INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

In the budding yeast Saccharomyces cerevisiae, cells of the a and  mating types secrete pheromones that bind to G-protein-coupled receptors on the surfaces of cells of the opposite mating type, initiating a signaling cascade in which the subunits of the G protein promote the activation of a mitogen-activated protein kinase (MAPK) cascade (4). This process appears to involve the binding of at least two proteins, the upstream kinase Ste20p and the scaffold protein Ste5p, to the liberated G subunits (11, 25, 40, 51). MAPK activation then promotes cell cycle arrest in G₁ and stimulates the expression of several genes, including FUS1, leading ultimately to fusion of the mating partners (22).

The small GTPase Cdc42p and its guanine nucleotide exchange factor Cdc24p are essential for polarity establishment and subsequent bud formation (1, 38). In addition to their roles in cell polarity, these proteins have been proposed to play roles in signal transduction in response to mating pheromones (46, 47, 53). Temperature-sensitive cdc42 and cdc24 mutants have defects in -factor-stimulated transcription of FUS1 and in maintaining G₁ arrest at the restrictive temperature (46, 53). In addition, an interaction was detected by two-hybrid analysis between G and Cdc24p (53), and Cdc42p-GTP was shown to bind to and activate Ste20p (46). These findings led to the hypothesis that G activated Cdc24p, causing GTP loading of Cdc42p and consequent activation of Ste20p, as an important part of the pheromone signaling pathway.

More recent experiments have cast doubt upon the existence of a G-Cdc24p-Cdc42p-Ste20p pathway. Mutations in CDC24 that abolished detectable interaction with G did not cause any defects in -factor-stimulated FUS1 transcription or G₁ arrest but rather were specifically defective in orientation of the mating projection towards the mating partner (33). Furthermore, mutations in STE20 that abolished detectable interaction with Cdc42p were also reported to be wild type with regard to -factor-stimulated FUS1 transcription, G₁ arrest, and mating (23, 37). Together, these studies indicated that neither the G-Cdc24p interaction nor the Cdc42p-Ste20p interaction was important for -factor signaling. Furthermore, the polarity defect exhibited by temperature-sensitive cdc24 and cdc42 mutants triggers the morphogenesis checkpoint to delay the cells in G₂ with abundant G₁ cyclins (26), a state known to render cells unresponsive to -factor (34). This raised the possibility that the signaling defect of these mutants might be an indirect consequence of their accumulation at a nonresponding stage of the cell cycle. Indeed, the transcriptional induction of FUS1 was found to be quite normal in cdc24 and cdc42 mutants that were first arrested in G₁ by deprivation of G₁ cyclins (35), raising the question of whether Cdc24p and Cdc42p play any role at all in -factor signaling.

As illustrated by these studies, the interpretation of cdc42 and cdc24 phenotypes is complicated by the possibility of indirect effects stemming from the well-characterized polarity defects caused by these mutants at the restrictive temperature. To circumvent these problems, we performed a screen to identify -factor-resistant alleles of CDC42 that could still perform polarization functions. In this paper we report the isolation and characterization of such mutants, supporting the notion that Cdc42p has some direct role in pheromone signaling. Our results further suggest that this signaling function operates through Cdc42p-dependent activation and localization of Ste20p.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast media and cell synchrony. Yeast media (YEPD rich medium, synthetic complete medium lacking specific nutrients, and sporulation medium) have been described previously (13). YEPG and YEPS are the same as YEPD but with 2% galactose or sucrose instead of dextrose. Centrifugal elutriation to isolate early-G₁-phase cells was performed as described previously (27).

Strains, plasmids, and PCR manipulations. Standard media and methods were used for plasmid manipulations (2) and yeast genetic manipulations (13). The S. cerevisiae strains used in this study are listed in Table 1, and the plasmids used are listed in Table 2.

