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Vol. 14, Issue 9, 3782-3803, September 2003
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* Department of Biology Queen's University, Kingston, Ontario K7L 3N6, Canada;
Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania 19104;
DiscoveRx Corp., Freemont, CA 94538;
|| Institute of Molecular Biology and Biochemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada;
¶ MDS Proteomics Inc., Toronto, Ontario M9W 7H4, Canada; and
# Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5S 1A8, Canada
Submitted January 17, 2003;
Revised May 9, 2003;
Accepted May 9, 2003
Monitoring Editor: Anthony Bretscher
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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). The temporal and spatial regulation of cell polarity and morphogenesis seems to be highly complex, involving the integration of multiple signaling networks, including those that regulate cell size and cell division (Knust, 2000; Rupes, 2002).
In the budding yeast Saccharomyces cerevisiae, multiple signaling networks coordinate bud emergence, polarized growth, and cell cycle progression (Pruyne and Bretscher, 2000a,b; Casamayor and Snyder, 2002). In response to favorable nutrient conditions, cell size, and initiation of cell cycle progression, a Ras-like GTPase signaling network becomes activated to specify the site of bud emergence. Coincident with initiation of DNA replication, the G1/S phase cyclin-dependent kinase (CDK) complex, Cln1/2p–Cdc28p, promotes bud emergence through activation of the Cdc42p GTPase cycle, which controls the assembly of macromolecular complexes containing multiple polarity determinant proteins (Lew and Reed, 1995; Pruyne and Bretscher, 2000a,b). In particular, the formin protein Bni1p controls the assembly of actin cables, which guide myosin-motor directed secretion (Evangelista et al., 2002; Pruyne et al., 2002; Sagot et al., 2002). During the initial stages of bud development, secretion is polarized to the bud tip, such that growth is focused to the bud apex. Later in the cell cycle, a switch is made from apical to isotropic growth, such that secreted materials are directed uniformly over the entire surface of the developing bud. This switch correlates with the coupling of CDK to the mitotic cyclins, Clb1/2p, and subsequent activation of a network involving the Nim1-like kinase Gin4p and the PAK-like kinase Cla4p, which terminate Cdc42p signaling (Gulli et al., 2000). At the end of mitosis, polarized cell growth is redirected from the bud cortex to the mother-daughter cell junction at the bud neck to facilitate septum formation and cell separation (Kilmartin and Adams, 1984).
The mechanisms involved in polarized growth in S. cerevisiae are also required for the formation of mating projections. Chemotropic growth of mating projections enables haploid cells of opposite mating types to make contact and subsequently form a diploid zygote through cell and nuclear fusion. Mating pheromone activates a cell surface G protein-coupled receptor, which stimulates a downstream mitogen-activated kinase kinase pathway and Cdc42p-signaling to induce the expression of mating genes, G1 cell cycle arrest, and polarized secretion, leading to the formation of a mating projection or shmoo (Leberer et al., 1997; Elion, 2000). Like polarized bud growth, formation of polarized mating projections requires Bni1p-mediated assembly of actin cables, which direct the secretion of plasma membrane and cell wall components to the growing tip of the cell (Evangelista et al., 2002; Pruyne et al., 2002; Sagot et al., 2002).
The S. cerevisiae Mob2p–Cbk1p kinase complex has been implicated in the regulation of polarized growth during budding and mating (Colman-Lerner et al., 2001; Weiss et al., 2002). Cbk1p kinase belongs to a conserved family of serinethreonine protein kinases, whose members include Dbf2p, a component of the S. cerevisiae mitotic exit network (MEN), S. pombe Orb6 and Sid2, and mammalian Ndr and LATS kinases (Verde et al., 1998; Racki et al., 2000; Bardin and Amon, 2001; Bidlingmaier et al., 2001). Cbk1p kinase activity peaks during periods of polarized bud growth and during late mitosis (Weiss et al., 2002). Cbk1p activity is dependent on Mob2p, a member of the Mob family of proteins that includes Mob1p, a Dbf2p binding protein and component of the S. cerevisiae MEN (Luca and Winey, 1998; Weiss et al., 2002). Cells deleted for either CBK1 or MOB2 are round, as are cells expressing a catalytically inactive form of Cbk1p (Racki et al., 2000; Bidlingmaier et al., 2001; Colman-Lerner et al., 2001; Weiss et al., 2002). Similarly, S. pombe mutant cells that lack CBK1 and MOB2 orthologs are round (Verde et al., 1998; Hou et al., 2003). The round cell morphology of cbk1
and mob2 mutants indicates a failure to establish or maintain apical bud growth. Consistent with a general role for the Mob2p–Cbk1p protein complex in polarized morphogenesis, mob2 and cbk1 mutants are defective in pheromone-induced projection formation (Bidlingmaier et al., 2001; Weiss et al., 2002). Moreover, the conditional inactivation of Cbk1p kinase results in the depolarization of the actin cytoskeleton at the growing tips of mating projections (Weiss et al., 2002).
