Molecular Biology of the Cell
Vol. 19, 2885–2896, July 2008
The Rho GDI Rdi1 Regulates Rho GTPases by Distinct
Christopher Tiedje, Imme Sakwa, Ursula Just, and Thomas Ho ¨fken
Institute of Biochemistry, Christian Albrecht University, 24098 Kiel, Germany
Submitted November 16, 2007; Revised March 31, 2008; Accepted April 9, 2008
Monitoring Editor: Daniel Lew
The small guanosine triphosphate (GTP)-binding proteins of the Rho family are implicated in various cell functions,
including establishment and maintenance of cell polarity. Activity of Rho guanosine triphosphatases (GTPases) is not
only regulated by guanine nucleotide exchange factors and GTPase-activating proteins but also by guanine nucleotide
dissociation inhibitors (GDIs). These proteins have the ability to extract Rho proteins from membranes and keep them in
an inactive cytosolic complex. Here, we show that Rdi1, the sole Rho GDI of the yeast Saccharomyces cerevisiae,
contributes to pseudohyphal growth and mitotic exit. Rdi1 interacts only with Cdc42, Rho1, and Rho4, and it regulates
these Rho GTPases by distinct mechanisms. Binding between Rdi1 and Cdc42 as well as Rho1 is modulated by the Cdc42
effector and p21-activated kinase Cla4. After membrane extraction mediated by Rdi1, Rho4 is degraded by a novel
mechanism, which includes the glycogen synthase kinase 3? homologue Ygk3, vacuolar proteases, and the proteasome.
Together, these results indicate that Rdi1 uses distinct modes of regulation for different Rho GTPases.
Small guanosine triphosphatases (GTPases) of the Rho fam-
ily control fundamental processes of cell biology common to
all eukaryotes, such as morphogenesis, polarity, movement,
and cell division (Jaffe and Hall, 2005). In the budding yeast
Saccharomyces cerevisiae, which encodes six Rho GTPases
(Cdc42 and Rho1 to Rho5), these proteins play a pivotal role
in the establishment of cell polarity (Park and Bi, 2007).
Budding yeast cells undergo polarized growth during vari-
ous phases of their life cycle, including budding during
vegetative growth, mating between haploid cells of opposite
mating types, and filamentous growth upon nutrient limi-
tation. Although Cdc42 and Rho1 are well characterized,
little is known about the other Rho proteins. Membrane
association of Rho-type GTPases is essential for their func-
tion, and it depends on a C-terminal prenyl moiety and an
adjacent polybasic region. Cdc42 localizes to internal mem-
branes and the entire plasma membrane, where it clusters at
sites of polarized growth, including the tips of mating pro-
jections, incipient bud sites, the tips and sides of growing
buds, and the bud neck region of large-budded cells (Ziman
et al., 1993; Richman et al., 2002). In addition to membrane-
bound Cdc42, a cytoplasmic pool has been found (Ziman
et al., 1993). Similar to Cdc42, Rho1 has been shown to
localize at sites of polarized growth (Yamochi et al., 1994).
Like other regulatory GTPases, they act as molecular
switches, cycling between an active guanosine triphosphate
(GTP)-bound state and an inactive guanosine diphosphate
(GDP)-bound state. This activity is highly regulated by gua-
nine nucleotide exchange factors (GEFs) and GTPase-acti-
vating proteins (GAPs). The exchange of GDP to GTP, and
thus the activation of Cdc42, is catalyzed by GEFs. In the
active GTP-bound state, Rho GTPases perform their regula-
tory function through a conformation-specific interaction
with effector proteins. GAPs stimulate the intrinsic GTPase
activity, leading to inactivation. However, the role of GAPs
might be more complex. For Cdc42, it has been suggested
that GTP hydrolysis, and thus a cycling between GDP and
GTP states, is necessary for the establishment of polarity,
septin assembly, and mating (Gladfelter et al., 2002; Caviston
et al., 2003; Irazoqui et al., 2003; Barale et al., 2006). Apart
from GEFs and GAPs, Rho GTPases are also regulated by
guanine nucleotide dissociation inhibitors (GDIs) (DerMard-
irossian and Bokoch, 2005; Dovas and Couchman, 2005;
Dransart et al., 2005). Three distinct biochemical activities
have been attributed to Rho GDIs. First, they inhibit the
dissociation of GDP from Rho GTPases. Second, they block
intrinsic and GAP-stimulated GTPase activity. Third, Rho
GDI extract Rho proteins from membranes and form high-
affinity cytosolic complexes, in which the C-terminal prenyl
group of the Rho protein inserts into the hydrophobic pocket
formed by the immunoglobulin-like ? sandwich of the GDI.
The dissociation of the GTPase–GDI complex is regulated by
various mechanisms. Several biologically relevant mem-
brane lipids have been reported to decrease the affinity of
Rho GDIs for GTPases (Chuang et al., 1993). Complex for-
mation and membrane extraction also depend on the phos-
phorylation status of the GDI, the GTPase, or both (Bourm-
eyster and Vignais, 1996; Mehta et al., 2001; Forget et al.,
2002; DerMardirossian et al., 2004). Furthermore, in higher
eukaryotes some membrane-associated proteins, such as
members of the ezrin/radixin/moesin family and the neu-
rotrophin receptor p75NTR, have the ability to disrupt the
GTPase–GDI complex (Takahashi et al., 1997; Yamashita and
This article was published online ahead of print in MBC in Press
on April 16, 2008.
Address correspondence to: Thomas Ho ¨fken (email@example.com-
Abbreviations used: GAP, GTPase-activating protein; GDI, guanine
nucleotide dissociation inhibitor; GEF, guanine nucleotide exchange
factor; PAK, p21-activated kinase; GSK-3?, glycogen synthase ki-
© 2008 by The American Society for Cell Biology 2885
In S. cerevisiae, the role of the sole Rho GDI Rdi1 is unclear.
Conflicting results have been reported for the overexpres-
sion of RDI1. Masuda et al. (1994) show that high levels of
RDI1 result in lethality, although the phenotype of these
cells has not been characterized (Masuda et al., 1994). In
contrast, others claim that RDI1 overexpression causes only
a slightly rounder cell morphology (Tcheperegine et al.,
2005). Deletion of RDI1 does not produce any detectable
phenotypes in budding yeast (Masuda et al., 1994), but in the
human fungal pathogen Candida albicans, cells lacking RDI1
exhibit reduced polarized growth (Court and Sudbery,
Rdi1 is found in the cytoplasm, but it also localizes to the
tips of small buds and the bud neck region, where it interacts
with Cdc42 (Koch et al., 1997; Richman et al., 2004; Cole et al.,
2007). Rdi1 has the ability to extract Cdc42 and Rho1 from
vacuolar membranes (Eitzen et al., 2001), and it can also
extract Cdc42 from the plasma membrane (Richman et al.,
2004; Tcheperegine et al., 2005). However, it has not been
tested whether Rdi1 interacts with other Rho GTPases.
