Ras- and Rho-type GTPases function as binary switches, cycling between an active GTP-bound state and an inactive GDP-bound state (Bourne et al., 1990). In multicellular organisms, both classes of GTPases are involved in signal transduction mediated through receptor-type tyrosine kinases. Although the roles of Ras- and Rho-type GTPases may be viewed somewhat separately, with the primary roles of Ras-type GTPases apparently being to control cell proliferation and differentiation (Bollag and McCormick, 1991) and the primary role of Rho-type GTPases apparently being to control cytoskeletal reorganizations (Ridley and Hall, 1992; Ridley et al., 1992; Johnson and Pringle, 1990), there is evidence that the activities of Ras- and Rho-type GTPases may be tightly coupled. For example, in addition to containing the Ras GEF ()domain (Cdc25 domain), both the Ras-type GEF mSOS and Ras-GRF (Boguski and McCormick, 1993) contain a putative GEF domain (the Dbl homology domain) for Rho-type GTPases. mSOS and Ras-GRF might therefore serve as activators of both Ras- and Rho-type GTPases. A second observation that suggests that the regulation of Ras- and Rho-type GTPases might be tightly coupled is that the protein p190, which was identified based upon its physical association with Ras-GAP, contains a Rho-GAP domain that can serve as a potent GTPase activator for various Rho-type GTPases (Settleman et al., 1992).

The process of bud emergence in Saccharomyces cerevisiae provides an opportunity to study the functional interplay between a Ras-type GTPase (Rsr1/Bud1, hereafter referred to as Rsr1) and a Rho-type GTPase (Cdc42). Rsr1, Bud5 (the GEF for Rsr1), and Bud2 (the GAP for Rsr1) are required for both the haploid (axial) and a/a diploid (bipolar) patterns of bud-site selection but not for bud-site assembly (Drubin, 1991). Mutations in any one of these proteins cause cells to display a random pattern of bud-site selection (Bender and Pringle, 1989; Chant and Herskowitz, 1991; Chant et al., 1991; Park et al., 1993; Bender, 1993). Cdc24, Cdc42, and Bem1 are required for bud-site assembly (Sloat et al., 1981; Adams et al., 1990). This essential requirement for Cdc24 is likely due, at least in part, to its interaction with Cdc42. Cdc24 contains a Dbl homology domain and has been shown to display GEF activity toward Cdc42 (Zheng et al., 1994a).

The observation that certain mutant alleles of CDC24 can randomize bud-site selection (Sloat et al., 1981) suggests that Cdc24 may serve as a link between proteins involved in bud-site selection and proteins involved in bud-site assembly. The observation that the temperature-sensitive (Ts) lethality of some cdc24 mutants can be suppressed by overexpression of RSR1 (Bender and Pringle, 1989) has raised the possibility that Cdc24 might bind to Rsr1.

Cdc24 can also bind to the SH3 domain-containing protein Bem1. The interaction between Cdc24 and Bem1 was initially identified during a screen for proteins that display two-hybrid interactions with Bem1 and was confirmed using bacterially expressed fusion proteins (Peterson et al., 1994). Given that mutant alleles of CDC24 have been identified during screens for mutations that display synthetic lethality with bem1 and that CDC24 and BEM1 both are involved in bud emergence (Hartwell et al., 1974; Bender and Pringle, 1991; Chant et al., 1991; Chenevert et al., 1992), the physical interaction between Bem1 and Cdc24 is almost certainly biologically important. However, the role of this interaction and the manner by which it is regulated are not yet known. One possibility is that Bem1 affects the interaction of Cdc24 with either Cdc42 or Rsr1. Given that mutations in CDC24 cause cells to be sensitive to high levels of Ca (Ohya et al., 1986), it is possible that Ca plays a role in the interaction between Cdc24 and Bem1.

In the present study, we have over-expressed Rsr1, Cdc42, Cdc24, and Bem1 in Escherichia coli or in insect Sf9 cells as GST fusion proteins and used various biochemical approaches to examine the possible interactions between these purified, recombinant bud-site selection and bud-site assembly gene products. One of the goals of the present study was to address whether Cdc24 can bind directly to Rsr1 and, if so, to determine whether this binding has any consequence on the GTPase cycle of Rsr1 or on the GEF activity of Cdc24 toward Cdc42. We also examined whether Ca affects the interaction of Cdc24 with Bem1 and if this interaction in turn influences the functional coupling of Cdc24 to Cdc42 or Rsr1.