GAL1p-GST-STE11N

ste20CRIB

STE20-URA3-ste20CRIB

ste20CRIB

ste20CRIB

Screen to identify -factor-resistant cdc24 mutants. The oligonucleotides DJL42-3 and DJL42-6 (see above) were used to amplify CDC42 (and TDH3 terminator sequences) by mutagenic PCR using pDLB643 as a template. Mutagenic PCRs were similar to standard PCR except for the addition of 0.2 mM MnCl₂ and the use of an unbalanced deoxynucleoside triphosphate mix (0.5 mM dTTP, 0.5 mM dGTP, 0.1 mM dATP, and 0.1 mM dCTP [final concentrations]). The PCR products were transformed into DLY3067 together with BglII-digested ("gapped" plasmid) pDLB644, and Ura⁺ transformants were selected on dextrose-containing plates (to repress transcription of the genomic GAL1p-CDC42 allele) coated with 10 µg of -factor (to select for -factor-resistant cdc42 mutants). This amount of -factor (custom synthesized by Research Genetics, Huntsville, Ala.) was more than 10 times the amount needed to completely inhibit growth of the bar1 parent strain. -Factor-resistant colonies could in theory arise due to genomic mutations in other genes. To distinguish cdc42 mutants, colonies were tested for -factor-resistant growth on galactose-containing plates, where the genomic (wild-type) CDC42 is expressed. Plasmids were isolated from colonies that were -factor resistant when grown in dextrose-containing medium but not when grown in galactose-containing medium and were then retransformed into fresh DLY3067 to confirm that the plasmids were responsible for the -factor-resistant growth phenotype. To generate the TRP1-containing plasmids pMOSB42 to -45, CDC42 alleles were transferred from URA3 plasmids (pMOSB36 to -38 and pMOSB55) as 2.1-kb BamHI/XhoI fragments to the corresponding sites of pRS314 (45).

Spot assays for growth arrest. Cells harboring plasmid-borne CDC42 alleles were grown to stationary phase in dextrose-containing medium (to repress genomic GAL1p-CDC42) lacking nutrients appropriate to select for plasmid maintenance. The number of cells was determined using a hemocytometer, and diluted stocks were generated to spot 2 µl containing ~1,250, ~250, ~50, and ~10 cells onto plates containing the same selective dextrose medium either with or without 10 µg of -factor per plate. Assuming a plate "volume" of 20 ml, this would translate to ~0.3 µM -factor.

Northern blot analysis. RNA extraction, formaldehyde-agarose gel electrophoresis, capillary transfer, probe hybridization, and wash procedures were performed as described previously (10, 41, 42). The FUS1 probe was made from a 880-bp internal EcoRI/NheI fragment from the FUS1 gene, and the ACT1 probe was made from the entire ACT1 open reading frame, using [-³²P]dCTP (ICN Pharmaceuticals, Costa Mesa, Calif.) and the Prime-it II kit (Stratagene) according to the manufacturer's recommendations.

Flow cytometry. Cells were processed for fluorescence-activated cell sorter (FACS) analysis as described (14), except that the DNA was stained with 1 µM Sytox (Molecular Probes, Eugene, Oreg.) in 50 mM Tris-HCl (pH 8.0) (instead of propidium iodide).

DIC microscopy. Cells were viewed on an Axioskop apparatus (Zeiss, Thornwood, N.Y.) equipped with epifluorescence and differential interference contrast (DIC) optics. Images were captured by using a cooled-model charge-coupled device camera (Princeton Instruments, Princeton, N.J.).

Analysis of FUS1-lacZ expression. -Galactosidase assays were performed as described previously (40). Transformants in cdc42-1 strains were grown overnight at 28°C in selective synthetic medium containing 2% raffinose and 0.1% dextrose to an optical density at 660 nm of 0.3 to 0.6, preincubated for 2 h at 38.5°C, and then induced for 4 h with 2% galactose with or without 0.01 µM -factor. Assays in CDC42 strains were performed at 30°C.

Production of recombinant proteins and binding assays. Wild-type and mutant myc-tagged CDC42 alleles were expressed in Escherichia coli BL21(DE3) (Stratagene). Extracts were prepared in bacterial lysis buffer (750 mM sucrose, 100 mM NaCl, 100 mM Tris-HCl [pH 8.0], 5 mM EDTA) containing the protease inhibitors aprotinin (7.5 µg/ml; Sigma, St. Louis, Mo.), pepstatin (5 µg/ml; Sigma), leupeptin (10 µg/ml; Boehringer Mannheim, Indianapolis, Ind.), and phenylmethylsulfonyl fluoride (0.5 mM; Sigma). The cells were treated with 2 mg of lysozyme/ml for 20 min on ice. To remove genomic DNA, MgCl₂ was added to 15 mM and DNase I was added to 50 µg/ml. The cells were lysed by 20 min of incubation at 4°C with 2 mg of deoxycholic acid/ml. Insoluble material was removed by centrifugation at 4°C for 10 min. GST-Ste20-CRIB was expressed in the protease-deficient E. coli BL21, extracts were prepared as described above, and the protein was purified using glutathione Sepharose 4B (Amersham Pharmacia Biotech, Piscataway, N.J.) as specified by the manufacturer.