Here, we show that Rdi1 interacts only with a subset of
Rho GTPases. Whereas Cdc42, Rho1 and Rho4 form a com-
plex with Rdi1 and can be extracted from membranes, Rdi1
does not bind to Rho2, Rho3, and Rho5. Furthermore, we
demonstrate that the p21-activated kinase (PAK) Cla4 dis-
rupts Rdi1–Rho1 and Rdi1–Cdc42 complexes. Finally, we
show that Rdi1 and the glycogen synthase kinase 3? (GSK-
3?) homologue Ygk3 promote Rho4 degradation by vacuo-
lar proteases and the proteasome.
MATERIALS AND METHODS
Growth Conditions, Yeast Strains, and Plasmids
All yeast strains are listed in Supplemental Table 1. The strains used in this
study were in the YPH499 background, with the exception of strains used for
filamentous growth. For these experiments strains in the ?1278b background
were used (CTY64, PC344, PPY966, THY697, THY705, and THY706). Yeast
strains were grown in yeast extract, peptone, dextrose (YPD) or synthetic
complete (SC) medium. For induction of the GAL1 promoter, yeast cells were
grown in yeast extract, peptone (YP) or SC medium with 3% raffinose instead
of glucose. Galactose (final concentration 2%) was added to induce the GAL1
promoter. SLAD medium contains 50 ?M ammonium sulfate, 2% glucose, 2%
Bacto-agar, and 0.67% YNB. Yeast strains were constructed using polymerase
chain reaction (PCR)-amplified cassettes (Longtine et al., 1998; Knop et al.,
1999; Janke et al., 2004).
All constructs used in this work are listed in Supplemental Table 2. For
N-terminal tagging of Rho proteins, these genes were first amplified by PCR
from genomic DNA and cloned into pRS315. A NotI site was introduced 3? of
the start codon by site directed mutagenesis. The plasmid GTEPI, which
contains a DNA fragment encoding three adjacent copies of the hemaggluti-
nin (HA) epitope cloned into the NotI site in the polylinker of pBluescript II,
was cut with NotI, and the liberated fragment was ligated in frame into the
NotI site of the pRS315 plasmids carrying the RHO genes. 3HA-tagged RHO
genes were transferred into pRS305. These genes were inserted at the genomic
LEU2 by transformation with EcoRV or KasI digested pRS305 carrying the
3HA-tagged RHO genes.
Immunoprecipitations and Membrane Extraction Assays
For immunoprecipitations and precipitations with glutathione-Sepharose (GE
Healthcare, Chalfont St. Giles, United Kingdom) cells were disrupted with
glass beads in lysis buffer (20 mM Tris, pH 7.5, 100 mM NaCl, 10 mM EDTA,
1 mM EGTA, 5% glycerol, 1% Triton X-100, and protease inhibitors) and
clarified by centrifugation at 13,000 rpm for 5 min. Protein concentration was
determined using Bradford protein assay solution (Roth, Karlsruhe, Ger-
many). Epitope-tagged proteins were immunoprecipitated by adding the
respective antibody and protein G-Sepharose (GE Healthcare). Resin was
washed three times with lysis buffer, resuspended in 2? SDS sample buffer,
and analyzed by immunoblotting. For membrane extraction assays, cells were
treated as for immunoprecipitations but without Triton X-100. Cleared lysates
were centrifuged for 1 h at 100,000 ? g. For dephosphorylation of 3HA-Rho4,
the protein was precipitated with anti-HA antibodies, beads were washed
three times with phosphatase buffer (20 mM Tris, pH 7.4, 10 mM NaCl, and
5% glycerol). Beads were then split into three and either no, 100 U of
?-phosphatase (New England Biolabs, Ispwich, MA), or 100 U of ?-phospha-
tase, which was heat-inactivated by treating it 30 min at 95°C, was added.
Samples were incubated 30 min at 30°C, and proteins were eluted with SDS
Monoclonal mouse anti-HA (12CA5) was obtained from Roche Diagnostics
(Mannheim, Germany), and goat anti-glutathione transferase (GST) was from
GE Healthcare. Mouse monoclonal anti-Myc (9E10), anti-Cdc42, anti-Cdc11,
and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were ob-
tained from Santa Cruz Biotechnology (Santa Cruz, CA). Secondary antibod-
ies were from Jackson ImmnuoResearch Laboratories (West Grove, PA).
F-actin of yeast cells was stained with rhodamine-phalloidin (Invitrogen,
Carlsbad, CA). One milliliter of cells was harvested and resuspended in
phosphate-buffered saline (PBS), 0.1% Triton X-100, and 3.7% formaldehyde.
After 30-min incubation at room temperature, cells were washed once with
PBS and fixed for 2 h at 30°C with PBS, 3.7% formaldehyde. Cells were
washed two times with PBS and resuspended in 90 ?l of PBS. Three units of
rhodamine-phalloidin was added, cells were incubated for 2 h in the dark,
and then they were washed three times with PBS. Cells were examined with
an Axiovert 200M fluorescence microscope (Carl Zeiss, Jena, Germany)
equipped with a 100? Plan oil-immersion objective, and images were cap-
tured using an AxioCam MRm charge-coupled device camera (Carl Zeiss).
Filamentous Growth Assays
For agar invasion assays, 105cells of an overnight culture were spotted on
YPD and grown for 2 d at 30°C. Plates were photographed before and after
being rinsed under a gentle stream of deionized water. For pseudohyphal
growth assays, cells were grown overnight, and 100 cells were spread on solid
SLAD medium. Plates were incubated for 4 d at 30°C, and images were taken
using a 10? objective.