EXPERIMENTAL PROCEDURES

Expression of Bud-site Selection and Bud-site Assembly Gene Products as GST Fusion Proteins

Complex Formation of Cdc24 with GST Fusion Proteins

GDP/GTP Exchange and GTPase Assays

RESULTS

To investigate whether Cdc24 can bind to Rsr1, we used recombinant Cdc24 protein purified via a baculovirus expression system from Sf9 cells and Rsr1 purified as a GST fusion protein from E. coli (see ``Experimental Procedures''). Fig. 1A shows the results of an experiment in which Cdc24 was incubated with different guanine nucleotide-bound states of GST-Rsr1 (i.e. guanine nucleotide-depleted, GDP-bound, and GTPS-bound forms), and then complex formation between Cdc24 and the different forms of GST-Rsr1 was assessed by co-precipitation of these proteins using glutathione-agarose. Immunoblot analysis using an anti-Cdc24 antibody indicated the presence of Cdc24 protein in the precipitated pellet under different conditions (lanes1-4); lane5 shows the purified Cdc24 used in the experiment (i.e. loaded directly on to the gel). As shown in lane1 of Fig. 1A, Cdc24 showed no detectable tendency to associate with GST alone. However, Cdc24 was able to effectively bind to GST-Rsr1-GTPS (lane4), less well to a GST-Rsr1-GDP (lane3), and to a much less extent to the guanine nucleotide-depleted form of Rsr1 (lane2).

Figure 1: Interactions of Cdc24 with Rsr1, Cdc42, and Bem1. Anti-Cdc24 immunoblot of the co-precipitates of GST fusion proteins immobilized on glutathione-agarose beads. A, 1 µg of Cdc24 was incubated with 5 µg of immobilized GST (lane1), GST-Rsr1 depleted of guanine nucleotide (lane2), GST-Rsr1-GDP (lane3), and GST-Rsr1-GTPS (lane4). 0.1 µg of Cdc24 was loaded on SDS-polyacrylamide gel electrophoresis as a positive control (lane5). Co-precipitation was performed as described under ``Experimental Procedures.'' B, co-precipitations of Cdc24 with GST-Cdc42. Lane1, GST alone; lane2, GST-Cdc42 depleted of guanine nucleotide; lane3, GST-Cdc42-GDP; and lane4, GST-Cdc42-GTPS. 0.1 µg of Cdc24 was used in lane5 as a control. C, co-precipitations of Cdc24 with GST-Bem1. Immobilized GST (lane1), GST-Bem1 with 2 mM CaCl (lane2), and GST-Bem1 with no extra Ca (lane3) were individually incubated with Cdc24. Lane4, 0.3 µg Cdc24 was loaded directly onto SDS-polyacrylamide gel.

These results can be compared with those obtained when assaying the interactions between Cdc24 and GST-Cdc42 (Fig. 1B). Previously, we demonstrated that Cdc24 acts as a potent GEF for the S. cerevisiae Cdc42 protein (Zheng et al., 1994a). The results presented in Fig. 1B show that Cdc24 binds most tightly to the guanine nucleotide-depleted form of Cdc42 (lane2) and to lesser extents to the GDP-bound (lane3) and GTPS-bound forms (lane4) of Cdc42 (lane5 again shows the Western blot of the Cdc24 protein used in the experiment). This pattern of interaction is virtually the same as that observed between the human Cdc42 protein and the DBL oncogene product (Hart et al., 1994) and is consistent with the fact that GEFs stabilize the guanine nucleotide-depleted state of GTP-binding proteins. The data in Fig. 1, A and B, suggest that Cdc24 does not serve as either a GEF or a GDP dissociation inhibitor for Rsr1 since in the former case it would be expected that Cdc24 would preferentially bind to the guanine nucleotide-depleted form of Rsr1, while in the latter case Cdc24 should preferentially bind to the GDP-bound form of Rsr1. However, the results shown in Fig. 1A are consistent with Cdc24 serving as a target for the GTP-bound form of Rsr1.

