% agarose gels. After excision and purification (Qiaexgel extraction kit; Qiagen), the DNA was ligated to BamHI-digested pRS316 (38). Approximately 18,000 independent Escherichia coli transformants were recovered and used to prepare plasmid DNA (Qiagen plasmid kit). Ten of 10 independent transformants contained plasmids with inserts of the expected sizes.
Microscopy techniques. Hog1-green fluorescent protein (GFP) and cell morphology were visualized by using a Nikon Microphot-SA microscope with a x60 objective lens and a Princeton Instruments cooled charge-coupled device camera (RTE/CCD-1300-V).
Microarray analysis. Protocols and material source information were obtained from http://microarrays.org/. DNA microarrays were fabricated at the University of California, San Francisco, by spotting full-length yeast ORFs (derived from PCR products) onto glass microscope slides. ORF DNA microarray analysis, total RNA isolation, and mRNA purification were performed as described previously (8), except that for each RNA sample to be assayed, 4 µg of mRNA was used for cDNA synthesis in the presence of amino-allyl dUTP. cDNA was then labeled with Cy3 or Cy5 (37).
Microarrays were scanned with GenePix 4000A or 4000B microarray scanners (Axon Instruments, Inc., Foster City, Calif.). Genes were assigned to the resulting microarray images with GenePix Pro software. After removal of data from gene spots that were damaged, data were uploaded to the database program AMAD (available at http://microarrays.org/software.html), which also normalized the data over the entire array. Red/green (r/g) expression ratios (the ratio of means) were used if the spot intensity was above 150 U for each channel; this strategy reduced variance due to a weak fluorescence signal. We further normalized the data by adjusting each of 16 sectors of the array to an average ratio of 1.0; this step decreased array position variation in the data set. Ultimately, expression data were manipulated by using FileMaker Pro, Microsoft Excel, Cluster, and TreeView. Cluster and TreeView (11) are available at http://rana.lbl.gov/EisenSoftware.htm.
For genes displayed in Fig. 2 and 3, we considered genes which were induced or repressed by a factor of twofold at two or more time points (in the time series of any strain displayed in either figure). We then calculated mutant effect (Me) ratios, which relate the expression of the mRNA in one strain to that in another strain, by dividing the r/g expression ratios of the means (for each time in the time series). The Me ratio is a quotient, with "versus" representing a division sign. For example, in Fig. 3C, the Me ratio of sho1 versus sho1 msb2 strains represents the expression value of the sho1 strain divided by the expression value of the sho1 msb2 strain. For gene expression to be considered defective (compared to wild-type expression) in the various mutant strains, a gene had to have at least two Me ratios which were 
3 or 
0.333 in the time series. In Fig. 2, genes were also considered defective if two Me ratios (compared to those of a hog1 strain) were 2 or 0.5 in the time series. Figure 2A displays a subset of the cross talk genes shown in Fig. ii at our website at http://www.sacs.ucsf.edu/home/HerskowitzLab/MSB2/. Data can also be downloaded for each microarray figure or for the entire genome from our website.
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RESULTS |
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We constructed a yeast genomic library in the low-copy-number plasmid pRS316 and isolated plasmids which restored expression to the FUS1-lacZ reporter. We attempted to clone the mutant gene(s) for 6 of the strains which displayed relatively little residual cross talk, and the remaining 15 strains were not studied further. Only SHO1- and FUS1-lacZ-containing plasmids were isolated for three of the mutants (both types of plasmids give an increased LacZ signal for trivial reasons). One of the remaining mutants was complemented by a STE7 plasmid and another mutant was complemented by a STE20 plasmid, indicating that these mutants may have reduced-function alleles in STE7 and STE20. These putative ste7 and ste20 strains were not studied further. The remaining mutant was complemented by a library plasmid containing the MSB2 gene and by a plasmid (3) that carries only the MSB2 gene. The mutation in this strain was allelic to an MSB2 deletion allele (as determined by linkage analysis), indicating that it contains a mutation at or near the MSB2 locus. Taken together, these data indicate that MSB2 is required for cross talk signaling in hog1 sho1 mutants.