Rdi1 Has a Role in Pseudohyphal Growth and Mitotic
To assess the function of Rdi1 in S. cerevisiae, we analyzed
the phenotype of cells deleted for and overexpressing RDI1,
respectively. The RDI1 deletion strain was indistinguishable
from wild-type cells in terms of growth, cell morphology,
and mating (data not shown). It has been reported that in
diploid C. albicans loss of the homologous gene RDI1 greatly
reduces filamentous growth (Court and Sudbery, 2007). In
addition, RDI1 deletion abrogates the enhanced filamenta-
tion of cells deleted for the Cdc42 GAPs BEM3 and RGA2
(Court and Sudbery, 2007). Therefore, we tested whether
similar phenotypes can be observed for S. cerevisiae, where
filamentous growth occurs in both, haploid (invasive
growth) and diploid (pseudohyphal growth) cells (Palecek
et al., 2002; Park and Bi, 2007). Haploid cells penetrate agar
in response to nutritional limitation. To analyze the role of
RDI1 in haploid invasive growth, we deleted this gene in
cells of the ?1278b background, which is necessary for tests
of filamentous growth. The haploid ?rdi1 strain invaded
agar like wild-type cells (Figure 1A). On nitrogen starvation,
diploid cells assume an elongated morphology and generate
chains of filamentous-form cells projecting from the main
colony of yeast-form cells (Figure 1B). The homozygous
?rdi1/?rdi1 diploid strain exhibited a defect in pseudohy-
phal differentiation when grown on low nitrogen medium
(Figure 1B). Notably, overexpression of CDC42 from a mul-
ticopy plasmid did not rescue this defect (Figure 1B). Thus,
it seems unlikely that Rdi1 regulates Cdc42 activity like the
GEF Cdc24. It is conceivable that the phenotype of the
?rdi1/?rdi1 strain reflects elevated, deregulated Cdc42 activ-
ity. Possibly Cdc42 removal from membranes by Rdi1 is
necessary to restrict active Cdc42 to the growing tip. In line
with this model is our observation that the constitutively
active CDC42G12Vmutant (Ziman et al., 1991) displayed a
severe pseudohyphal growth defect (Figure 1C).
Cdc42 and its effectors Ste20, Cla4, and Gic1 promote exit
from mitosis via at least three independent mechanisms
(Ho ¨fken and Schiebel, 2002; Jensen et al., 2002; Seshan et al.,
C. Tiedje et al.
Molecular Biology of the Cell2886
2002; Ho ¨fken and Schiebel, 2004; Bosl and Li 2005) (Figure
2A). The small GTPase Tem1 and its putative GEF Lte1 are
the most upstream components of the mitotic exit network,
the signaling cascade that controls exit from mitosis (Bosl
and Li, 2005). Cells in which LTE1 is deleted grow normally
at 30°C, but arrest in anaphase with large buds, two sepa-
rated 4,6-diamidino-2-phenylindole staining regions, and an
elongated mitotic spindle when grown at low temperatures
(Bosl and Li, 2005). We used this mitotic exit defect of ?lte1
cells to assay the activity of Cdc42 regulators. Previously, it
has been demonstrated that the deletion of LTE1 in a tem-
perature-sensitive mutant of CDC24, the sole GEF of Cdc42,
is lethal at all temperatures (Ho ¨fken and Schiebel, 2002).
Here, we show that the individual deletion of the Cdc42
GAPs BEM3, RGA1, and RGA2 suppressed the cold-sensi-
tive growth defect of ?lte1 (Figure 2B). Interestingly, the
double mutant ?lte1?rdi1 also grew like the wild-type strain
at 10°C (Figure 2B). These results are consistent with a role
for Cdc24 as a positive regulator and for Rdi1, Bem3, Rga1,
and Rga2 as negative regulators of Cdc42. We then asked
whether deletion of RDI1 and the GAPs rescued the mitotic
exit defect of the ?lte1 strain. Cells were arrested in G1 with
?-factor and released at 10°C. In addition to the percentage
of large-budded cells, the release of Cdc14-green fluorescent
protein (GFP) from the nucleolus was monitored through
the cell cycle. Cdc14 is released from the nucleolus during
anaphase and thus serves as a reliable marker for mitotic exit
(Bosl and Li, 2005). At 10°C, wild-type cells released Cdc14
and underwent cytokinesis at ?6 h (Figure 2C). In contrast,
most ?lte1 cells arrested in anaphase with Cdc14-GFP
trapped in the nucleolus (Figure 2C). ?lte1 cells in which
either RDI1 or the Cdc42 GAPs were deleted did not arrest
in anaphase and released Cdc14 from the nucleolus with the
same kinetics as wild-type cells (Figure 2C).
RDI1 overexpression has been reported to either result in
lethality (Masuda et al., 1994) or produce slightly rounder
cells (Tcheperegine et al., 2005). Therefore, we tested the
consequences of higher RDI1 levels in our background. RDI1
under control of its own promoter on a multicopy plasmid
had no effect on cell growth and morphology (Supplemental
Figure S1A; data not shown). The same result was obtained
for a strain in which a single copy of genomic RDI1 was
under control of the strong GAL1 promoter (Supplemental
Figure S1B). In contrast, RDI1 under control of the GAL1
promoter expressed from a 2 ?m-based multicopy plasmid
(Masuda et al., 1994) resulted in lethality (Supplemental
Figure S1C). To examine the cause of this lethality, we
monitored cell morphology over time after RDI1 overex-
pression. Four hours after induction of GAL1-RDI1 from a
multicopy plasmid, ?90% of cells arrested as slightly bigger
round, unbudded cells (Supplemental Figure S1, D and E).
The actin cytoskeleton was polarized in only 6% of these
round cells, whereas 70% of unbudded cells with normal
Rdi1 levels had an polarized actin cytoskeleton (Supplemen-
tal Figure S1E).
Rdi1 Specifically Interacts with Cdc42, Rho1, and Rho4
The most likely reason for the failure of cells to polarize after
strong RDI1 overexpression is the removal of Rho GTPases
from sites of polarized growth by Rdi1. It has been shown
that Rdi1 is able to extract Cdc42 and Rho1 from membranes
(Koch et al., 1997; Eitzen et al., 2001; Richman et al., 2004;
Tcheperegine et al., 2005), but it has not been tested whether
Rdi1 interacts with other Rho GTPases as well. Therefore, we
performed immunoprecipitation experiments with strains in
which Rho GTPases were N-terminally tagged with the HA
and Rdi1 C-terminally with the myc epitope. Coimmuno-
precipitation of Rho1 and Rho4, but not of Rho2, Rho3, and
Rho5 with Rdi1 was observed (Figure 3A). The multiple
bands observed for 3HA-Rho4 are due to phosphorylation
of this protein (see below). In a similar immunoprecipitation
experiment using antibodies against Cdc42, it was demon-
strated that Cdc42 binds to Rdi1 (Figure 3B). Because all
proteins were expressed from their own promoters for
this experiment, Rho protein levels might not be suffi-
ciently high for detection of a Rdi1–Rho complex. To
circumvent this problem, RHO genes were fused with
GST and placed under the control of the strong GAL1
promoter. Although all Rho GTPases were expressed at
high levels upon induction, again only Cdc42, Rho1 and
Rho4, but not the other Rho proteins, formed a complex
with Rdi1 (Supplemental Figure S2).