Localization of GFP-Ste20p. Cells containing pRL116 or pPP828 were propagated for at least 24 h in selective liquid medium containing 2% dextrose and 0.008% adenine (to inhibit accumulation of red pigment in ade1 cells) at 30°C (GAL1p-CDC42 strains) or at 36°C (cdc42-1 strains). The cells were examined without fixation using a Nikon E600 epifluorescence microscope with 100× Plan Fluor and 50× Plan oil-immersion objectives. Images were collected using a cooled black and white charge-coupled device camera (DAGE-MTI).

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Identification of recessive -factor-resistant alleles of CDC42. A hypothetical cdc42 mutant specifically defective in pheromone signal transduction should retain all functions necessary for cell polarity and proliferation but would fail to arrest in G₁ upon exposure to -factor. Thus, cells expressing such an allele as the sole source of Cdc42p should proliferate and form colonies on plates containing a growth-inhibitory dose of -factor. We employed an error-prone PCR mutagenesis strategy to generate random mutations in CDC42 and recombined the resulting alleles into a low-copy-number plasmid in cells whose endogenous CDC42 was transcriptionally repressed (using the regulatable GAL1 promoter; see Materials and Methods for details). Transformants that promoted proliferation on -factor-containing plates were selected. Plasmids containing putative CDC42 mutants were isolated from the rare -factor-resistant colonies and retransformed into the starting strain to confirm that the -factor resistance was due to the plasmid and not to a fortuitous genomic mutation. Three independently derived mutants (cdc42-md1, cdc42-md2, and cdc42-md12 [md for mating pathway defective]) were selected for further characterization (Fig. 1).

View larger version (33K): [in this window] [in a new window]   FIG. 1.   Isolation of -factor-resistant cdc42 alleles. The results of growth arrest assays of strain DLY3067 (GAL1p-CDC42) containing plasmid pMOSB55 (CDC42), pMOSB36 (cdc42-md1), pMOSB37 (cdc42-md2), or pMOSB38 (cdc42-md12) are shown. The photographs show growth after 3 days at 30°C with (+) or without () -factor.

View larger version (41K): [in this window] [in a new window]   FIG. 2.   FUS1 induction and cell cycle arrest in early-G1-phase cells. Cells containing two copies of wild-type CDC42 (DLY1 plus pMOSB55) or cdc42-md1 (MOSY0178 plus pMOSB36) were grown to exponential phase in sucrose-containing medium lacking uracil at 30°C (using two gene copies improved the morphology of cdc42-md1 cells, making the elutriation more effective). Small G1-phase cells were isolated by centrifugal elutriation and resuspended in YEPD medium at 30°C. Following a 10-min recovery period, -factor was added to the indicated final concentrations and the incubation was continued with vigorous shaking. (A) Northern blot of FUS1 mRNA accumulation following 30 min of -factor treatment. cdc42-md1 cells display severely reduced induction compared to wild-type cells. ACT1 mRNA on the same blots provides an internal control for loading and transfer. (B) FACS analysis demonstrating that both wild-type and cdc42-md1 cells were still in G1 phase after 40 min in YEPD medium without -factor (the same time point used for the FUS1 mRNA analysis: a 10-min recovery period plus a 30-min -factor treatment). The FACS profile of asynchronous wild-type cells (top) was used to determine the fluorescence signal equivalent to G1 (1 N) and G2 (2 N) DNA content. FACS profiles are shown for the indicated strains immediately following elutriation (t = 0') or 40 min later in the absence of -factor (t = 40'). (C) cdc42-md1 cells were not arrested in G1 phase in response to -factor. Aliquots of cells were withdrawn at the indicated times, fixed, and examined by phase-contrast microscopy to determine the percentage of budded cells (n = 200). , no -factor; , 0.015 µM -factor; , 0.059 µM -factor.