In addition to their role in polarized growth, Cbk1p and Mob2p are critical for regulating the localization and activity of Ace2p transcription factor, which mediates mother-daughter cell separation at the end of mitosis (Colman-Lerner et al., 2001; Weiss et al., 2002). In wild-type cells, Ace2p localizes to daughter cell nuclei during mitotic exit and activates a set of genes that are essential for mother-daughter cell separation, such as CTS1 and SCW11, which encode proteins involved in septum degradation (Dohrmann et al., 1992; Bidlingmaier et al., 2001; Colman-Lerner et al., 2001; Doolin et al., 2001; Weiss et al., 2002). Consistent with their dual roles in cell polarity and Ace2p regulation, Cbk1p and Mob2p localize to sites of cortical growth during G1 through mid-mitosis and to the bud neck region and the daughter cell nucleus at the end of mitosis (Colman-Lerner et al., 2001; Weiss et al., 2002). Cells deleted for ACE2, CBK1, or MOB2 display similar cell separation defects; however, unlike cbk1 and mob2 mutant cells, ace2 cells are not defective in polarized morphogenesis (Racki et al., 2000; Bidlingmaier et al., 2001). Thus, the role of the Mob2p–Cbk1p protein complex in Ace2p regulation is distinct from its role in polarized morphogenesis.
The mechanism for Mob2p–Cbk1p kinase activation remains to be elucidated. However, given the sequence similarities between Mob1p and Mob2p and between their respective kinase partners, Dbf2p and Cbk1p, it is likely that the Mob2p–Cbk1p complex is activated within a signaling pathway whose circuitry resembles that of the S. cerevisiae MEN and S. pombe septation initiation network (SIN) (Bardin and Amon, 2001). So far, Hym1p, an ortholog of the Aspergillus nidulans hyphal growth protein hymA and the mouse MO25 protein (Miyamoto et al., 1993; Karos and Fischer, 1999), and Tao3p (Pag1p), a 270 kDa protein conserved from yeast to humans of undefined molecular role, are the only known proteins that seem to function within the Mob2p-Cbk1p pathway (Dorland et al., 2000; Bidlingmaier et al., 2001; Du and Novick, 2002). However, Hym1p- and Tao3p-like proteins have not been implicated in MEN or SIN signaling.
Herein, we used a variety of approaches, including microarray analysis, protein–protein interaction methodologies, live cell microscopy, and in vitro kinase assays to investigate the functional relationships between Cbk1p, Mob2p, Tao3p, and Hym1p. We conducted genetic and two hybrid screens to identify additional genes involved in both polarized morphogenesis and cell separation. We present evidence that the serine-threonine kinase Kic1p (Sullivan et al., 1998) and a novel leucine-rich repeat (LRR)-containing protein Sog2p collaborate with Cbk1, Mob2p, Tao3p, and Hym1p to regulate Ace2p transcriptional activity and polarized morphogenesis. Each of these proteins localizes similarly to sites of polarized growth and displays a complex array of physical interactions and interdependencies for subcellular localization, indicating an intimate functional relationship. Notably, we establish that Mob2p–Cbk1p activity is dependent on Kic1p, Tao3p, Hym1p, and Sog2p, and we provide evidence that Hym1p, Sog2p, and Kic1p interact to form a functional unit. Given the sequence similarity of Kic1p to the MEN kinase Cdc15p, which activates the Mob1p–Dbf2p kinase complex directly (Mah et al., 2001), it is probable that Kic1p activates Mob2p-Cbk1p for regulating Ace2p and cellular morphogenesis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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) or S288C (Roberts et al., 2000; Weiss et al., 2002) and were cultured as described previously. Strains deleted for BNI1 (Y3314), CBK1 (Y1747), MOB2 (Y3424), TAO3 (Y3152), HYM1 (Y1560), KIC1 (Y3323), and SOG2 (FLY1492), were created using a polymerase chain reaction (PCR)-based targeted gene replacement strategy, as described previously (Longtine et al., 1998).
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Cells expressing C-terminal–tagged proteins (3 x hemagglutinin [HA], 13 x Myc, or green fluorescent protein [GFP]) were constructed via PCR-based gene fusion, as described previously (Longtine et al., 1998). These alleles did not cause any observable phenotypes, indicating that the tags did not interfere with protein function. Double mutant combinations were constructed through standard genetic crossing methods.
