Copyright 1999 by the Genetics Society of America Activation of the Saccharomyces cerevisiae Filamentation/Invasion Pathway by Osmotic Stress in High-Osmolarity Glycogen Pathway Mutants K. D. Davenport, K. E. Williams, B. D. Ullmann and M. C. Gustin Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005-1892 Manuscript received January 7, 1999 Accepted for publication July 6, 1999 ABSTRACT Mitogen-activated protein kinase (MAPK) cascades are frequently used signal transduction mechanisms in eukaryotes. Of the ®ve MAPK cascades in Saccharomyces cerevisiae, the high-osmolarity glycerol response (HOG) pathway functions to sense and respond to hypertonic stress. We utilized a partial loss-of-function mutant in the HOG pathway, pbs2-3, in a high-copy suppressor screen to identify proteins that modulate growth on high-osmolarity media. Three high-copy suppressors of pbs2-3 osmosensitivity were identi®ed: MSG5, CAK1, and TRX1. Msg5p is a dual-speci®city phosphatase that was previously demonstrated to dephosphorylate MAPKs in yeast. Deletions of the putative MAPK targets of Msg5p revealed that kss1D could suppress the osmosensitivity of pbs2-3. Kss1p is phosphorylated in response to hyperosmotic shock in a pbs2-3 strain, but not in a wild-type strain nor in a pbs2-3 strain overexpressing MSG5. Both TEC1 and FRE::lacZ expressions are activated in strains lacking a functional HOG pathway during osmotic stress in a ®lamentation/invasion-pathway-dependent manner. Additionally, the cellular projections formed by a pbs2-3 mutant on high osmolarity are absent in strains lacking KSS1 or STE7. These data suggest that the loss of ®lamentation/invasion pathway repression contributes to the HOG mutant phenotype. EAST cells, like many other eukaryotes, utilize mito- rate activating signal and physiological response (for Y gen-activated protein kinase (MAPK) cascades to review see Gustin et al. 1998), it is becoming increas- transmit signals from plasma-membrane-associated sen- ingly clear that the MAPK pathways interact with each sory complexes to the nucleus, where transcriptional other. For example, the HOG pathway MAPK Hog1p is responses are elicited (for review see Banuett 1998; rapidly dephosphorylated in response to decreases in Gustin et al. 1998). MAPK cascades are composed of extracellular osmolarity in a Slt2p-dependent manner three conserved families of protein kinases: the MAPK, (Davenport et al. 1995), suggesting that the HOG path- the MAPK and ERK kinase (MEK), and the MEK kinase way is negatively regulated by the cell integrity pathway. (MEKK; for review see Davis 1993). The MEKK receives The phosphorylation of the pheromone response path- a signal from an upstream pathway component and way MAPK Fus3p in response to hypertonic stress is phosphorylates a conserved threonine and serine resi- enhanced by the deletion of HOG1 or the HOG pathway due within the MEK's activation domain (Kyriakis et al. MEK gene PBS2 (Hall et al. 1996). The observation 1992; Lange-Carter et al. 1993). Once phosphorylated, that the pheromone response pathway is activated in the activated MEK phosphorylates a threonine and a response to hypertonic stress in the absence of Hog1p tyrosine residue within the activation domain in the is further supported by evidence that a transcriptional MAPK (Crews and Erikson 1992). The phosphorylated target of the pheromone response pathway, FUS1, is also MAPK is then able to phosphorylate the targets of the induced by osmotic stress in the absence of catalytically MAPK cascade, including transcription factors (Gille et active Hog1p (Hall et al. 1996; O'Rourke and Hers- al. 1992; Seth et al. 1992) and other regulatory proteins kowitz 1998). It has been suggested that these data (Cook et al. 1996). indicate that Hog1p prevents the inappropriate activa- The budding yeast Saccharomyces cerevisiae contains ®ve tion of the pheromone response pathway by osmotic MAPK cascades, each with its own unique MAPK: Fus3p stress. in the pheromone response pathway, Kss1p in the ®la- These cross-pathway interactions may provide a mech- mentation/invasion pathway, Hog1p in the high-osmo- anism for establishing and maintaining signaling speci- larity-growth (HOG) pathway, Slt2p in the cell integrity ®city. The question is whether these interactions are pathway (Figure 1), and Smk1p in the spore wall assem- physiologically signi®cant or merely introduced by the bly pathway. Although these pathways each have a sepa- genetic manipulations of the organism. For example, the activation of Fus3p by high osmolarity in the absence of Hog1p (Hall et al. 1996) may indicate an important Corresponding author: Mike Gustin, Department of Biochemistry and Cell Biology, MS140, Rice University, 6100 S. Main, Houston, TX role for Hog1p in the maintenance of signal speci®city, 77005-1892. E-mail: [email protected] or it may be an artifact of the experimental overexpres- Genetics 153: 1091±1103 (November 1999) 1092 K. D. Davenport et al. mutant, pbs2-3. Prevention of Kss1p phosphorylation by the overexpression of the MAPK phosphatase MSG5, the deletion of KSS1, or