View larger version (72K): [in this window] [in a new window]   FIG. 3.   Intragenic-complementation analysis of the cdc42-md mutants. Plasmids containing the indicated pairs of CDC42 alleles were transformed into strain DLY3067. (A) Pheromone resistance of cdc42-md mutants is recessive. The photographs show growth after 3 days at 30°C with (+) and without () -factor. The plasmid pairs were (in order) pMOSB55 plus pMOSB45, pMOSB36 plus pMOSB42, pMOSB55 plus pMOSB42, pMOSB37 plus pMOSB43, pMOSB55 plus pMOSB43, pMOSB38 plus pMOSB44, and pMOSB55 plus pMOSB44. wt, wild type. (B) cdc42-md mutants form a single complementation group with regard to -factor resistance. Spot assays were performed as for panel A (except that fewer cells were spotted in this experiment). The plasmid pairs were (in order) pMOSB55 plus pMOSB45, pMOSB36 plus pMOSB42, pMOSB37 plus pMOSB43, pMOSB36 plus pMOSB43, pMOSB38 plus pMOSB44, pMOSB36 plus pMOSB44, and pMOSB37 plus pMOSB44. (C) cdc42-md mutants form two complementation groups with regard to cell morphology. Cells were grown to exponential phase and photographed using DIC optics. cdc42-md1 mutants exhibit broad necks and elongated buds (48% of cells), while cdc42-md2 mutants exhibit large round cells (>70% of cells); although some abnormal cells are present in the cdc42-md1 plus cdc42-md2 population (with generally less severe defects, perhaps due to plasmid loss), the majority (73%) exhibit normal morphology. The morphologies were similar in the presence and absence of -factor. The plasmid pairs were pMOSB55 plus pMOSB45 (wt + wt), pMOSB36 plus pMOSB42 (md1 + md1), pMOSB37 plus pMOSB43 (md2 + md2), and pMOSB36 plus pMOSB43 (md1 + md2).

Intragenic complementation analysis of the cdc42-md mutants. We took advantage of the recessive nature of these alleles to determine whether they had defects in the same or different functions required for the -factor response. Cells containing all pairwise combinations of plasmids bearing different cdc42-md alleles were uniformly resistant to -factor (Fig. 3B), indicating that no mutant could provide the signaling function(s) defective in another and suggesting that all three alleles were defective for the same function.

Epistasis analysis of the cdc42-md mutants. To determine where in the pheromone response pathway Cdc42p plays a role, we examined whether the cdc42-md mutants could block signaling that was initiated at various steps in the pathway. Induction of a FUS1-LacZ reporter construct was used as a readout of pathway activation. Like signaling induced by -factor, the signals initiated by overexpression of the G subunit Ste4p, or by a membrane-targeted Ste5p scaffold protein, were substantially blocked by cdc42-md mutants (Fig. 4). In contrast, signals initiated by the activated MEKK gene allele STE11-4 or by overexpression of the transcription factor Ste12p were unaffected by cdc42-md mutants (Fig. 4). This suggests that Cdc42p is required after Ste5p membrane recruitment for the activation of Ste11p. Previous studies have shown that this is precisely the step at which Ste20p is required (40).

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Epistatis analysis of cdc42-md mutants. Strain PPY911 (cdc42-1) was transformed with pRS314 (vector), pMOSB45 (wild type [WT]), pMOSB42 (md1), pMOSB43 (md2), pMOSB44 (md12), or pMOSB47 (V36A), as indicated. Each of these strains was then transformed with the mating-pathway-activating plasmids pL19 (GAL1p-STE4 [GAL::STE4]), pL-GS5N-CTM (GAL1p-STE5N-CTM [GAL::STE5N-CTM]), pG11-4 (GAL1p-STE11-4 [GAL1::STE11-4]), or pNC252 (2µm GAL1p-STE12 [2µm GAL1::STE12]) as indicated on the right. Transformants grown at restrictive temperature to inactivate cdc42-1 were induced with 2% galactose (or 2% galactose plus 0.01 µM -factor; top). The bars indicate the means ± standard deviation for four to six independent transformants. Results similar to those shown with STE5N-CTM were also seen with STE5-CTM, and results similar to those shown with GAL1p-STE11-4 were also seen with GAL1p-STE11N (data not shown).

Sequence analysis of cdc42 mutants. To determine the natures of the mutations that rendered CDC42 signaling defective, we recovered the mutant plasmids from yeast and sequenced the open reading frame of each mutant. As shown in Fig. 5A, all three mutants contained single-amino-acid changes in the "effector" domain of Cdc24p: a valine-to-alanine substitution at residue 36 in cdc42-md1 and a tyrosine-to-cysteine substitution at position 40 in both cdc42-md2 and cdc42-md12. In addition, cdc42-md1 contained substitutions at residues 149 and 177, and cdc42-md12 contained a valine-to-alanine substitution at residue 77 (Fig. 5A). Because the Y40C substitution was the only change in cdc42-md2, we assume that the -factor resistance of cdc42-md12 also derives from this same change. Presumably the V77A mutation confers the somewhat-improved growth properties observed in cdc42-md12 compared to those of cdc42-md2. In a recent study, cdc42^Y40C was reported to be nonviable (18). The discrepancy appears to be due to the fact that in our case the mutant is expressed from a low-copy-number (CEN ARS) plasmid while in that study it was integrated into the genome. To determine which changes were responsible for the -factor resistance of the cdc42-md1 mutant, we constructed a mutant that contained only the V36A substitution. This mutant also conferred an -factor-resistant phenotype (although not as strong as that conferred by cdc42-md1 [Fig. 4 and 5B]). We conclude that a mutation at either V36 (to A) or Y40 (to C) in the effector domain of Cdc42p renders the protein signaling defective.