Quantitative analysis of the role of MSB2 in cross talk signaling.
To characterize the role of MSB2 in signaling, we measured cross talk to FUS1-lacZ. As shown in Fig. 4A, a hog1 strain exhibits substantial activation of FUS1-lacZ in response to 1 M sorbitol (40 U of ß-galactosidase activity after 5 h of induction). As observed previously (27), inactivation of STE20, STE50, or STE11 completely abolished cross talk in the hog1 strain (less than 1.0 U of activity). Mutation of SLN1 or STE4 still allowed robust cross talk, indicating that the osmosensor Sln1 and the pheromone receptor upstream of Ste4 (Ste2) are not responsible for providing inputs to this response pathway. In contrast, inactivation of SHO1 or MSB2 in the hog1 strain resulted in a marked reduction of cross talk signaling (Fig. 4A, black bars). Moreover, a hog1 sho1 msb2 strain exhibited even less cross talk (2.7 U) than the hog1 sho1 (5.2 U) or hog1 msb2 (6.4 U) strain at 5 h of induction with 1 M sorbitol. These results are consistent with the idea that Sho1 and Msb2 each independently provide input to Ste20 and Ste50. The remaining cross talk in the hog1 sho1 msb2 strain is apparently due to the activity of at least one additional osmosensor which stimulates Ste11. We tested this set of strains for the induction of FUS1-lacZ by 
-factor as well (Fig. 4A, white bars). This analysis demonstrated that the msb2 and sho1 mutations did not affect signaling through the pheromone response pathway initiated from the -factor receptor.
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Msb2 promotes cell elongation in hog1 mutants. During mating, yeast cells polarize their cytoskeleton and grow toward the mating partner to form a pear shape or shmoo. In addition to activating the FUS1-lacZ reporter, cross talk in hog1 mutants also induces cells to exhibit a shmoo-like morphology (Fig. 4C) (27). This morphology is largely dependent on Sho1 but not on upstream elements of the pheromone response pathway, such as Ste4 (27). However, some residual cell elongation remains in hog1 sho1 mutants, especially after long incubation in the presence of 1 M sorbitol (24 h) (Fig. 4C). We hypothesized that MSB2 might also contribute to this response and thus examined the effect of the deletion of MSB2 on cell elongation. Indeed, mutation of MSB2 reduced cell elongation and did so to a greater extent than mutation of SHO1 (Fig. 4C), although some cells still formed shmoo-like structures. Strikingly, a hog1 strain deficient in both SHO1 and MSB2 exhibited little, if any, morphological change in response to high osmolarity. In fact, the morphology of hog1 sho1 msb2 cells was similar to that of hog1 ste11 cells, which exhibit no cross talk, with the exception that the hog1 ste11 strain frequently produced abnormally large cells. These observations on high-osmolarity-induced morphological changes reinforce the prior conclusion that Msb2 and Sho1 are partially redundant for providing inputs to Ste11 in a Hog1-deficient strain background.