Identification of Bilateral Mating Mutants
Cells were engineered to select for mutants with reduced mating efficiency. Mutant ade2 cells grown in medium containing low levels of adenine are red, due to the accumulation of a red biosynthetic precursor of adenine (Ugolini and Bruschi, 1996). ade2 ade8 double mutants fail to accumulate the red pigment due to a blockage at an earlier step in biosynthesis and therefore grow as white colonies. In haploid cells, 
2 encodes a transcriptional repressor of MATa-specific genes. In diploid cells, a1-2 represses haploid-specific genes, such as FUS1 and HO. FUS1pr-ADE8 (ADE8 behind the FUS1 promoter) was used as a haploid-specific pheromone response reporter. When integrated into the genome of a haploid ade2 ade8 cells, the basal activation of FUS1 expression is strong enough to produce adequate amounts of Ade8p, thus yielding red colonies. Colonies that successfully mate will produce diploid cells that will repress the production of FUS1pr-ADE8 (due to a1-2 repression of haploid-specific genes) and therefore look white, due to a lack of production of Ade8p. Mutations in the pheromone response cascade repress the basal signaling of FUS1 and thus yield white colonies. We selected for more subtle bilateral mating mutants that cannot mate efficiently and therefore continue to express FUS1pr-ADE8. Colonies of such mutants are red or contain red and white sectors when grown on adenine-deficient plates.
Strain Y282 (MATa ade2 ade8 GAL1pr-MAT2, FUS1pr-ADE8) was transformed with p1240, a YCp50 (CEN, URA3) plasmid containing HO, which encodes an endonuclease that catalyzes mating type conversion. Cells were grown in synthetic medium lacking uracil and containing 2% galactose and plated onto rich medium containing minimal amounts of adenine and 2% glucose. They were then immediately mutagenized with UV light to a 10% survival rate. Of the 20,000 colonies screened, 262 were chosen for further analysis based on a red sectoring quality. Colonies were streaked onto 5-fluoroorotic acid medium to counterselect for HO plasmid and assayed for mating type. MATa isolates were assayed for G1 arrest, by using a modified protocol from (Fink and Styles, 1972) and for pheromone production and response as described previously (Boone et al., 1993). Of 262 colonies, 75 were found to be normal for these assays and subjected to further analysis. FAR1 was transformed into these strains and found to complement 11, which were subsequently removed from further analysis. One of the mutants that showed a round cell morphology, and a cell separation defect (Y871) was transformed with a YCp50 (CEN, URA3)-based genomic library. Approximately 14,000 colonies were screened for restored mating ability. Plasmids were recovered and sequenced from cells exhibiting an increased mating efficiency. A single complementing plasmid clone was isolated (p1319) that contained the HYM1 gene and flanking sequences. HYM1 was subcloned and confirmed to complement the mutant phenotypes of Y871.
Isolation of Cell Separation Mutants
Y2862, which contains an integrated copy of OCH1pr-HIS3 and OCH1pr-lacZ, was transformed with an mTn-3 x HA/GFP mutagenized library (Burns et al., 1994) and plated onto synthetic medium lacking uracil and histidine and containing 15 mM 3-amino triazole. Colonies (340,000) were screened for growth on medium containing 15 mM 3-amino triazole, and 59 colonies were selected. Eight colonies of the 59 also exhibited a cell separation phenotype and round cell morphologies. These mutants were placed into four complementation groups and direct genomic sequencing was used to determine the identity and transposon insertion site of each of the mutants (Horecka and Jigami, 2000). Four alleles of TAO3, two alleles of KIC1, and single alleles of MOB2 and CBK1 were recovered. All mutations showed elevated OCH1 expression (approximately twofold above wild type) by lacZ measurement (Horecka, unpublished data). Only cbk1 cells showed elevated levels of OCH1 expression (twofold) by DNA microarray analysis, which illustrates the greater sensitivity of the lacZ-reporter for analysis of gene expression.
Suppression of Cell Separation Mutants
Y1614 (hym1::LEU2), which is defective for cell separation, was transformed with the YEp24-based (URA3, 2 µ) genomic library. Approximately 18,000 colonies were pooled and placed vertically at room temperature for 40 min and allowed to sediment. A small volume (0.25 ml) of liquid was transferred from the culture and transferred to 50 ml of fresh SD-URA and grown at 30°C to saturation. This enrichment for nonsedimenting cells was repeated for two additional rounds. Liquid was taken from the surface of the culture and plated onto SD-URA at a density of 
200 colony-forming units/plate. Forty-nine round, smooth colonies were chosen for microscopic analysis to confirm cell separation. Plasmids were extracted and examined by restriction analysis, one was sequenced and found to contain a 10.8-kb insert containing ACE2 (p3679).
DNA Microarrays
Yeast cultures were grown for three generations to mid-logarithmic phase in synthetic complete medium. Cells were pelleted and frozen in liquid nitrogen. RNA extractions and hybridizations were performed as described previously (Roberts et al., 2000).
Assays for Development of Mating Projections
Logarithmically growing MATa cells were exposed to 50 nM synthetic -factor for 2 h and stained with rhodamine-phalloidin to visualize filamentous actin. Cells were scored for ability to polarize filamentous actin patches to discrete sites at the tips of cells.