View larger version (43K): [in this window] [in a new window]   FIG. 5.   Defective interaction of cdc42-md mutants with Ste20p. (A) Altered residues in cdc42-md mutants. (B) The V36A substitution confers -factor resistance. Growth arrest assays of strain DLY3067 (GAL1p-CDC42) containing plasmid pMOSB55 (CDC42), pMOSB36 (cdc42-md1), or pMOSB177 (cdc42-V36A) were performed. The photographs show growth after 3 days at 30°C with (+) or without () -factor. (C) Binding of cdc42 mutants to the Ste20p CRIB domain. The binding conditions were as described in Materials and Methods. The top row shows the amount of the indicated myc-tagged Cdc42p mutant that bound to immobilized GST-CRIB, the second row shows the amount of each Cdc42p mutant that bound to immobilized GST (to control for nonspecific adsorption), and the third row shows the amount of each Cdc42p mutant added to the binding reaction. India ink staining of the blots confirmed that equal amounts of GST or GST-CRIB were present in each lane. Similar results were obtained in four independent experiments. Recombinant Cdc42p was generated from pDLB1234 (D57Y, mimicking GDP-bound Cdc42p), pDLB1235 (Q61L, mimicking GTP-bound Cdc42p), pDLB1240 (V36A and Q61L), or pDLB1242 (Y40C and Q61L).

Defective interaction of Ste20p with cdc42-md mutants. The Y40C mutation has been described in human CDC42 (which is 80% identical to yeast CDC42 and complements a cdc42 null mutation in yeast [44]), where it was found to render Cdc42p defective in binding to and activating p21-activated kinase (PAK), a mammalian Ste20p homologue (19). Combined with epistasis analysis (Fig. 4), this suggested that the cdc42-md mutants might be defective in binding to Ste20p. Binding of PAK family kinases to Cdc42p is mediated by the CRIB domain in these proteins (8). We produced the Ste20p CRIB domain as a GST fusion protein in E. coli and purified it using glutathione Sepharose beads. The wild type or V36A or Y40C mutants of Cdc42p were produced as myc-tagged proteins in E. coli and tested for binding to the GST-Ste20p-CRIB beads or to GST beads as a negative control. A Q61L substitution, which inhibits Cdc42p GTPase activity, was also engineered into the constructs to ensure that the proteins were GTP bound (a prerequisite for CRIB domain binding). As shown in Fig. 5C, the V36A substitution conferred a mild defect in Ste20p binding while the Y40C substitution conferred a severe defect.

cla4

ste20

View larger version (54K): [in this window] [in a new window]   FIG. 6.   Synthetic lethality of cdc42-md mutants with cla4 but not ste20 mutants. Strains DLY3067 (GAL1p-CDC42; left), MOSY0106 (GAL1p-CDC42 ste20; middle), and MOSY0023 (GAL1p-CDC42 cla4; right) were transformed with pMOSB55 (CDC42), pMOSB36 (cdc42-md1), pMOSB37 (cdc42-md2), or pMOSB38 (cdc42-md12), as indicated. Cells were streaked out on dextrose-containing plates (to repress the genomic GAL1p-CDC42) lacking uracil (to select for plasmid maintenance) and incubated at 30°C for 3 days. WT, wild type.

Defective localization of Ste20p in cdc42-md mutants. Ste20p is concentrated at the bud tips of many cells during vegetative growth, and at the shmoo tips of cells responding to -factor, in a manner that depends to a large extent on the CRIB domain (23, 37). GFP-Ste20p localization to bud tips was greatly reduced in all of the cdc42-md mutants (Fig. 7). Even cells containing both cdc42-md1 and cdc42-md2, which exhibited a wild-type growth rate and cell morphology, failed to localize GFP-Ste20p to the bud tip in most cells (see Fig. 11 below). We were unable to address the question of whether shmoo tip localization was affected because these cells were resistant to -factor and did not make shmoos.