Msb2 and Sho1 control the expression of a subset of genes in hog1 mutants. To learn more about the role of Msb2 in the cross talk pathway of signal transduction, we monitored gene expression with DNA microarrays. We sought genes with expression patterns that differed between wild-type and hog1 strains or between hog1 and hog1 sho1, hog1 msb2, or hog1 sho1 msb2 strains. We examined RNA levels over a relatively long time period, after 0, 30, 60, 120, and 180 min of 1 M sorbitol treatment, and with additional intermediate time points for the wild type. To focus on genes with altered regulation in a multiple-mutant strain relative to the regulation in a hog1 strain, we calculated Me ratios (see Materials and Methods), which relate the RNA level in one strain to that in another. There were 462 genes in six major classes with altered expression in hog1 mutants compared to the wild-type or between hog1 mutants (Fig. ii at our website). The different patterns of gene expression in hog1 mutants will be more fully described elsewhere (S. M. O'Rourke and I. Herskowitz, unpublished data). However, two classes included cross talk-induced genes (genes induced in hog1 mutants but not in the wild type) in response to increased osmolarity. Class I was dependent on Sho1 and Msb2 for induction, while class II was not, as revealed by mutation of SHO1 and MSB2. Figure 2A displays the response profiles for 58 class I and class II genes. On average, class I genes were induced 9-fold in hog1 mutants and class II genes were induced 3.8-fold in hog1 mutants after 1 h of sorbitol treatment (Fig. 2B). The average expression of class I and class II genes in the wild type was near basal levels after 1 h (0.92- and 0.85-fold, respectively).
The induction of class I genes was reduced when SHO1, MSB2, or SHO1 and MSB2 were deleted. Indeed, this class contains the native FUS1 gene, which displays regulation similar to that seen with the FUS1-lacZ reporter gene, as described for Fig. 4A. Strikingly, all of the class I genes were also induced by -factor. Genes involved in pheromone reception and signaling (STE2, SST2, BAR1, and FUS3) as well as genes involved in cell-cell adherence, cell fusion, and nuclear fusion (AGA2, FUS2, PRM1, KAR4, and KAR5) are likewise found in class I. The effect of a SHO1 mutation on these genes is clear, but significant residual induction is still present in a hog1 sho1 strain (Fig. 2B). For example, the average induction of the class I genes at 180 min in a hog1 strain is 6.6-fold, but in a hog1 sho1 strain, the induction is 2.7-fold. Mutation of MSB2 has a similar effect at 180 min: the average induction in a hog1 msb2 strain is 2.7-fold. Finally, a hog1 sho1 msb2 strain induces this set of genes only 1.8-fold. Thus, the induction of a variety of -factor-induced cross talk genes is similar to the results obtained for the FUS1-lacZ reporter gene: Msb2 and Sho1 provide partially redundant inputs to the cross talk pathway. An interesting observation is that mutation of SHO1 or MSB2 altered the expression of identical gene sets. This observation suggests that these two transmembrane proteins indeed activate the same pathway.
Despite the fact that class II genes are cross talk induced, they do not depend on SHO1 or MSB2 for their induction (Fig. 2A, Me ratios). These genes show an average induction of three- to fourfold in any strain where hog1 is deleted (Fig. 2B). In addition, they are not generally -factor inducible. Many of these genes code for enzymes involved in metabolic processes. Two are involved in cell wall metabolism (AMS1 and GSC2), three are involved in vitamin B1 and pyrimidine biosynthesis (THI5, THI11, and THI12), and several function in energy production (GPM2, NDI1, and MDH2). Many of these genes are induced during nitrogen starvation and stationary phase (13). It is possible that these genes are induced in response to secondary stress damage due to a deficient osmotic response or that a different cross talk circuit is activated which is unrelated to the pheromone response pathway. At any rate, these genes clearly form an SHO1- and MSB2-insensitive class of cross talk genes.
Msb2 controls osmosensitivity in HOG1 strains. The studies above indicate that Msb2 functions in parallel to Sho1 in a signaling pathway that is operative in a hog1 mutant background, the cross talk pathway (Fig. 1). To determine whether Msb2 functions in a HOG1 strain, we examined the contribution of MSB2 to growth under conditions of high osmolarity. It was shown previously that inactivation of the two inputs to Pbs2 and Hog1 by mutations in both SSK1 and STE11 leads to osmosensitivity, whereas mutation in either SSK1 or STE11 alone does not (23, 30) (Fig. 3A). Strains defective in both SSK1 and SHO1 also exhibit greater osmosensitivity than strains defective in only SSK1, but an ssk1 sho1 strain is not as osmosensitive as an ssk1 ste11 strain (Fig. 3A). We therefore tested whether MSB2 contributes to the osmoresistant phenotype of the ssk1 sho1 strain by examining the properties of an ssk1 sho1 msb2 strain; the triply defective strain is more osmosensitive than the ssk1 sho1 MSB2 strain and is almost as sensitive to high osmolarity as the ssk1 ste11 strain (Fig. 3A) (unpublished data). We interpret these observations to indicate that both Sho1 and Msb2 may activate Ste11 and consequently Hog1 and thus promote the osmoresistance of wild-type strains. However, the ssk1 msb2 strain is not osmosensitive (unlike the ssk1 sho1 strain) (Fig. 3A), suggesting that Msb2 provides a relatively smaller contribution to osmotolerance and presumably to Ste11 activity.