Analysis of Budding Patterns
Strains expressing a BUD4-containing plasmid (p2698; a gift from Silvia Sanders, Massachusetts Institute of Technology, Cambridge, MA) or the vector pRS316 were grown in medium lacking uracil to mid-logarithmic phase. Cells were sonicated for cell separation and Calcofluor White was added to 2 µg/ml concentration. Bud scars were visualized by fluorescence microscopy and bud patterns were defined as described previously (Chant and Pringle, 1995). Only cells containing three or more bud scars were counted. Bud scars residing on opposite thirds of the cells were considered to be bipolar. Bud patterns were considered axial only if scars resided immediately adjacent to one another. Cells were considered to have random patterns if bud scars resided in the middle third of the cell.
Two-Hybrid Screens and Assays
Two-hybrid bait and prey plasmids are listed in Table 2 and were based on pEG202, which encodes the LexA-DNA binding domain, and pJG4-5, which encodes the B42 transcriptional activation domain (Gyuris et al., 1993). DNA fragments of various genes were amplified by the PCR with primers that incorporated 5'-BamHI and 3'-NotI restriction sites (unless otherwise noted in Table 2) for insertion into the two-hybrid vectors.
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Two-hybrid assays were performed as described previously (Phizicky and Fields, 1995) by mating Y1026, which contains a LexA DNA-binding domain plasmid, and Y860, which contains a prey plasmid. Diploids were grown on selective media and subjected to 
-galactosidase expression analysis (Sheu et al., 2000). Strains qualitatively more reactive than the vector control were classified as positive. p2139 was used as bait to identify genes encoding Hym1p-binding proteins from a yeast cDNA library derived from pACT (Durfee et al., 1993). Plasmids encoding positive interactors were recovered and sequenced. One plasmid, designated p3153, contained a fragment of YOR353c (SOG2).
Plasmid Construction
To construct yeast strain Y282, several integrating plasmids (p40, p65 and p89) were created. The plasmid p40 introduces a frame-shift mutation at codon 45 of ADE8 and was created in a three-step process. First, a 2-kb EcoRI-HindIII ADE8 fragment was cloned into YIplac211 (URA3; Gietz and Sugino, 1988) to create p23. p23 was cut with EcoRI and SnaBI, treated with Klenow and dNTPs, and religated to remove an MscI site. The resultant plasmid, designated p38, was cut with Eco47III-HpaI and ligated to a BamHI linker (5'-CGGGATCCCG-3') to create an ADE8 frame-shift mutation. This plasmid (p40) was cut with MscI to target integration to the ADE8 locus. p65 was used to integrate GAL1-2 at the HO locus, thereby disrupting HO after codon 83. It was constructed by cutting YI-plac211 with PvuII to remove the multicloning site. A HindIII linker (5'-CCCAAGCTTGGG-3') was then ligated to create p41, which was subsequently cut with HindIII and ligated to a HindIII fragment carrying HO and its promoter, creating p53. p53 was cut with PstI-BamHI and ligated with an oligonucleotide linker containing EcoRI-SstI sites; the resultant plasmid was cut with EcoRI and SstI and ligated to a EcoRI-SstI fragment containing the GAL1 promoter (p113, Boone laboratory collection) and a BamHI-SstI fragment containing a MAT2 PCR product. The MAT2 fragment was PCR amplified from genomic DNA by using primers AAAGGATCCA AAATGAGAAC GGCCGTA and AAAGAGCTCT TGGAAAAATC CATTAACT, which incorporate BamHI and SstI sites at the 5' and 3' ends, respectively.
p89 was used to introduce the FUS1pr-ADE8 reporter was constructed as follows. A YIp plasmid containing a 4.8 Kb LYS2 genomic fragment (pCP6; provided by Eric Foss, Fred Hutchinson Cancer Research Center, Seattle, WA), was digested with HpaI and ligated to linkers to introduce HindIII-SrfI-XbaI sites within the LYS2 gene. The resultant plasmid (p72) was cut with HindIII and XbaI and ligated to the HindIII-NheI fragment of FUS1pr-ADE8 from a pUC13-based plasmid (p51), to create p89.
The human MST3 gene was cloned from a pool of infant brain, placenta, spinal chord, and colon total RNAs (BD Biosciences Clonetech, Palo Alto, CA) by reverse transcription-PCR by using the primers CGCGGATATC ACCATGGCTC ACTCCCCGGT GCA and CGCGGTCGAC GTGGGATGAA GTTCCTCCAC CACT. The PCR fragment was ligated into EcoRV and SalI-digested pCMV-TAG 4A (Stratagene, La Jolla, CA), which resulted in a C-terminal FLAG-tagged MST3 cDNA under the control of the cytomegalovirus promoter, creating pMST3-FLAG.