To examine the amount of membrane-associated Rho GT-
Pases, cell lysates were submitted to cell fractionation by
high-speed centrifugation. Interestingly, the ratio between
protein in soluble supernatant and the membrane pellet was
quite different for the various Rho GTPases (Supplemental
Figure S3). To characterize which Rho proteins could be
extracted from membranes by Rdi1, lysates of cells with and
without RDI1 overexpression, respectively, were fraction-
not affect haploid invasive growth. Cells (105) of the indicated
strains were spotted on a YPD plate and incubated for 2 d at
30°C. Pictures were taken before and after gentle rinsing with
water. ?ste20 cells, which fail to invade the agar, served as control.
(B) ?rdi1/?rdi1 cells exhibit reduced diploid pseudohyphal growth.
The indicated strains were grown on low nitrogen SLAD medium
for 4 d at 30°C. ?ste20/?ste20 cells, which are defective in filamen-
tous growth, were used as control. (C) Constitutively active Cdc42
impairs pseudohyphal growth. 3HA-CDC42 and 3HA-CDC42G12V
under control of the CDC42 promoter were integrated at the URA3
locus by using an integrative plasmid. The indicated strains were
treated as described in B. The results shown in this figure are
representative of two independent experiments.
Rdi1 and filamentous growth. (A) RDI1 deletion does
Regulation of Rho GTPases by Rdi1
Vol. 19, July 2008 2887
ated and analyzed by immunoblotting. After overexpression
of RDI1, the amount of cytosolic protein increased for Cdc42
and Rho1 (Figure 4A). Consistently, less Rho1 and Cdc42
protein was found in the membrane fraction of cells over-
expressing RDI1 (Figure 4A). However, because Rho pro-
teins predominantly associate with membranes, these
changes can be most easily observed in the cytosolic fraction.
Higher levels of RDI1 had no effect on the distribution of
Rho2, Rho3, and Rho5 (Figure 4A). Interestingly, the total
amount of Rho4 dropped following RDI1 overexpression
(Figure 5A). Because Rho4 protein level were only moder-
ately reduced in cells lacking PEP4 after RDI1 overexpres-
sion (see below), the membrane extraction assay for Rho4
was performed in the ?pep4 background. Higher RDI1 levels
caused an increase of Rho4 in the supernatant and a reduc-
tion of membrane-associated Rho4 (Figure 4B). The ability of
Rdi1 to extract Rho4 from membranes also was examined by
fluorescence microscopy. GFP-Rho4 under control of its own
promoter could be detected by immunoblotting, but it was
too weak to be visualized by fluorescence microscopy.
Therefore, we expressed GFP-RHO4 from the inducible
GAL1 promoter. GFP-Rho4 was associated with internal
membranes and the entire plasma membrane (Figure 6A).
The protein clustered at the presumptive bud site and the
bud neck of large buds. Unlike most other proteins that
have a role in polarity, Rho4 was not or only weakly
enriched at the tip of small buds and absent from mating
projections (Figure 6, A and B). As expected, RDI1 over-
expression resulted in the loss of membrane-bound Rho4
(Figure 6, C and D).
We next asked whether Rdi1 preferentially extracts the
GDP- or GTP-bound form of Cdc42, Rho1, and Rho4 from
membranes. The mutations CDC42G12Vand CDC42D118Alock
Cdc42 in the GTP- and GDP-bound state, respectively (Zi-
man et al., 1991). Therefore, we compared the intracellular
distribution of these mutated proteins and also of the para-
digmatic mutant proteins Rho1G19V, Rho1D125A, Rho4G81V,
and Rho4D197Aafter RDI1 overexpression. Unexpectedly,
Rdi1 extracted the GTP-bound Cdc42G12Vand Rho1G19Vlike
wild-type proteins from membranes, whereas the cytosolic
levels of GDP-bound Cdc42D118Aand Rho1D125Adid not
increase in cells overexpressing RDI1 (Figure 4, A and C). In
contrast, wild type Rho4, Rho4G81V, and Rho4D197Aseemed
to be equally well extracted from membranes by Rdi1 (Fig-
ure 4, B and C). However, the effect of Rdi1 on Rho4 was less
pronounced; therefore, it was more difficult to assess.
Because Rdi1 extracts Rho1 and Cdc42 from membranes,
we examined whether these Rho proteins were present in
the cytosol of cells deleted for RDI1. Unexpectedly, the
cytoplasmic pool of Rho1 and Cdc42 was the same in wild
type and ?rdi1 cells (Figure 4D).
In summary, our data indicate that Rdi1 only interacts
with Cdc42, Rho1, and Rho4, but not with other Rho
mitotic exit defect of ?lte1 cells. (A) The role of
Cdc42 in mitotic exit. Cdc42 is activated by its
GEF Cdc24, whereas the GAPs Bem3, Rga1,
and Rga2 stimulate GTP hydrolysis. Activated
Cdc42 triggers mitotic exit by three indepen-
dent mechanisms. Gic1 disrupts the interac-
tion between the small GTPase Tem1 and its
two-component GAP Bub2-Bfa1, which re-
sults in the activation of Tem1. Cla4 stimulates
Lte1, the putative GEF of Tem1, which causes
Tem1 activation. Ste20 promotes mitotic exit
independently of Gic1 and Cla4 by an un-
known mechanism. (B) Suppression of the
?lte1 growth defect by deletion of either RDI1
or Cdc42 GAPs. Serial dilutions (1:10) of the
indicated strains grown on YPD at 10 and
30°C, respectively. (C) Deletion of either RDI1
or Cdc42 GAPs suppresses the mitotic exit
defect of ?lte1 cells. The indicated strains with
CDC14-GFP were arrested in G1 with ?-factor.
Cells progressed synchronously through the
cell cycle at 10°C upon removal of ?-factor by
washing with YPD medium. The number of
cells with large buds and nucleolar Cdc14-
GFP (n ? 100 at each time point) was deter-
mined over time. Because RDI1 and Cdc42
GAP single deletion strains grew like wild-
type cells at 10°C (Figure 1B), these strains
were not included for this analysis. The results
shown in this figure are representative of two
Deletion of RDI1 suppresses the
C. Tiedje et al.
Molecular Biology of the Cell 2888
PAK-Family Kinase Cla4 Regulates Association of Rdi1
with Cdc42 and Rho1
The association of Rho GTPases with Rho GDIs can be
modulated by phosphorylation of these proteins (Bourmey-
ster and Vignais, 1996; Mehta et al., 2001; Forget et al., 2002;
DerMardirossian et al., 2004). Because the Cdc42 effectors
Cla4 and Ste20 play a key role in cell polarity, we asked
whether elevated levels of these kinases would reduce the
efficiency of the coimmunoprecipitation of Cdc42 and Rdi1.