The contribution of Msb2 to resistance to high osmolarity in HOG1 strains suggests that Msb2 activates Hog1. Prior studies showed that Hog1 is rapidly phosphorylated by Pbs2 after a shift to high-osmolarity medium (4) and that Hog1 protein rapidly translocates to the nucleus after exposure of cells to high osmolarity (12, 33). To investigate whether Msb2 functions in osmoregulation by activating Hog1, we examined whether Msb2 governs the phosphorylation and nuclear localization of the Hog1 protein. In one set of experiments, we assayed tyrosine phosphorylation of Hog1 in wild-type, ssk1, ssk1 sho1, ssk1 msb2, and ssk1 sho1 msb2 strains (Fig. 5A). Although Hog1 was phosphorylated in wild-type, ssk1, and ssk1 msb2 strains in response to a variety of high-osmolarity conditions, we could not detect phosphorylation of Hog1 in ssk1 sho1 and ssk1 sho1 msb2 strains. In another set of experiments, we used a Hog1-GFP fusion (shown to have Hog1 activity; see Materials and Methods) to monitor Hog1 localization. As reported previously, the Hog1 protein localized to the nucleus in response to increased osmolarity in the wild-type strain. Some Hog1 was localized to the nucleus in the ssk1 sho1 strain, and similar localization was seen in the ssk1 sho1 msb2 strain (Fig. 5B). These observations indicate that the activity of Msb2 for stimulating the bona fide osmotic response pathway may be too weak to assay by Hog1 phosphorylation or nuclear localization (under the conditions that we tested). Alternatively, Msb2 may stimulate tolerance to high osmolarity in a manner that does not depend on Hog1.
A search for genes regulated by MSB2 in HOG1 strains. In Fig. 2 we identified genes whose expression was regulated by MSB2 in hog1 strains. These were a subset of genes in the pheromone response pathway that were induced by the cross talk pathway. We next sought to determine whether Msb2 controls the expression of any osmoregulated genes in a HOG1 strain. We carried out two sets of experiments. The first set compares genes induced by 0.5 M KCl in strains in which the SLN1-SSK1 branch is operative; the second set compares genes induced by 0.5 M KCl in strains in which SSK1 is inactive. In the SSK1 set, we compared the wild type with msb2, sho1, and msb2 sho1 mutants (Fig. 3B). The Me plots in Fig. 3C show that the inactivation of these genes had little effect on the expression of 82 genes whose expression is induced by the HOG pathway. We have noted that a significant induction of HOG pathway-dependent genes still occurs in mutants which block the HOG pathway (O'Rourke and Herskowitz, unpublished). Furthermore, an Me plot comparing sho1 versus sho1 msb2 strains demonstrated that Msb2 plays little, if any, role in the sho1 strain (Fig. 3C, right panel). In the analysis of ssk1 strains, we analyzed ssk1, ssk1 msb2, ssk1 sho1, and ssk1 msb2 sho1 mutants (Fig. 3B). We observed a set of genes whose expression was dependent on SHO1 (that is, whose expression decreased in the ssk1 sho1 strain). However, mutation of MSB2 had little effect on the expression of these genes in either an ssk1 or an ssk1 sho1 background. A direct comparison of ssk1 sho1 with ssk1 sho1 msb2 demonstrated that the patterns were essentially identical, indicating that Msb2 did not contribute to the expression of any of these genes (Fig. 3C, right panel).