Yeast Immunoprecipitation and Protein Kinase Assays
For coprecipitation experiments, 1-liter culture of yeast cells was grown to early log phase in rich media, and extracts were prepared by grinding frozen cell pellets in lysis buffer (0.1% Triton X-100, 50 mM Tris-HCl, 100 mM NaCl, 10 mM EDTA) containing a protease inhibitor cocktail (Roche Diagnostics, Indianapolis, IN). Immunoprecipitations were carried out with 1.5-ml extracts, containing 20–25 mg/ml total protein, monoclonal antibody HA.11 (Babco, Richmond, CA), or monoclonal antibody c-Myc (Babco) and G-Sepharose beads (Pharmacia, Peapack, NJ). Total yeast extract (25 µg) was analyzed on immunoblots, and 10 and 90% of the immunoprecipitated material was analyzed for detection of the immunoprecipitated and coprecipitating proteins, respectively. Immunoblot analysis was performed as described previously (Peter et al., 1993) and probed using rabbit polyclonal HA antibody (Babco) and rabbit polyclonal c-Myc antibody (Santa Cruz Biotechnology, Santa Cruz, CA).
For Cbk1p protein kinase assays, cells were grown to mid-log phase and synchronized in G1 phase with mating pheromone. Cell extracts were prepared by glass bead lysis as described previously (Weiss et al., 2002). Cell extracts were normalized to 5 mg/ml and immunoprecipitated by incubating in 4 µg of anti-Myc antibody (9E10) for 45 min at 4°C followed by 25 µl of Gamma-bind G-Sepharose (Pharmacia) for 45 min at 4°C. Immunoprecipitated material was washed four times in lysis buffer and three times in kinase buffer (50 mM HEPES pH 7.4, 60 mM sodium acetate, 10 mM MgCl2, 1 mM dithiothreitol). Half the immunoprecipitated protein was processed for SDS-PAGE and immunoblotted. The other half was incubated in kinase buffer containing 5 µg of histone H1, 10 µM ATP, and 10 µCi of 32P-ATP for 30 min at 30°C. Kinase reactions were terminated by addition of protein sample buffer and were processed for PAGE and autoradiography. Relative kinase activity was determined using a GS-525 PhosphorImager (Bio-Rad, Hercules, CA) and Multianalyst software.
Fluorescence Microscopy
Live cell microscopy was performed as described previously (Luca et al., 2001; Weiss et al., 2002).
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONACKNOWLEDGMENTSREFERENCES |
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cells (Dorland et al., 2000; Bidlingmaier et al., 2001). Second, we screened for other mutations that cause cell separation and morphogenesis defects and identified alleles of CBK1, MOB2, TAO3, and KIC1. KIC1 encodes a protein kinase that interacts with centrin-related Cdc31p and controls polarized growth and cell wall integrity (Sullivan et al., 1998; Vink et al., 2002). HYM1, CBK1, MOB2, TAO3, and KIC1 are all essential for viability in the S288C-derived strain that was used by the yeast deletion consortium (Winzeler et al., 1999). However, in our laboratory isolates of S288C (Luca laboratory) and W303 yeast strains (Luca and Boone laboratories), none of the genes is essential, which is likely due to a mutation in the SSD1 gene (Du and Novick, 2002; Jorgensen et al., 2002). We found that viable hym1, cbk1, mob2, tao3, and kic1 deletion cells exhibit indistinguishable round cell morphology and cell separation defects (Figure 1) and fail to mate efficiently (our unpublished data).
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Yeast cell separation requires the activation of Ace2p transcription factor, which controls the daughter cell-specific expression of genes required for septum degradation, such as CTS1 and SCW11 (Dohrmann et al., 1992; Racki et al., 2000; Colman-Lerner et al., 2001; Doolin et al., 2001). Ace2p localizes to the daughter cell nucleus at the end of mitosis (Colman-Lerner et al., 2001; Weiss et al., 2002), and its activation and daughter-specific localization are dependent on Mob2p and Cbk1p (Colman-Lerner et al., 2001; Weiss et al., 2002). In tao3, hym1 and kic1 cells, we found that Ace2p-GFP was not restricted to the daughter cell nucleus at the end of mitosis, but localized weakly to both mother and daughter cell nuclei (Figure 1), as observed in mob2 and cbk1 cells (Weiss et al., 2002; Figure 1). These findings suggest that Mob2p, Cbk1p, Tao3p, Hym1p, and Kic1p function within the same signaling network, which we have designated the RAM network for regulation of Ace2p activity and cellular morphogenesis.