View larger version (50K): [in this window] [in a new window]   FIG. 7.   Ste20p localization in cdc42-md mutants. Strain PPY911 (cdc42-1) was transformed with pRL116 (GFP-STE20 [GFP-Ste20p]) and then with pMOSB45 (CDC45), pMOSB42 (cdc42-md1), pMOSB43 (cdc42-md2), pMOSB44 (cdc42-md12), or pMOSB47 (cdc42-V36A) as indicated. The cells were grown at 36°C to inactivate cdc42-1, and the localization of GFP-Ste20p was examined by fluorescence microscopy of living cells. Representative fields are shown (top), and the percentage of budded cells displaying GFP-Ste20p concentrated at the bud tips is quantitated (bottom left). The same set of plasmids was transformed into DLY3067 (GAL1p-CDC42), and the cells were grown on dextrose (to repress GAL1p-CDC42) and analyzed as above (bottom right). Several images were captured for each transformant, and all cells within those images were scored for whether GFP-Ste20 was detectably concentrated at the bud tip. n = 80 to 138 (PPY911 transformants) or n = 55 to 91 (DLY3067 transformants).

Suppression of the cdc42-md signaling defect by overexpression of Ste20p. If the -factor resistance phenotype of the cdc42-md mutants is due to defective interaction with Ste20p, then overexpression of Ste20p might suppress that phenotype. Indeed, overexpression of STE20 substantially suppressed the -factor-resistant growth of cdc42-md mutants (Fig. 8). In contrast, overexpression of the related CLA4 did not suppress the cdc42-md signaling defect, although it did suppress the growth defect of cdc42-md2 mutants in the absence of -factor (Fig. 8). These findings could be accommodated by at least two models: the observed suppression may be due to a bypass of the cdc42-md defects by the overexpressed kinases or to a restoration of a sufficiently effective interaction between the mutant Cdc42p and the kinases. In the latter model, the CRIB-binding defect of cdc42-md2 causes a vegetative growth defect due to impaired interaction with Cla4p and a signaling defect due to impaired interactions with Ste20p.

View larger version (40K): [in this window] [in a new window]   FIG. 8.   Overexpression of STE20 suppresses the cdc42-md signaling defect. Strain DLY3067 (GAL1p-CDC42) was transformed with pMOSB45 (wild type [WT]), pMOSB42 (md1), pMOSB43 (md2), or pMOSB44 (md12), and each strain was then transformed with pRS426 (vector), pVTU-Ste20 (STE20), or pDLB722 (CLA4). The photographs show growth after 3 days at 23°C with (+) or without () -factor.

Restoration of pheromone signaling in cdc42-md mutants by the ste20CRIB allele. The findings presented above are consistent with the simple hypothesis that the Cdc42p-Ste20p interaction is a critical step in the -factor signaling pathway: the cdc42-md mutants are defective for this interaction in vitro and in vivo, epistasis analysis suggests that Cdc42p acts at the same level of the pathway as Ste20p, and overexpression of Ste20p suppresses that the cdc42-md signaling defect. Nevertheless, the hypothesis that the Cdc42p-Ste20p interaction is important for signaling is contradicted by previous studies showing that a ste20 mutant lacking the CRIB domain (and therefore defective in Cdc42p interaction) did not affect signaling (23, 37). A possible resolution of this apparent paradox is suggested by recent evidence that the CRIB domain in PAK family kinases is part of an autoinhibitory domain, leading to a model in which Cdc42p (or Rac) proteins activate PAK family kinases by "relief of autoinhibition" (3, 49, 52, 54). For example, in the Pak1 kinase of Schizosaccharomyces pombe, a region overlapping the CRIB domain of Pak1 binds intramolecularly to the catalytic domain, yielding a "closed" conformation that can be "opened" either by binding to Cdc42p or by mutations in the CRIB domain (49). By analogy, the ste20CRIB mutant may be competent for signal transduction because it no longer requires a normally critical interaction with Cdc42p for its activation. This hypothesis predicts that the ste20CRIB mutant should bypass the requirement for Cdc42p in signaling, rendering cdc42-md strains sensitive to pheromone. Indeed, we found that ste20CRIB restored significant -factor signaling to the cdc42-md mutants, as assayed by growth inhibition (Fig. 9A) or FUS1 induction (Fig. 9B). This suggests that the ste20CRIB allele encodes an activated form of Ste20p and supports the hypothesis that the Cdc42p-Ste20p interaction is normally important for pheromone signaling. We noticed, however, that ste20CRIB did not completely restore -factor sensitivity to cdc42-md mutants. This suggests that cdc42-md mutants have some defect in addition to the Ste20p activation defect.