Using bacterially expressed fusion proteins, the C-terminal 75 amino acids of Cdc24 have been found to be sufficient for the binding to Bem1 (Peterson et al., 1994). To investigate the possible effects of Bem1 on the interactions of Cdc24 with Rsr1 and Cdc42, we first tested whether full-length Cdc24 can bind to full-length Bem1. As shown in Fig. 1C, full-length Cdc24 can indeed bind to a GST-Bem1 fusion protein (lane3), and this interaction is inhibited by 2 mM Ca (lane2) (lane1 shows the results obtained with GST alone, and lane4 shows the Cdc24 standard). When comparing the interactions of Cdc24 with Rsr1, Cdc42, and Bem1, we find that Cdc24 binds most tightly to Bem1; virtually stoichiometric binding of Cdc24 to Bem1 is observed even after extensive washing of the precipitated glutathione-agarose GST-Bem1 beads (i.e. >6 times). The interaction between Cdc24 and Rsr1 appears to be the least stable; four or more washes of the precipitated glutathione-agarose GST-Rsr1 beads eliminate most of the detectable binding of Cdc24.

The ability of Cdc24 to bind to Rsr1 raises the question of whether Cdc24 has any effect on the GTP-binding/GTPase cycle of Rsr1. Phenotypic, sequence, and genetic data argue strongly that the GDP-GTP exchange activity of Rsr1 is regulated by Bud5 (Chant et al., 1991; Powers et al., 1991; Bender, 1993). To test whether Cdc24 has any effect on the ability of Bud5 to regulate the guanine nucleotide exchange activity of Rsr1, we first examined whether purified, recombinant Bud5 can in fact serve as a GEF for Rsr1. To do this, we expressed and purified a GST-Bud5 protein from Sf9 cells and then assayed the ability of this fusion protein to stimulate the dissociation of GDP from GST-Rsr1. The results presented in Fig. 2A demonstrate that Bud5 strongly stimulates the rate of GDP dissociation from Rsr1. Specifically, the half-time for the dissociation of GDP from Rsr1 was >35 min at 25 °C compared with 5 min in the presence of GST-Bud5. These findings provide direct biochemical evidence that Bud5 does indeed serve as a GEF for Rsr1. Identical rates were measured for the Bud5-stimulated dissociation of GDP from Rsr1 in the presence and absence of excess Cdc24 (Fig. 2A), suggesting that Cdc24 has no effect on the interaction between Bud5 and Rsr1. Bem1 was also seen to have no effect on the GEF activity of Bud5 toward Rsr1, whether in the presence or absence of Cdc24 (data not shown).

Figure 2: A, effect of Cdc24 on the intrinsic and Bud5-stimulated GDP dissociation from Rsr1. The time course for the dissociation of GDP from [H]GDP-bound Rsr1 (0.5 µg) was measured after the addition of 2 µg of GST, 0.2 µg of GST-Bud5, 2 µg of Cdc24, or 0.2 µg of GST-Bud5 plus 2 µg of Cdc24. The percentage of GDP that remained bound was calculated relative to subtracting the amount of [H]GDP bound to Rsr1 prior to any additions (i.e. time zero). B, effects of Bem1 and Rsr1 on the Cdc24-stimulated dissociation of GDP from Cdc42 (0.6 µg). GDP dissociation was measured at different times after the addition of 2 µg of GST, 0.4 µg of Cdc24, 0.4 µg of Cdc24 plus 2 µg of GST-Bem1, 0.4 µg of Cdc24 plus 2 µg of GST-Rsr1-GTPS, or 0.4 µg of Cdc24 plus 2 µg of GST-Rsr1-GDP.

We next examined whether Rsr1 could influence the ability of Cdc24 to act as a GEF for Cdc42. As shown in Fig. 2B, the addition of excess amounts of the GDP-bound or GTPS-bound forms of Rsr1 had no effect on the rate of the Cdc24-stimulated dissociation of GDP from Cdc42. Similarly, the addition of excess Bem1 had no effect on the GEF activity of Cdc24 toward Cdc42. These results suggest that independent (and not overlapping) domains of Cdc24 may be responsible for the interactions with Rsr1, Cdc42, and Bem1.