CBK1, MOB2, TAO3, HYM1, and KIC1 Are Essential for Ace2p-dependent Transcription
Ace2p is required for the daughter cell-specific transcription of a specific subset of genes (Colman-Lerner et al., 2001). The gene expression profiles of cbk1 cells were shown to be similar to ace2 cells (Bidlingmaier et al., 2001). To determine whether Mob2p, Tao3p, Hym1p, and Kic1p control the expression of a similar set of genes, we monitored global changes in gene expression for cbk1, mob2, tao3, hym1, and kic1 cells during vegetative growth by microarray analysis (DeRisi et al., 1997; Roberts et al., 2000). We compared the gene expression profiles of the RAM network deletion mutants to a compendium of >300 reference gene expression profiles (Hughes et al., 2000). We found that the expression profiles associated with cbk1, mob2, tao3, hym1, and kic1 were highly similar and formed a unique cluster (Figure 2). In particular, several Ace2p-regulated genes, such as CTS1, SCW11, and DSE1, DSE2, DSE3, and DSE4 (Colman-Lerner et al., 2001; Doolin et al., 2001) were coregulated across numerous experiments and were dependent on Cbk1p, Mob2p Tao3p, Hym1p, and Kic1p for normal expression (Figure 2). Thus, all of these proteins function in a common signaling pathway that controls Ace2p function.
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CBK1, MOB2, TAO3, HYM1, and KIC1 Are Required for Polarized Growth
The round cell morphologies of cbk1, mob2, tao3, hym1, and kic1 cells suggest that, in addition to Ace2p regulation, Cbk1p, Mob2p, Tao3p, Hym1p, and Kic1p have roles in polarized morphogenesis. To examine the roles of these proteins in pheromone-induced morphogenesis, we exposed cbk1, mob2, tao3, hym1, and kic1 cells to saturating levels of pheromone for 2 h and stained with rhodaminephalloidin to visualize the filamentous actin cytoskeleton. Each of the five mutants formed broader and less pronounced mating projections than wild-type cells (Figure 3A), as observed for cbk1 and mob2 cells (Bidlingmaier et al., 2001; Weiss et al., 2002). Only 70% of the mutant cells, in contrast to 98% of the wild-type cells, displayed a polarized distribution of cortical actin patches (Figure 3, A and B). Double mutant cells carrying different combinations of cbk1, mob2, tao3, hym1, and kic1 displayed defects in mating projection formation that were comparable to single mutants (Figure 3C). In contrast, similarly treated cells lacking the formin gene BNI1 exhibited more severe defects in mating projection formation than cbk1, mob2, tao3, hym1, and kic1 single mutants or double mutants (Figure 3B). Significantly, cells lacking one of the RAM genes in combination with bni1 exhibited more severe defects in mating projection formation than single mutants (Figure 3D). Thus, Cbk1p, Mob2p, Tao3p, Hym1p, and Kic1p seem to collaborate to control cell polarity and likely function independently from Bni1p, which is required for actin cable assembly during polarized morphogenesis.
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During budding, the cyclin-dependent kinase Cdc28p promotes polarized apical growth when coupled to the G1 cyclins and isotropic growth when coupled to mitotic cyclins (Lew and Reed, 1995). The apical growth phase can be prolonged by G1 cyclin overexpression, resulting in hyperelongated buds. Cells deleted for genes encoding cell polarity proteins, such as Bni1p, Bud6p and Spa2p (Chenevert et al., 1994; Amberg et al., 1997; Evangelista et al., 1997; Sheu et al., 1998), do not form hyperelongated buds in response to overexpression of the G1 cyclin CLN1, presumably due to a defect in actin-dependent polarized secretion to the bud tip (Sheu et al., 2000). To test Cbk1p, Mob2p, Tao3p, Hym1p, and Kic1p for a role in apical growth during budding, we overexpressed CLN1 in deletion strains and scored for the presence of hyperelongated buds. With prolonged CLN1 expression, wild-type cells showed a steady increase in the percentage of hyperelongated buds, reaching 55% of the total population after 4 h (Figure 4). In contrast, large budded cbk1, mob2, tao3, hym1, and kic1 cells resembled bni1, bud6, or spa2 cells, showing no apparent hyperelongation of buds and displayed a random distribution of actin patches. Immunoblot analysis revealed that the mutants and wild-type cells expressed comparable levels of galactose-induced Cln1p (our unpublished data). Thus, Cbk1p, Mob2p, Tao3p, Hym1p, and Kic1p are all required for the establishment or maintenance of apical bud growth.
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HYM1, CBK1, TAO3, KIC1, and MOB2 Affect Budding Patterns
Cells that are defective in actin-based polarized morphogenesis often display errors in bipolar bud site selection (Casamayor and Snyder, 2002). We examined the budding patterns of diploid cbk1, mob2, tao3, hym1, or kic1 cells that contained three or more calcofluor-stained bud scars. For wild-type diploids, 76% of the cells displayed bipolar budding patterns, whereas only 5–9% of diploid cbk1, mob2, tao3, hym1, and kic1 cells showed a bipolar pattern (Table 3). We also examined the budding patterns of haploid cells. The yeast strain W303 exhibits an axial budding defect, which can be rescued by introducing a plasmid carrying BUD4 (our unpublished data). Approximately 83% of haploid W303 cells that were transformed with pBUD4 showed an axial pattern (Table 3). In contrast, all five RAM deletion mutants exhibited reduced axial budding (Table 3). Only 55, 44, 56, 64, and 40% of cbk1, mob2, tao3, hym1, and kic1 cells, respectively, displayed axial budding patterns. Thus, Cbk1p, Mob2p, Tao3p, Hym1p, and Kic1p are not essential for axial budding but seem to participate in this process.