We have argued that both Sho1 and Msb2 provide inputs to Ste11. If they provide the only inputs to Ste11, then the ssk1 sho1 msb2 strain should exhibit a pattern similar to that of the ssk1 ste11 strain. A comparison of these two strains reveals that there are several genes whose RNA levels are reduced more in the ste11 ssk1 strain than in the ssk1 sho1 msb2 strain (Fig. 3B and C, right panel). This observation suggests that Ste11 has osmoregulatory inputs in addition to Sho1 and Msb2.
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DISCUSSION |
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Msb2 and Sho1 provide partially redundant inputs to Ste20-Ste50-Ste11. Several observations indicate that Msb2 and Sho1 are partially redundant and provide inputs to Ste11. Mutation of either MSB2 or SHO1 reduces the osmotically induced expression of FUS1-lacZ and other genes (in class I) (Fig. 2A) and the induction of the shmoo-like morphology in hog1 mutants; inactivation of both virtually eliminates FUS1-lacZ expression and the morphological response. Msb2 contributes to the residual tolerance of high osmolarity of an ssk1 sho1 strain (Fig. 3A). Thus, deletion of both SHO1 and MSB2 results in a phenotype like that resulting from an STE11 deletion for both cross talk signaling and osmoresistance. An interesting feature of Sho1 and Msb2 in signal transduction is that mutations in either gene reduce cross talk signaling (Fig. 4A and B). However, mutations in both genes together reduce cross talk even further. These results are puzzling but may indicate that Sho1 and Msb2 have a partially interdependent relationship in responding to increased osmolarity. For example, the Sho1 and Msb2 proteins may interact with each other for maximal activity. Alternatively, these two proteins may constitute a linear pathway with (as yet) unidentified and redundant components at both the Msb2 and the Sho1 steps in the pathway. Although we propose that Msb2 is an osmosensor, we cannot exclude the possibility that it is required for the activity, localization, or trafficking of another protein that is the true osmosensor. In fact, biochemical evidence for Sln1 or Sho1 directly sensing increased osmolarity is also lacking.
Structure of Msb2 and possible functional features. The structure of Msb2 differs considerably from those of Sho1 and Sln1 (Fig. 1B). It is a putative integral membrane protein with a single putative membrane-spanning domain (positions 1186 to 1208) and an overall serine-threonine content of 43%. It is proposed to have a large extracellular domain of 1,166 amino acids and a short intracellular tail of 98 amino acids but to lack any clear functional domains. In contrast, Sho1 has four proposed transmembrane segments and an intracellular SH3 domain which is used to bind downstream signaling proteins, notably Pbs2; Sln1 has two proposed transmembrane segments and a histidine kinase domain which is used for the regulation of Ypd1 and Ssk1. Msb2 functions in the Ste11 branch of the HOG pathway and thus should contain binding sites for some or all of Ste20, Ste11, and Ste50 and perhaps for Pbs2 as well. Msb2 lacks similarity to Sho1 and also lacks a recognizable SH3 domain, by which Sho1 associates with Pbs2 (23). One notable feature of the Msb2 protein is that its presumed extracellular domain contains seven 17-amino-acid repeated sequences, which are serine and threonine rich. The yeast protein with the greatest similarity to Msb2 (26.7% identity over its entire length) is Hkr1, which is also a type I membrane protein and has 1,802 amino acids. Hkr1 has a transmembrane domain at positions 1486 to 1506; its large presumptive extracellular domain also contains 13 repeats of a 26-amino-acid serine- and threonine-rich sequence. Hkr1 is a cell surface protein whose overexpression confers resistance to the killer toxin of Hansenula mrakii (15) and controls ß-glucan synthesis and the axial budding pattern (39). Hkr1 and Msb2 thus may have similar localizations. Whether Hkr1 plays a role in osmoregulation remains to be determined. We suggest that Msb2 may link the plasma membrane and the cell wall through an interaction of the large extracellular domain, possibly the repeat sequences, with components of the cell wall. Such a linkage could be used to monitor mechanical stress between the plasma membrane and the cell wall.