View larger version (45K): [in this window] [in a new window]   FIG. 9.   ste20CRIB largely restores -factor sensitivity to cdc42-md strains. (A) Growth arrest. Strains DLY3067 (GAL1p-CDC42 STE20) and MOSY0268 (GAL1p-CDC42 ste20CRIB) were transformed with pMOSB55 (wild type [WT]), pMOSB36 (md1), pMOSB37 (md2), or pMOSB38 (md12), as indicated. The photographs show growth after 3 days at 23°C with (+) or without () -factor. (B) FUS1-lacZ induction. The same strains as in panel A harbored the FUS1-lacZ plasmid pSB231 plus either pMOSB45 (WT) or pMOSB42 (md1) and were assayed in glucose medium after 90 min with (+) or without () 0.01 µM -factor. The bars indicate the means ± standard deviations of four transformants.

Contribution of Bem1p to Cdc42p- and Ste20-dependent signaling. The BEM1 gene was discovered through its key role in polarity establishment (5, 9, 38). Subsequent studies showed that Bem1p can also modulate the pheromone response pathway, as Bem1p overexpression increases signaling while Bem1p removal decreases signaling (17, 29). Because the role of Bem1p in polarity establishment involves Cdc42p and Cdc24p (5, 38), we speculated that its role in pheromone-responsive signaling may also involve Cdc42p and therefore that the residual signaling defect observed in cdc42-md ste20CRIB mutants might reflect reduced Bem1p function resulting from the cdc42-md mutations. Consistent with this hypothesis, we found that like cdc42-md ste20CRIB mutants, bem1 ste20CRIB mutants also displayed partial -factor resistance (Fig. 10A) and reduced FUS1 induction (Fig. 10B) compared to ste20CRIB single mutants. The signaling defect in bem1 ste20CRIB mutants was also evident when the pathway was activated by membrane-targeted Ste5p but not when it was activated by the activated Ste11p derivative Ste11N (Fig. 10C). This indicates that like Cdc42p, Bem1p acts downstream of Ste5p membrane recruitment to promote activation of Ste11p by Ste20p.

View larger version (48K): [in this window] [in a new window]   FIG. 10.   BEM1 is important for signaling in ste20CRIB mutant strains. (A) Growth arrest. The photographs show growth after 2 days at 30°C with (+) and without () -factor. (B) FUS1p-lacZ induction by -factor. Strains harboring a FUS1p-lacZ reporter plasmid (p3058) were treated with and without 0.1 µM -factor in duplicate for 1 and 2 h with similar results; the data were expressed as percent mean induced -galactosidase activity in the wild-type (WT) strain (427 U at 1 h; 835 U at 2 h) and combined, with each bar representing the mean ± standard deviation (SD) of four measurements. (C) FUS1p-lacZ induction by Ste5N-CTM or Ste11N was assayed in strains harboring p3058 and either pGFP-GS5N-CTM or pGS11N-T after 4 h of induction with galactose; the bars indicate the means ± SD of four to six transformants, expressed as percent mean induced -galactosidase activity in the WT strain (1,024 U for Ste5N-CTM; 644 U for Ste11N). (D) FUS1p-lacZ induction in strains harboring p3058 and either pGFP-GS5N-CTM (Ste5N-CTM) or pGFP-GS5N-Sec22 (Ste5N-Sec22) was assayed after 4 h of induction with galactose; the bars indicate the means ± SD of eight transformants. The strains in all panels were DLY1 (WT), MOSY0252 (ste20CRIB), JMY1128 (bem1), and MOSY0270 (bem1 ste20CRIB).

ste20CRIB

ste20CRIB

ste20CRIB

View larger version (59K): [in this window] [in a new window]   FIG. 11.   Overproduction of BEM1 restores Ste20p localization and signaling competence to cdc42-md strains. Strain PPY911 (cdc42-1) containing pL-GS5N-CTM (for FUS1 induction) or pPP828 (for Ste20p localization) was transformed with the CDC42 plasmids pMOSB45 (wild type [WT]), pMOSB42 (md1), or pMOSB43 (md2) and then with YEp24 (vector), pMOSB36 (md1), pMOSB37 (md2), pPB321 (2µm BEM1), or pDLB722 (2µm CLA4) as indicated. FUS1-lacZ induction was measured as for Fig. 4; the bars indicate means ± SD of six independent transformants. GFP-Ste20p localization was quantitated as for Fig. 7 (200 cells counted), and representative cells are shown below.