Given that Cdc24 interacted more effectively with the GTPS-bound form of Rsr1 than with the guanine nucleotide-depleted or GDP-bound forms, we sought to investigate whether Cdc24 could affect the intrinsic or GAP-stimulated GTPase activities of Rsr1. We found that Cdc24 strongly inhibits the intrinsic GTPase activity of Rsr1 (compare the opensquares and soliddiamonds in Fig. 3A). Rsr1 is similar in sequence to the mammalian Rap1a protein (Bender and Pringle, 1989), and the GTPase activities of Rsr1 and Rap1a can be activated by the same GAP from bovine brain cytosol (Holden et al., 1991) and by recombinant Rap-GAP. ()Fig. 3A further illustrates the ability of the recombinant Rap-GAP to stimulate the GTPase activity of Rsr1 (compare the opendiamonds and opensquares) and shows that Cdc24 can inhibit this GAP-stimulated activity (compare the opendiamonds and opentriangles). Control experiments using [-P]GTP indicate that the loss of radioactivity being measured in these experiments in fact reflects the release of P due to GTP hydrolysis and is not the outcome of the dissociation of radiolabeled GTP from Rsr1 (see Fig. 3A). These results indicate that there is a functional consequence of the direct binding of Cdc24 to Rsr1, namely an inhibition of the Rsr1 GTPase activity. Thus, Cdc24 has the potential to influence simultaneously the GTP-binding/GTPase cycles of a Rho-subtype protein (acting as a GEF for Cdc42) and a Ras-subtype protein (acting as a GTPase-inhibitor protein (GIP) for Rsr1). The results presented in Fig. 3B show that the GIP activity of Cdc24 is not influenced by Bem1 or GDP-bound Cdc42, further supporting the view that Rsr1, Cdc42, and Bem1 bind to distinct sites on Cdc24.

Figure 3: A, the effects of Cdc24 on the GTPase activity of Rsr1. 0.6 µg of Rsr1 was preloaded with [-P]GTP or [-P]GTP, and the time course of GTP hydrolysis or dissociation was followed in the presence of 2 µg of GST, 1 µg of Cdc24, 2 µg of Rap1-GAP, or 1 µg of Cdc24 plus 2 µg of Rap1-GAP. B, the effects of Bem1 and Cdc42 on the GIP activity of Cdc24. [-P]GTP hydrolytic activity of Rsr1 was monitored in the presence of GST, Cdc24, Rap1-GAP, Cdc24 plus Rap1-GAP, Cdc24 plus Rap1-GAP and GST-Bem1, or Cdc24 plus Rap1-GAP and GST-Cdc42-GDP.

DISCUSSION

The ability of RSR1 to serve as a multicopy suppressor of Ts mutations in CDC24 suggests that there is a functional relationship between the Rsr1 and Cdc24 proteins (Bender and Pringle, 1989; Ruggieri et al., 1992). In the present study, we have provided biochemical evidence for a direct physical association between the two proteins. Specifically, we found that Cdc24 can bind to glutathione-agarose beads coated with GST-Rsr1 (but not to control beads coated with GST alone) and with preference to the GTP-bound state of Rsr1. Also, we have shown that Cdc24 can interfere with both the intrinsic and GAP-stimulated GTPase activity of Rsr1. The observations that Cdc24 binds most tightly to the GTP-bound form of Rsr1 in the direct binding assays and that Cdc24 can stabilize the GTP-bound state of Rsr1 (i.e. serve as a GIP) are consistent with a model in which Cdc24 is a target for activated (GTP-bound) Rsr1, along the lines of recently identified targets for the human Cdc42 protein (Manser et al., 1993; Manser et al., 1994; Zheng et al., 1994b) that preferentially bind to the GTP-bound form of the GTP-binding protein and in some cases stabilize the GTP-bound state by acting as GIPs (Manser et al., 1993, 1994). Thus, we propose that the activation of Rsr1 by the Bud5 protein primes Rsr1 for binding to Cdc24 and that the interaction stabilizes the GTP-bound state of Rsr1 (Fig. 4).

Figure 4: A model for the interactions among proteins involved in the GTPase cycles of Rsr1 and Cdc42.

One possible outcome of the interaction between Rsr1 and Cdc24 would be that Rsr1 activates the exchange factor activity of Cdc24 toward Cdc42. However, two lines of evidence argue against this model. First, we were unable to detect any effect of Rsr1 on the GEF activity of Cdc24 toward Cdc42. Second, since mutations that are predicted to interfere with the GEF activity of Cdc24 toward Cdc42 are lethal (Ziman and Johnson, 1994), if Rsr1 were required for the activation of that GEF activity, then Rsr1 would be expected to also be essential for bud emergence. However, cells lacking Rsr1 form apparently normal buds and are healthy (Bender and Pringle, 1989).