Anti-myc epitope antibodies not only efficiently precipitated
Rdi1–9myc but also Cdc42 (Figures 3B and 7A). Overexpres-
sion of CLA4 abolished binding between Rdi1 and Cdc42
(Figure 7A). Importantly, higher expression of either STE20
or SKM1, which are both Cdc42 effectors and PAK-family
kinases as well, had no effect on the Cdc42–Rdi1 interaction
(Figure 7, A and B). Thus, Cla4, but not related proteins, can
specifically disrupt binding between Cdc42 and Rdi1. We
also tested, whether CLA4 overexpresssion has the same
effect on the Rho1–Rdi1 complex. However, although the
coimmunoprecipitation of Rho1 and Rdi1 was highly repro-
ducible, we came to varying results for the Rho1–Rdi1 com-
plex after CLA4 overexpression (data not shown). Therefore,
using coimmunoprecipitations, it cannot conclusively be
demonstrated whether Cla4 has an effect on the Rho1–Rdi1
complex. To circumvent this problem, we tested whether
high levels of CLA4 change the intracellular distribution of
Rho1 and Cdc42. Overexpression of CLA4 decreased the
amount of cytosolic Rho1 and Cdc42 (Figure 7C). Impor-
tantly, this effect was not observed in the absence of RDI1
(Figure 7C). These results suggest that Cla4 disrupts the
binding between Rdi1 and Cdc42 as well as between Rdi1
and Rho1. Consistently, in ?cla4 cells a slightly higher por-
tion of Rho1 was found in the cytosol compared with the
wild-type strain, whereas the cytosolic amount of Rho1 in
the ?cla4 ?rdi1 double mutant was similar to the wild-type
and ?rdi1 (Figure 4D). For Cdc42, there was only a weak or
no effect for Cdc42 in the CLA4 deletion strain (Figure 4D).
Finally, using the membrane extraction assay it was tested
whether Cla4 antagonizes Rdi1. Whereas higher Rdi1 levels
increased the cytosolic pool of Cdc42 and Rho1 (Figures 4A
and 7D), simultaneous overexpression of CLA4 and RDI1
reversed this effect (Figure 7D). Under these conditions,
cytosolic amounts of Cdc42 and Rho1 are comparable with
the wild-type situation. Next, we tested whether the kinase
activity of Cla4 is required for its effect on Rdi1 complexes.
To this end, we examined the localization of 3HA-Cdc42
after overexpression of wild-type CLA4 and the kinase-dead
CLA4K594A. As described above, overexpression of wild-type
CLA4 results in a reduced cytosolic pool of 3HA-Cdc42
compared with the wild-type strain (Figure 7, C and E). In
contrast, this effect was much weaker in cells overexpressing
the kinase-dead CLA4 (Figure 7E).
Interestingly, Cla4 did not affect the amount of Rho4 in the
supernatant and pellet after high-speed centrifugation (Sup-
plemental Figure S4, A and B). Thus, the regulatory role of
Cla4 is specific for Cdc42 and Rho1.
Rdi1 and the GSK-3? Homologue Ygk3 Promote
Degradation of Rho4
As mentioned above, RDI1 overexpression reduces the total
amount of Rho4 protein (Figure 5A). Conversely, higher
protein levels of Rho4 were observed in ?rdi1 cells com-
pared with the wild-type strain (Figure 5B). This could be
achieved by two different mechanisms. Either Rdi1 targets
Rho4 for degradation or, alternatively, Rdi1 regulates Rho4
protein levels through gene expression. Because Rho4 levels
dropped markedly after only 2 h of RDI1 overexpression, a
regulation at the transcriptional or translational level would
only have an effect if Rho4 were a short-lived protein. To test
this, we blocked protein synthesis with cycloheximide. Even
after 2-h incubation with cycloheximide, Rho4 protein levels
remained unchanged (Figure 5C), suggesting that Rho4 is a
relatively stable protein. Thus, Rdi1 seems to regulate Rho4
levels through proteolysis. Because usually multiple bands
were observed for 3HA-Rho4 in immunoblots, it was exam-
ined whether these are phospho-forms of 3HA-Rho4. In fact,
slower migrating bands disappeared following phosphatase
treatment (Figure 5D).
In a proteomic approach, phosphorylation of Rho4 by
Ygk3, a homologue of GSK-3? kinase, has been demon-
strated previously (Ptacek et al., 2005). Furthermore, Ygk3
has a role in ubiquitin-dependent proteolysis (Andoh et al.,
2000). Therefore, we tested whether Ygk3 is involved in the
degradation of Rho4. In fact, YGK3 overexpression resulted
other Rho proteins. (A) Rdi1 coimmunoprecipitates with Rho1 and
Rho4. Cells expressing RDI1–3myc, and RDI1–3myc with 3HA-
tagged RHO1, RHO2, RHO3, RHO4, and RHO5 were lysed, and
equal amounts of protein extract were precipitated with anti-HA
antibodies. Immunoprecipitates were analyzed by immunoblotting
with antibodies against the myc and HA epitopes. 3HA-Rho1 and
3HA-Rho2 run at the same height as the light chain. However, a
stronger signal indicates that these proteins were precipitated, com-
pared with the heavy chain, which occurs with the same intensity in
all samples. (B) Coimmunoprecipitation of Rdi1 with Cdc42. Cells
expressing RDI1–9myc and wild-type cells were lysed, and equal
amounts of protein were precipitated with anti-myc antibodies.
Precipitates were examined with antibodies raised against Cdc42
and the myc epitope, respectively. The results shown in this figure
are representative of two independent experiments.
Rdi1 interacts with Cdc42, Rho1, and Rho4, but not with
Regulation of Rho GTPases by Rdi1
Vol. 19, July 2008 2889
in reduced Rho4 levels (Figure 8A), indicating that this
protein, like Rdi1, promotes Rho4 proteolysis. Importantly,
in the absence of RDI1 Ygk3 fails to degrade Rho4 (Figure
8A), suggesting that Rdi1 and Ygk3 act in the same pathway.