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Cbk1p Kinase Activity Is Dependent on Mob2p, Tao3p, Hym1p, and Kic1p
Cbk1p kinase activity is critical for cell separation and maintenance of polarized growth (Racki et al., 2000; Colman-Lerner et al., 2001; Weiss et al., 2002) and thus may be a marker for RAM network activation. In agreement, Cbk1p kinase activity is stimulated during periods of polarized growth and peaks during late mitosis (Weiss et al., 2002). Moreover, Cbk1p activation requires Mob2p (Weiss et al., 2002). To test whether other RAM proteins are important for Cbk1p kinase activation, we immunoprecipitated Cbk1-Myc from pheromone-treated ace2, mob2, tao3, hym1, kic1, and wild-type cells and performed in vitro kinase reactions by using histone H1 as substrate. Immunoblots revealed that similar amounts of Cbk1-Myc immunoprecipitated from ace2, tao3, hym1, kic1, and wild-type cells, whereas the amount of Cbk1-Myc immunoprecipitated from mob2 cells was more variable, ranging from equivalent to twofold less than that from wild-type cells (Figure 5). In vitro kinase assays showed that there was 5- to 20-fold less kinase activity associated with immunoprecipitated Cbk1-Myc from either mob2, tao3, hym1, or kic1 cells than from ace2 and wild-type cells (Figure 5). We obtained similar data from immunoprecipitants of asynchronous cells (our unpublished data). Thus, Mob2p, Tao3p, Hym1p, and Kic1p are essential for maximal Cbk1p kinase activity, suggesting that they function in concert with Cbk1p or before Cbk1p in RAM network signaling. In contrast, Cbk1p kinase activation does not require Ace2p, which is consistent with Ace2p being a downstream target of RAM signaling.
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Network of Hym1p, Cbk1p, Tao3p, Kic1p, and Mob2p Protein–Protein Interactions
To test for pairwise protein interactions between RAM network components, we performed two-hybrid assays on various fragments of these proteins (Figure 6A). We confirmed previously demonstrated Mob2p–Cbk1p and Tao3p–Cbk1p interactions (Racki et al., 2000; Du and Novick, 2002; Weiss et al., 2002) and observed Cbk1p–Cbk1p, Cbk1p–Kic1p, Tao3p–Kic1p, and Hym1p–Kic1p interactions (Figure 6A). We corroborated the Hym1p–Kic1p two-hybrid interactions by coimmunoprecipitating epitope-tagged proteins from asynchronous cells (Figure 6B).
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Additional protein interactions were identified in a parallel study (Ho et al., 2002). In these experiments, overproduced FLAG epitope-tagged Hym1p, Cbk1p, and Mob2p were immunoprecipitated from yeast extracts and the coprecipitated proteins were identified by mass spectroscopy. Tao3p, Mob2p, Ace2p, and Ssd1p, a protein implicated in Sit4p phosphatase function and polarized morphogenesis (Sutton et al., 1991), copurified with Cbk1p, and Cbk1p copurified with Mob2p. The Cbk1p–Ssd1p interaction is likely to be functionally relevant, because deletion of the SSD1 gene suppresses the lethality associated with disruption of RAM network signaling in certain strain backgrounds (Du and Novick, 2002; Jorgensen et al., 2002). The various interaction data are summarized in Figure 6C. This complex network of interactions is reminiscent of other signal transduction pathways, such as the yeast pheromone response pathway (Schultz et al., 1995; Elion, 2000; Dohlman and Thorner, 2001).
Sog2p Is a Novel RAM Component
To identify additional RAM-associated proteins, we conducted a two-hybrid screen by using Hym1p as bait and identified YOR353c as a gene encoding a Hym1p-interacting protein. YOR353c is registered as SOG2 in the Saccharomyces Genome Database (http://www.yeastgenome.org) and is designated as such throughout this manuscript. SOG2 encodes an 87-kDa protein that contains leucine-rich repeats and shares weak similarity with the NH2-terminal region of S. cerevisiae adenylate cyclase. In parallel studies, Sog2p was found to coprecipitate with Cbk1p and Hym1p by large-scale precipitation methods (Ho et al., 2002) and to interact with Cbk1p in independent two-hybrid screens (Uetz et al., 2000; Ito et al., 2001).