the deletion of upstream ®la- mentation/invasion pathway MAPK cascade genes is suf- ®cient to suppress not only the osmosensitivity of pbs2-3, but also one of the morphological phenotypes associ- ated with HOG pathway mutants on high-osmolarity media: the formation of long projections. These data indicate that the ®lamentation/invasion pathway is in- appropriately activated by hyperosmotic stress in cells lacking a functional HOG pathway. MATERIALS AND METHODS Strains, media, and general methods: The yeast strains and plasmids used in this study are listed in Table 1. The bacterial strain DH5a was used for all plasmid ampli®cations and isola- tions. Growth media (YEPD, supplemented SD, and LB) were prepared as described (Kaiser et al. 1994). Common proce- dures (DNA manipulations, bacterial propagation, etc.) were followed as described (Sambrook et al. 1989). Techniques used for genetic crosses, sporulation, dissection, and propaga- tion of S. cerevisiae are described elsewhere (Kaiser et al. 1994). Yeast were transformed by the one-step method (Chen et al. Figure 1.ÐMAPK cascades in yeast. 1992). Isolation, cloning, and sequencing of pbs2-3: Wild-type strain MG159B was mutagenized with ethyl methanesulfonate (EMS) sion of FUS3. Other examples, such as the activity of and screened for colonies that failed to grow on YEPD supple- mented with 900 mm NaCl or 1.5 m sorbitol and that showed Fus3p in the repression of the ®lamentation/invasion a reduced production of glycerol in high-osmolarity media. pathway and the maintenance of pheromone response Four complementation groups of recessive mutations were pathway signal speci®city (Madhani et al. 1997), seem identi®ed and designated hog1±hog4 (Brewster et al. 1993). to indicate that some interactions are physiologically The mutants in the hog4 complementation group (alleles of signi®cant. However, in general, the prevalence and PBS2; Brewster et al. 1993) were then screened for the ability to grow at an intermediate osmolarity of 400 mm NaCl (YEPD physiological signi®cance of these cross-pathway interac- plus 400 mm NaCl). Only one mutant, hog4-3 (renamed pbs2- tions are not yet well known. 3), was able to grow at this osmolarity. Thus, the phenotype The HOG pathway mutants hog1 and pbs2 were ®rst of pbs2-3 was intermediate between a pbs2D strain and the wild- isolated in a screen for yeast unable to grow or produce type strain. pbs2-3 was backcrossed to W303-1A four times to glycerol in high-osmolarity media (Brewster et al. produce strain KDY1. Genomic DNA was extracted (Hoffman 1993). Additionally, budding and growth defects have and Winston 1987) from strain KDY1 and used as a template to amplify the PBS2 locus by PCR. PCR products from three also been described for these mutants (Brewster and independent reactions were cloned using the TA cloning kit Gustin 1994). In high-osmolarity media, hog1D or pbs2D (Invitrogen, San Diego) to produce pKD10, pKD11, and cells will abandon a small bud and grow a new bud. pKD12. One clone, pKD10, was sequenced (Seqwrite) and This double-budded phenotype may indicate a defect compared to the Saccharomyces Genome Database and pre- in cell cycle regulation in HOG pathway mutants that viously published sequences (Boguslawski and Polazzi 1987). Discrepancies between the pbs2-3 sequence and published se- are exposed to high-osmolarity media (Brewster and quences of PBS2 were veri®ed by sequencing portions of Gustin 1994). A second growth defect observed in pKD11 and pKD12 to ensure that the mutations identi®ed in HOG pathway mutants grown in high-osmolarity media pKD10 were not introduced by PCR. Two mutations were is the production of long cellular projections (Brew- identi®ed, which are predicted to code for the following substi- ster and Gustin 1994), indicating stimulated or unreg- tutions in the polypeptide sequence: proline for serine at position 168 (S168P) and aspartate for glycine at position 509 ulated polarized growth in HOG mutants exposed to (G509D). Restriction fragments from pbs2-3 containing either high osmolarity. However, the precise cause of these one or both of the identi®ed mutations were used to replace morphological defects is not known. the corresponding restriction fragments of a plasmid con- In this article, we provide evidence that part of the taining PBS2 to produce pKD13, pKD14, and pKD15. A strain HOG pathway mutant phenotype is a consequence of deleted for PBS2 (KDY9) was transformed with these plasmids the loss of HOG-pathway-dependent inhibition of a sec- and assayed for growth on high-osmolarity media (YEPD plus 400 or 900 mm NaCl) to identify which of the mutations were ond MAPK pathway, the ®lamentation/invasion path- responsible for the pbs2-3 growth phenotype. way. Kss1p is shown here to be phosphorylated in re- High-copy suppressor screen: A pbs2-3 strain (KDY1) was sponse to hyperosmotic shock in a HOG pathway MEK transformed with two high-copy plasmid yeast genomic DNA Osmotic Activation of the MAPK Kss1 1093 TABLE 1 Strains and Plasmids Genotype Reference or source Strain MG159B MATa ura3-52 Brewster et al.