In contrast YGK3 overexpression had no effect on protein
levels of Cdc42 and Rho1 (Figure 8B), which are also ex-
tracted from membranes by Rdi1. Because the ubiquitin
ligase Rsp5 is involved in GSK-3?–mediated proteolysis
(Andoh et al., 2000), we examined the role of Rsp5 in Rho4
degradation using a temperature-sensitive mutant. At both,
the permissive (23°C) and restrictive (37°) temperature,
Rho4 degradation after RDI1 overexpression in rsp5-1 cells
was indistinguishable from the wild type (Supplemental
Figure S5). Thus, the ubiquitin ligase Rsp5 is not required for
Rho4 degradation. Eukaryotic cells contain two distinct sys-
tems for protein degradation: the vacuolar system and the
proteasome. To find out whether Rho4 is degraded in a
proteasome-dependent manner, Rho4 protein levels after
RDI1 overexpression were examined in the presence and
absence of the proteasome-specific inhibitor MG132 (Lee
and Goldberg, 1996). Treatment with MG132 stabilized Rho4
(Figure 8C), indicating an involvement of the proteasome in
Rho4 degradation. We also examined whether the vacuolar
system plays a role in Rho4 degradation. To this end, RDI1
was overexpressed in ?pep4 cells, which lack most vacuolar
proteases (Ammerer et al., 1986). Unexpectedly, Rho4 was
not degraded in the absence of vacuolar proteases (Figure
8D), suggesting that Rho4 proteolysis depends on the vac-
uolar system as well as the proteasome.
Next, we examined whether Rho4 was degraded in a cell
cycle-dependent manner. However, no major changes were
observed at different stages of the cell cycle (Figure 9A). It is
conceivable, that Rho4 degradation is necessary to restrict
the protein to a certain membrane domain. Nevertheless,
GFP-Rho4 is enriched at the presumptive bud site in wild-
type cells and in the absence of RDI1 (Figure 6E). Therefore,
it seems unlikely, that the polarized localization of Rho4 is
achieved by its proteolysis. It is also possible, that only
active Rho4 is extracted from the membrane and subse-
quently degraded to terminate its function. To test whether
3HA-tagged Rho proteins and carrying either GAL1-RDI1 on a plasmid or the empty vector were grown in selective medium with 3%
raffinose. RDI1 was overexpressed for 3 h by addition of galactose. Cells were lysed and equal amounts of protein extract were separated by
centrifugation at 100,000 ? g to obtain the soluble supernatant and membrane pellet fractions. Rho proteins in different fractions were
detected by Western blotting with antibodies against the HA epitope. (B) RDI1 extracts Rho4 from membranes. The experiment was
performed as described in A but in a ?pep4 background to counteract Rho4 degradation caused by RDI1 overexpression (C), Rdi1
preferentially extracts GTP-bound forms of Rho GTPases from membranes. The experiment was carried out as described in A. For Rho4
proteins the assay was performed in cells lacking PEP4. (D) Effect of RDI1 and CLA4 deletion on cytosolic levels of Cdc42 and Rho1. Cells
of the indicated strains were lysed and protein extracts were fractionated by centrifugation at 100,000 ? g. Cdc42 and Rho1 were detected
by immunoblotting using anti-HA antibodies. The results shown in this figure are representative of two independent experiments.
Rdi1 specifically extracts Rho1 and Cdc42 from membranes. (A) Rdi1 extracts Cdc42 and Rho1 from membranes. Cells expressing
C. Tiedje et al.
Molecular Biology of the Cell2890
hyperactive Rho4 causes any damage to cells, we overex-
pressed RHO4G81V. As described above, the paradigmatic
rho4G81Vmutant is locked in the active GTP-bound state due
to a defect in GTP hydrolysis. This constitutively active form
of Rho4 reduces cell growth markedly (Figure 9B). Unfortu-
nately, no morphological or other changes were observed in
these cells (data not shown). Together, our data show that
Rdi1 and Ygk3 promote Rho4 degradation, a process which
depends on both, vacuolar proteases and the proteasome.
Further, the degradation of Rho4 may be important to ter-
minate its activity.
In this work, we show that the regulation of Rho proteins by
the sole budding yeast Rho GDI Rdi1 is rather complex. Rdi1
interacts only with Cdc42, Rho1, and Rho4, but not with the
other Rho proteins (summarized in Figure 10). We demon-
strate that Rdi1 extracts Cdc42 and Rho1 from membranes
and forms a soluble complex with these proteins, a process
that is regulated by the PAK-family kinase Cla4. In contrast,
membrane extraction of Rho4 by Rdi1 results in the degra-
dation of this Rho protein. We found that this proteolytic
pathway includes the proteasome, vacuolar proteases, and
the GSK-3? homologue Ygk3.
Functions of Rdi1
Very few phenotypes for the overexpression or deletion of
RDI1 are known. Conflicting results have been reported for
RDI1 overexpression. Tcheperegine et al. (2005) show that
high levels of RDI1 cause only slightly rounder cell mor-
phology, whereas others claim that RDI1 overexpression
results in lethality (Masuda et al., 1994). In our hands, effects
of RDI1 overexpression were clearly dose dependent. Only
RDI1 expressed from the GAL1 promoter of a 2-?m plasmid
was lethal. The actin cytoskeleton was depolarized in these
cells and consequently buds were no longer present. This
lack of polarization can be explained by the extraction of the
essential Rho proteins Cdc42 and Rho1 from the plasma
membrane by Rdi1.
To date, no phenotype has been described for ?rdi1 cells.
Here, we show that deletion of RDI1 suppresses the mitotic
exit defect of ?lte1 cells at low temperatures, possibly due to
sion results in lower Rho4 protein levels. 3HA-RHO4 cells carrying
either GAL1-RDI1 on a vector or the empty plasmid were grown in
YP medium with 3% raffinose. RDI1 was overexpressed for 2 h by
addition of galactose. Subsequently, cells were lysed, and equal
amounts of protein extract were examined by immunoblotting us-
ing antibodies against the HA epitope and Cdc11 (loading control).
(B) ?rdi1 cells have higher Rho4 protein levels. Cells expressing
3HA-RHO4 in a wild-type and ?rdi1 background were analyzed by
immunoblotting with the indicated antibodies. (C) Rho4 is a rela-
tively stable protein. Exponentially growing 3HA-Rho4 cells were
incubated with 50 ?g/ml cycloheximide. At the indicated times,
samples were taken and analyzed by immunoblotting. (D) Rho4 is
a phosphoprotein. 3HA-tagged Rho4 was immunoprecipitated with
anti-HA antibodies and treated with buffer, active, or heat-inacti-
vated ?-phosphatase. Samples were analyzed by immunoblotting.