To investigate whether Sog2p functions similarly to other RAM proteins, we analyzed the phenotypes of sog2 cells. SOG2 and the RAM genes are essential for viability in the S288C-derived strains used for the Saccharomyces genome deletion project (Winzeler et al., 1999). However, SOG2 and the RAM genes were not essential for viability in our laboratory isolates of S288C and W303 strains, which carry the defective ssd1-d allele (Figure 7; our unpublished data). Using these strains, we found that cells lacking SOG2 exhibited round morphologies and failed to separate upon completion of mitosis (Figure 7A). Moreover, sog2 cells were unable to restrict Ace2p to the daughter cell nucleus (Figure 7A). To determine whether Sog2p is important for Cbk1p kinase activity, as are Mob2p, Hym1p, Tao3p, and Kic1p (Figure 5), we conducted in vitro kinase assays of immunoprecipitated Cbk1p derived from sog2 cells. We found that Cbk1p kinase activity was greatly diminished in the sog2 cells, indicating that Mob2p–Cbk1p function was dependent on Sog2p (Figure 7B). These data indicate that Sog2p is an essential component of the RAM network.
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Tao3p, Hym1p, Sog2p, and Kic1p Localize to Sites of Polarized Growth and Control Mob2p–Cbk1p Nuclear Localization
We examined the subcellular localizations of GFP-tagged RAM proteins to gain insight to their functional specificity. Indeed, consistent with roles in both polarized growth and Ace2p regulation, Cbk1p and Mob2p localize to the cortex during apical bud growth and to the bud neck and daughter cell nucleus during late mitosis (Colman-Lerner et al., 2001; Weiss et al., 2002). Tao3p was also shown to localize to sites of polarized growth but was not observed in the daughter nucleus (Du and Novick, 2002). We confirmed these observations by monitoring cells expressing Tao3-GFP (Figure 8A). Similarly, Kic1-GFP, Hym1-GFP, and Sog2-GFP localized prominently to incipient bud sites and small buds and weakly to the cortex and bud necks of large budded cells (Figure 8A). Tao3p, Hym1p, Sog2p, and Kic1p also localized to the growing tips of mating projections (Figure 8B), as shown for Mob2p and Cbk1p (Weiss et al., 2002). We did not detect Tao3p, Hym1p, Sog2p, or Kic1p in the nucleus. Thus, because Mob2p and Cbk1p are the only proteins of the network that are detected in the daughter cell nucleus, it is likely that Mob2p and Cbk1p function downstream of the other RAM proteins with respect to Ace2p regulation.
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Interdependency of RAM Protein Localization
To determine whether Cbk1p, Mob2p, Tao3p, Hym1p, Sog2p, and Kic1p are interdependent for localization, we systematically examined the localization of each protein in various RAM deletion strains. The results are summarized in Table 4.
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We asked whether Mob2p and Cbk1p localization was dependent on other RAM proteins. As observed previously (Weiss et al., 2002), Cbk1p and Mob2p localized interdependently at the cortex, bud neck, and daughter cell nucleus. In contrast, both Cbk1p and Mob2p were able to localize to the bud cortex and bud neck in tao3, hym1, sog2, and kic1 cells (Figure 9, A and B). Nevertheless, the nuclear localization of Mob2p and Cbk1p was distinctly aberrant in these cells. Mob2p was not detected in any kic1 or hym1 cell nucleus but was weakly detectable in (9%) daughter nuclei or in (9%) both mother and daughter nuclei of large budded sog2 cells (Figure 9B, arrows). Cbk1p was also undetectable in kic1 and sog2 cell nuclei but localized weakly to the daughter cell nucleus (7%) or to both mother and daughter cell nuclei (15%; see arrows Figure 9A) in some large budded hym1 cells. Cbk1p and Mob2p also localized to both mother and daughter cell nuclei in 25–46% of the large budded tao3 cells (Figure 2A, inset) and to the daughter nucleus in only 15% of the large budded tao3 cells. In particular, Mob2p was detectable in some late G1 tao3 cell nuclei (see arrowhead, Figure 9B, inset), which is in contrast to its localization in wild-type cells where Mob2p disappears from the daughter nucleus in early G1, well before bud emergence (Weiss et al., 2002). Together, these findings support a model in which Kic1p, Sog2p, Hym1p, and Tao3p function upstream of Mob2p-Cbk1p to control the catalytic activity and proper nuclear localization of the Mob2p–Cbk1p complex.
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There were subtle, but significant changes in RAM protein localization in mob2 and cbk1 cells. In these cells, Tao3-GFP, Hym1-GFP, Sog2-GFP, and Kic1-GFP seemed brighter at the cortexes of small and large buds than in wild-type cells (Figure 9, C,Figure 9, D–F). Moreover, there were significantly fewer large budded mob2 and cbk1 cells with Kic1-GFP (0%), Hym1-GFP (11–14%), or Sog2-GFP (4–6%) localized to bud necks than in wild-type cells (49%, n 
50 large budded cells). Immunoblots of the RAM deletion strains reveal no significant difference in protein levels of the remaining RAM proteins (our unpublished data). Thus, these data suggest that Mob2p and Cbk1p influence the localized concentration or abundance of RAM proteins at the sites of polarized growth.
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