(Received for publication, September 13, 1994; and in revised form, October 27, 1994)
From the
Bud formation in yeast involves the actions of the Ras-type
GTPase Rsr1, which is required for the proper selection of the bud
site, and the Rho-type GTPase Cdc42, which is necessary for the
assembly of cytoskeletal structures at that site. The Cdc24 protein is
required both for proper bud-site selection and bud-site assembly and
has been recently shown to display guanine-nucleotide-exchange factor
(GEF) activity toward Cdc42. Here, we demonstrate, using recombinant
proteins, that Cdc24 can also bind directly to Rsr1. This binding has
no effect on the ability of Rsr1 to undergo intrinsic or GEF-stimulated
GDP-GTP exchange. However, Cdc24 can inhibit both the intrinsic and
GTPase-activating protein-stimulated GTPase activity of Rsr1 and
thereby acts as a GTPase-inhibitor protein for Rsr1. Cdc24 thus appears
to bind preferentially to the activated form of Rsr1. The SH3
domain-containing bud-site assembly protein Bem1 also binds directly to
Cdc24, and we show here that this interaction is inhibited by
Ca. Neither Bem1 nor Cdc42 affects the
GTPase-inhibitor protein activity of Cdc24 toward Rsr1, and neither
Bem1 nor Rsr1 affects the GEF activity of Cdc24 toward Cdc42. Taken
together, these results suggest that Cdc24 enables the direct
convergence of a Ras-like protein (Rsr1) and a Rho-like protein (Cdc42)
with the SH3-domain-containing protein (Bem1) and that independent
domains of Cdc24 are responsible for these different interactions.
These results also suggest that rather than directly controlling the
GEF activity of Cdc24, the primary roles of Rsr1 and Bem1 might be to
control the positioning of Cdc24 within the cell.
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.
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-GTP
S (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-GTP
S.
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-GTP
S, 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.
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.