View larger version (41K): [in this window] [in a new window]   FIG. 12.   Bem1p and Ste20pCRIB cooperate to restore -factor sensitivity to a cdc42-md1 strain. Strains DLY3067 (GAL1p-CDC42 STE20) and MOSY0268 (GAL1p-CDC42 ste20CRIB) were transformed with pMOSB42 (cdc42-md1) and either pRS426 (vector) or pPB321 (2µm BEM1), as indicated. The photographs show growth after 3 days at 30°C with (+) and without () -factor.

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

A role for Cdc42p in signal transduction in response to -factor. We have isolated cdc42 mutants that display recessive defects in -factor signaling while retaining the ability to proliferate. All of these cdc42-md mutants had morphogenesis defects in addition to their signaling defect. However, the signaling defect is unlikely to be an indirect result of the morphogenesis defects for the following reasons. First, overexpression of CLA4 suppressed the morphogenesis defects of cdc42-md mutants but not the signaling defect. Second, two pairwise combinations of cdc42-md alleles displayed intragenic complementation for their morphogenesis defects but not for their signaling defects. Third, a synchronous early-G₁-phase population of cdc42-md1 mutant cells isolated by centrifugal elutriation displayed a signaling defect even during G₁ phase, a time when cells are uniformly sensitive to -factor and Cdc42p-dependent polarity has yet to be established. It is difficult to see how a signaling defect in this synchronous culture could arise due to any indirect cell cycle or morphogenesis problems. In summary, these results strongly suggest that Cdc42p plays a role in -factor-induced signaling that is separate from its roles in morphogenesis, confirming the suggestions from earlier studies with temperature-sensitive cdc42 mutants (46, 47, 53).

Ste20p is a key Cdc42p target involved in pheromone signaling. By several criteria, the cdc42-md mutants are defective in interacting with Ste20p: they are defective (to varying extents) in binding to the Ste20p CRIB domain in vitro, they fail to localize Ste20p to bud tips in vivo, and they display synthetic lethality with cla4 mutants. Furthermore, epistasis analysis suggests that cdc42-md mutants are defective in signal transduction at the same step in the pathway as ste20 mutants (i.e, the activation of Ste11p following membrane targeting of Ste5p). Finally, the signaling defect of cdc42-md mutants is largely suppressed by overproduction of Ste20p. These data strongly support the hypothesis that the Cdc42p-Ste20p interaction is important for efficient signaling in the pheromone response pathway, as originally proposed (46, 53). However, more recent studies have argued that the Cdc42p-Ste20p interaction is dispensable for pheromone response, based on the signaling competence of a version of Ste20p (Ste20pCRIB) that cannot bind to Cdc42p. We found that Ste20pCRIB largely restored signaling competence to strains containing the cdc42-md mutants. This observation suggests that Ste20pCRIB is an activated version of Ste20p that no longer requires interaction with Cdc42p for its activation, consistent with current models for PAK activation which involve a relief of autoinhibition mechanism (3, 49, 54). We therefore conclude that interaction of Cdc42p with Ste20p is normally required for Ste20p to participate in the pheromone response pathway. Importantly, however, we do not find it necessary to propose that pheromone regulates either GTP loading of Cdc42p, the Cdc42p-Ste20p interaction, or Ste20p kinase activity. Instead, the simplest model consistent with available data is that access of Ste20p to its substrates is the pheromone-regulated step, with the effect of Cdc42p on Ste20p kinase activity being a preexisting condition that is independent of pheromone exposure (see reference 40 for further discussion).

Cdc42p may play a second role together with Bem1p in pheromone signaling. Although Ste20p appears to be the major Cdc42p target for signal transduction, our results suggest the existence of a second role for Cdc42p in this process, because cdc42-md mutants displayed a residual signaling defect even in the presence of the activated ste20CRIB allele. Given the strong links between Cdc42p and the SH3-domain-containing scaffold protein Bem1p that have been established through studies of cell polarity in yeast (5, 9, 38), we suspected that this second role might involve Bem1p (which has also been shown to modulate the strength of -factor signaling [17, 29]). Consistent with this hypothesis, Bem1p overexpression partially suppressed the signaling defect of cdc42-md mutants and Bem1p overexpression together with ste20CRIB could fully suppress the -factor-resistant growth of cdc42-md mutants.

ste20CRIB