A second model for the role of the interaction between Rsr1 and Cdc24 is that Rsr1 serves to position Cdc24 at the site where a bud is supposed to form. In one version of this model, the GEF for Rsr1 would be predicted to be localized to that site first, causing Rsr1 to be in the GTP-bound state only at that site. A variety of phenotypic, sequence, and genetic results strongly suggest that the GEF for Rsr1 is encoded by the BUD5 gene. In this study, we demonstrated that Bud5 can indeed stimulate GDP-GTP exchange on Rsr1. The observation that Cdc24 does not interfere with the GEF activity of Bud5 on Rsr1 is consistent with the view that Cdc24 interacts preferentially with the GTP-bound form of Rsr1.

We were unable to detect any effects of Bem1 on the interaction of Cdc24 with either Rsr1 or Cdc42. It remains possible, however, that Bem1 might affect the GEF activity of Cdc24 in vivo, for example in response to phosphorylation events on Cdc24 or Bem1. Alternatively, the main role of Bem1 might be to bring Cdc24 into the vicinity of one or more of the (as yet unidentified) targets of Cdc42. Although the role of the interaction between Cdc24 and Bem1 remains unclear, the observation that Ca can inhibit the in vitro interaction between Cdc24 and Bem1 may help to explain why some mutant alleles of CDC24 cause cells to be sensitive to high concentrations of Ca (Ohya et al., 1986). Given that mutations in BEM1 display synthetic lethality with partial loss-of-function alleles of CDC24, it is possible that the Ca sensitivity of some cdc24 mutants is due to the inhibition by Ca of the interaction between Cdc24 and Bem1.

The model that Rsr1 and Bem1 both play roles in the spatial positioning of (or the assembly of complexes involving) Cdc24 could help to explain the observation that mutations in BUD5 display synthetic lethality with mutant alleles of BEM1 (Chant et al., 1991). The synthetic lethality between bud5 and bem1 might be due to the combined effects of these mutations on Cdc24 function. For example, either Rsr1 or Bem1 alone may be sufficient to allow for the appropriate localization of Cdc24. However, Cdc24 may not be efficiently localized when both Bem1 and Rsr1 are missing or are not functioning correctly (as in the case of a bem1 bud5 mutant).

Another situation in which improper regulation of Rsr1 is deleterious to cell growth is when not all of the G1 cyclins are expressed. During a screen for mutations that are lethal in the absence of the G1 cyclins Cln1 and Cln2, mutations in the BUD2 gene (which encodes the GAP for Rsr1) were identified (Benton et al., 1993; Cvrckova and Nasmyth, 1993). An activated allele of RSR1 was also shown to be lethal in the absence of Cln1 and Cln2 (Benton et al., 1993), suggesting that the GTP-bound form of Rsr1 can interfere with an essential Cln-dependent kinase-regulated event. Since cln1 cln2 bud2 mutants appear to be defective in bud emergence, it is likely that activated Rsr1 interferes with the phosphorylation by Cln1/Cln2-dependent kinase of some protein that is required specifically for bud emergence. Given our results that suggest that Rsr1-GTP binds to Cdc24, a model that could explain these genetic observations is that Cdc24 is directly regulated by a G1 cyclin-dependent kinase and that the binding of the GTP-bound form of Rsr1 blocks the access of the kinase to Cdc24.

Overall, the ability of Cdc24 to bind directly to Rsr1 and Cdc42 provides an interesting example of the convergence of Ras-type (Rsr1) and Rho-type (Cdc42) pathways. It is likely that similar types of convergence will be observed in other biological systems, and in particular in mammalian cells, as potentially mediated by the Ras-GEF or mSOS, which contains both Ras- and Rho-type exchange domains, and/or the p190 protein, which binds to the Ras-GAP but also serves as a GAP for Rho-subtype proteins. In the future, it will be important to delineate the binding domain for Rsr1 on Cdc24 (the Dbl domain serves as the functional site for Cdc42 interactions) as well as to determine how the interaction between Cdc24 and Rsr1 or Cdc24 and Bem1 might be regulated by Ca and/or by phosphorylation-dephosphorylation events as governed by cell-cycle progression.

FOOTNOTES

*

This work was supported by National Institutes of Health Grants GM47458 (to R. A. C.) and GM46271 (to A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

()

The abbreviations used are: GEF, guanine nucleotide exchange factor; GAP, GTPase-activating protein; GIP, GTPase-inhibitor protein; GST, glutathione-S-transferase; GTPS, guanosine 5`-3-O-(thio)triphosphate; kb, kilobase(s).

()

P. Polakis, personal communication.

ACKNOWLEDGEMENTS

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