The results shown in this figure are representative of two indepen-
Rdi1 targets Rho4 for degradation. (A) RDI1 overexpres-
calization during budding. GAL1-GFP-RHO4
of exponentially growing cells was induced
for 120 min by the addition of galactose. Cells
were fixed with formaldehyde and analyzed
by fluorescence microscopy. (B) Rho4 is not
enriched at the tip of mating projections. Log-
arithmically growing GAL1-GFP-RHO4 cells
were incubated with 1 ?g/ml ?-factor and 2%
galactose for 150 min. Fixed cells were exam-
ined by fluorescence microscopy. (C) Rdi1 ex-
tracts Rho4 from plasma membranes. Loga-
rithmically growing GAL1-GFP-RHO4 cells
carrying either GAL1-RDI1 on a plasmid
(pKT10-GAL) were induced with galactose for
120 min. (D) Quantification of C. The percent-
age of cells with membrane-associated Rho4 is
given as the mean of three independent exper-
iments with SD bars (n ? 100 for each exper-
iment). (E) RDI1 deletion does not affect po-
larized Rho4 localization. GFP-Rho4 of wild
type and ?rdi1 cells was induced for 120 min.
Localization of Rho4. (A) Rho4 lo-
Regulation of Rho GTPases by Rdi1
Vol. 19, July 2008 2891
Cdc42 activation. Cdc42 regulates exit from mitosis via at
least three independent pathways involving the Cdc42 ef-
fectors Cla4, Ste20, and Gic1 (Figure 2A) (Ho ¨fken and
Schiebel, 2002; Jensen et al., 2002; Seshan et al., 2002; Ho ¨fken
and Schiebel, 2004). Previously, we could demonstrate that a
temperature-sensitive allele of the Cdc42 GEF CDC24 in a
?lte1 background is lethal at all temperatures (Ho ¨fken and
Schiebel, 2002). Here, we observed that deletion of either
RDI1 or of one of the Cdc42 GAPs results in suppression of
the mitotic exit defect of ?lte1 cells. Consistently, overex-
pression of either STE20, GIC1, or GIC2 has the same effect
on cells deleted for LTE1 (Ho ¨fken and Schiebel, 2002;
Ho ¨fken and Schiebel, 2004). This suggests that deletion of
the GAP genes or RDI1 leads to a slight activation of Cdc42
and its effectors, which trigger exit from mitosis indepen-
dently of Lte1 (Figure 2A).
Interestingly, we observed a pseudohyphal growth defect
for the diploid homozygous RDI1 deletion strain. Because
CDC42 overexpression does not rescue this phenotype, it is
unlikely that Rdi1 activates Cdc42. Because the deregulated
CDC42G12Vmutant exhibited a similar filamentation defect,
Rdi1 may rather be necessary to restrict active Cdc42 to the
growing tip. At first sight removal of Rho GTPases from the
plasma membrane seems to suggest that Rdi1 acts as an
inhibitor. However, Rho proteins could be targeted back to
the membrane, where they are required, e.g., to sites of
polarized growth. Furthermore, the extraction of Rho GT-
Pases by Rdi1 from the membrane could play an important
role in the maintenance of polarized distribution of these
molecules. Cdc42 and other polarity proteins are not immo-
bilized via scaffolds, but they are instead maintained in
dynamically polarized states (Wedlich-Soldner et al., 2004).
Endocytosis is required for the maintenance of cell polariza-
tion, e.g., by counteracting lateral diffusion of polarized
proteins within the membrane (Valdez-Taubas and Pelham,
2003; Marco et al., 2007). Therefore, Rdi1-dependent extrac-
tion of Rho GTPases from membranes and return transport
could be equally important for their polarized localization.
It has been reported that the expression of the constitu-
tively active CDC42G12Vor CDC42Q61Lresults in enhanced
pseudohyphal growth (Mo ¨sch et al., 1996; Roberts et al.,
1997). For these experiments, the CDC42 alleles were over-
expressed from the inducible GAL1 promoter, whereas in
our analysis CDC42 was placed under the control of its own
promoter. In both cases, the endogenous copies of CDC42
were also present in the cell. Therefore, the protein levels
seem to be critical. Possibly, physiological levels of
Cdc42G12Vmay interfere with Cdc42 signaling pathways,
causing a defect in pseudohyphal growth, whereas high
amounts due to overexpression of constitutively active
forms of Cdc42 cause enhanced pseudohyphal growth. Sim-
ilarly, dose dependent polarity phenotypes were observed
for other Cdc42 mutants which were attributed to differ-
ences in the capacity for GTP hydrolysis by Cdc42 of yeast
cells after expression of physiological or supraphysiological
levels (Irazoqui et al., 2004). Further work is needed to
analyze whether molecular mechanisms along this line play
a role for Cdc42G12Veffects on pseudohyphal growth.
In contrast to the diploid homozygous ?rdi1/?rdi1 strain,
which exhibited a marked filamentation defect, haploid
?rdi1 cells invaded agar like the wild-type strain. The reason
for this difference is unclear. Notably, RDI1 deletion in the
Cdc42 and Rho1. (A) CLA4 overexpression
inhibits complex formation between Cdc42
and Rdi1. The indicated strains were grown
in YP medium with raffinose. We added 2%
galactose to induce expression of STE20 and
CLA4, respectively. Cells were then lysed and
equal amounts of protein were precipitated
with anti-myc antibodies. Immunoblots were
probed with antibodies raised against Cdc42
and the myc epitope. (B) SKM1 overexpres-
sion has no effect on the Cdc42–Rdi1 complex.
3HA-CDC42 RDI1–3myc cells carrying either
GAL1-GST-SKM1 on a plasmid or the empty
plasmid were induced with galactose for 120
min. Cells were subjected to immunoprecipi-
tation with anti-HA antibodies. Immunoblots
were analyzed with anti-HA and anti-myc
antibodies. (C) CLA4 overexpression de-
creases the cytoplasmic pool of Rho1 and
Cdc42. Cells expressing the indicated 3HA-
tagged Rho GTPase and carrying either
GAL1-myc-CLA4 on a plasmid or the empty
plasmid, respectively, were induced for 120
min. Cells were lysed, and equal amounts of
protein extract were separated by centrifuga-
tion at 100,000 ? g. Rho proteins were de-
tected by immunoblotting using antibodies
against the HA epitope. (D) CLA4 overexpres-
sion reverses RDI1-dependent membrane ex-
traction of Cdc42 and Rho1. 3HA-CDC42,
3HA-CDC42 GAL1–3HA-RDI1, 3HA-RHO1,
and 3HA-RHO1 GAL1–3HA-RDI1 cells carry-
ing either an empty plasmid or a plasmid
encoding GAL1-myc-CLA4 were induced with
Cla4 regulates binding of Rdi1 to
galactose for 2 h. Subsequently, lysed cells were fractionated by centrifugation at 100,000 ? g and analyzed by immunoblotting. (E) Cla4
kinase activity is required for its effect on Cdc42-Rdi1. 3HA-CDC42 cells carrying either an empty plasmid, GAL1-myc-CLA4, or GAL1-myc-
CLA4K594Awere treated as described in C. The results shown in this figure are representative of two independent experiments.