MSB2 was discovered because its overexpression partially suppresses a cdc24-ts mutation in the presence of 1 M sorbitol (2, 3), but its function has otherwise been obscure. Cdc24 is a guanine nucleotide exchange factor for the small G protein Cdc42, which plays a role in the activation of Hog1 and Pbs2 through the Sho1 branch of the HOG pathway (32, 34). In particular, it is thought that Cdc42 regulates the PAK-like kinase Ste20, which subsequently activates Ste11. Moreover, Cdc42 is partially responsible for recruiting Pbs2 to sites of polarized growth during osmotic stress (34). Cdc24 is known to have a variety of binding partners, including Bud1/Rsr1 and Far1 (5, 25, 26, 28), which link the localization of Cdc24 to internal and external polarity signals. If Msb2 also regulates Cdc24, an osmolarity-sensitive input to the Cdc24-Cdc42 module would be created. Interestingly, ßPix (a human guanine nucleotide exchange factor which regulates Cdc42) activates the p38 MAPK cascade via PAK1 (19), and the activities of both Cdc42 and PAK1 are stimulated by increased osmolarity (6, 20). Another potential link between Msb2 and elements of the HOG pathway is the recent finding (9) that Msb2 interacts in a two-hybrid assay with Cla4, a PAK-like kinase that is related to Ste20 and that also functions in the HOG pathway (32). Thus, Msb2 can potentially provide input to upstream elements of the HOG pathway via multiple mechanisms.
Role of Msb2 in HOG1 strains. Msb2 plays a clear role in regulating gene expression through the cross talk pathway, that is, in a hog1 background. We have also observed that MSB2 contributes to osmoresistance in a HOG1 background: ssk1 sho1 MSB2 strains are more tolerant to high osmolarity than are ssk1 sho1 msb2 strains. The simplest interpretation of this result is that either Msb2 or Sho1 can signal to Pbs2 and Hog1. Our attempts to observe the regulation of Hog1 directly, by tyrosine phosphorylation or by nuclear localization of Hog1 protein, however, yielded no positive results. In addition, HOG1-dependent gene expression was not influenced by mutation of MSB2. We can propose two different explanations for this behavior of MSB2. Perhaps the assays used to detect Msb2 function in a HOG1 strain were not sufficiently sensitive. Another possibility is that Msb2 does not stimulate Hog1 activity. According to this explanation, Msb2 activates Ste11 in a way that does not lead to Pbs2 activation but that does activate Ste7 (note that Msb2 lacks an SH3 domain for binding to Pbs2). Thus, Msb2 may play a role in the weak cross talk activation of the pheromone response pathway that occurs in HOG1 cells (14) or in the activation of the pseudohyphal growth pathway. Why then would ssk1 sho1 msb2 strains be more sensitive to high osmolarity than ssk1 sho1 MSB2 strains? We suggest that perhaps weak expression of genes in the pheromone response pathway and/or the pseudohyphal growth pathway under the control of Msb2 contributes to tolerance to high osmolarity. Indeed, Cullen et al. found that the Sho1 protein signals to pheromone response pathway components for the monitoring of cell wall conditions (7). Thus, Msb2 may also utilize the pheromone response pathway for physiologically relevant processes.
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ACKNOWLEDGMENTS |
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This work was supported by National Institutes of Health (NIH) grant GM59466 (to I.H.). S.M.O. was supported by an NIH training grant, the Markey Program in Biological Sciences, the Herbert W. Boyer Fund, and a UCSF Chancellor's Fellowship.
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FOOTNOTES |
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