C. Tiedje et al.
Molecular Biology of the Cell 2892
diploid fungal pathogen C. albicans shows a similar defect in
filamentation, when grown on solid medium (Court and
Sudbery, 2007). Therefore, the role of Rdi1 might be well
Complex formation between Rdi1 and Rho GTPases
Although the association of Rdi1 with Cdc42 and Rho1 has
been demonstrated (Koch et al., 1997; Eitzen et al., 2001;
Richman et al., 2004; Tcheperegine et al., 2005), it remained
unclear whether Rdi1 also interacts with other Rho GTPases.
Therefore, we systematically examined the binding specific-
ity of Rdi1. By using coimmunoprecipitations and mem-
brane extraction assays, we could demonstrate that Rdi1
only binds to Cdc42, Rho1, and Rho4. Even after strong
overexpression of RHO2, RHO3, and RHO5, the correspond-
ing proteins did not form a complex with Rdi1. In line with
these data, Rdi1 failed to extract the Rho GTPases Rho2, -3,
and -5 from membranes. It seems likely that the specific
association between Rdi1 and Rho GTPases depends on
protein–protein interactions. X-ray crystallography revealed
the binding of Rho GDIs to the switch I and II regions of Rho
proteins (Hoffman et al., 2000; Scheffzek et al., 2000). Specific
residues in these regions might determine whether Rdi1
binds to a Rho protein. Furthermore, Rho2 and Rho3 not
only carry a prenyl moiety but also were identified as being
palmitoylated (Roth et al., 2006). This additional lipid group
could possibly prohibit the release of Rho2 and Rho3 from
membranes by Rdi1.
Unexpectedly, we found that Rdi1 preferentially extracts
the GTP-bound state of Cdc42 and Rho1 from membranes.
In contrast, Rdi1 extracted comparable amounts of wild-
type, GDP- , and GTP-associated Rho4 from membranes.
However, it cannot be excluded that the point mutations,
that were introduced to lock the Rho proteins either in the
GDP- or GTP-bound state, also affect the interaction with
Rdi1. Whether Rho GDIs form a complex with the GDP- or
GTP-bound form of Rho GTPases is still controversial. Using
fluorescence spectroscopy, it has been demonstrated that
RhoGDI binds both forms of human Cdc42 equally well
Rho4 degradation. Cells in the wild-type and ?rdi1 background,
respectively, expressing 3HA-RHO4 and carrying either an empty
plasmid or a plasmid encoding GAL1-GST-YGK3 were induced for
2 h. Equal amounts of proteins were separated by SDS-PAGE, and
Rho4 was detected by antibodies against the HA epitope. Cdc11 was
used as loading control. (B) Overexpression of YGK3 has no effect on
protein levels of Cdc42 and Rho1. The experiment was performed as
described in A. (C) Rho4 degradation depends on the proteasome.
GAL1-RDI1 was overexpressed from a 2-?m plasmid (pKT10-GAL-
RDI1) in 3HA-RHO4 cells in the presence of the proteasome-specific
inhibitor MG132 (50 ?M) or with dimethyl sulfoxide alone. The
experiment was carried out in a ?erg6 background, because these
cells have enhanced permeability for MG132 (Lee and Goldberg,
1996). (D) Rho4 is degraded by vacuolar proteases. Wild-type and
?pep4 cells with HA-tagged Rho4 carrying an empty plasmid and a
plasmid with GAL1-RDI1, respectively, were incubated for 2 h with
galactose to overexpress RDI1. Rho4 was detected by immunoblot-
ting using anti-HA antibodies. GAPDH served as loading control.
The results shown in this figure are representative of two indepen-
Degradation of Rho4. (A) YGK3 overexpression leads to
(A) Rho4 protein levels remain constant throughout the cell cycle.
GAL1-CDC20 3HA-RHO4 cells were arrested in metaphase by incu-
bating cells in YP raffinose medium (no expression of CDC20).
Galactose was added to the synchronized cells to induce CDC20
expression and to trigger anaphase onset. At the indicated times,
samples were taken, cells were lysed, and equal amounts of protein
were analyzed by immunoblotting with antibodies against the HA
epitope and GAPDH (loading control). Cell cycle progression was
determined by following the number of cells without, with a small
and with a large bud by microscopy (n ? 100 at each time point). (B)
Overexpression of constitutively active RHO4 inhibits growth. Cells
carrying either GAL1-GST-RHO4G81Vor the corresponding empty
plasmid pEG(KT) were spotted on SC-Ura plates with glucose and
galactose, respectively, and they were grown for 2 d at 30°C. The
results shown in this figure are representative of two independent
Rho4 protein levels do not change during the cell cycle.
Regulation of Rho GTPases by Rdi1
Vol. 19, July 20082893
(Nomanbhoy and Cerione, 1996). In contrast, budding yeast
Rho1 exclusively binds in the GDP-bound state to Rdi1
(Koch et al., 1997). The reason for these discrepancies is
unclear, and further experiments are needed to reconcile
these conflicting results.
We and Koch et al. (1997) found Cdc42 and Rho1 in the
cytosol of ?rdi1 cells to a similar extent as in the wild-type
strain. Possibly another protein can bind to the prenyl group
of these Rho GTPases and forms a soluble complex. In
addition, the observed cytosolic protein might be newly
synthesized, which is not yet prenylated and therefore re-
mains in the cytosol.
We also presented data, which suggest that the PAK-
family kinase and Cdc42 effector Cla4 negatively regulates
binding between Rdi1 and Cdc42 as well as Rho1 (Figure
10). Several lines argue against the idea that Cla4 simply
competes with Rdi1 for binding with Rho proteins. First, this
effect is specific for Cla4. Skm1 and Ste20 belong to the same
kinase family as Cla4, and they act as direct downstream
effectors of Cdc42 as well (Park and Bi, 2007). However,
these related proteins did not interfere with Rdi1. Second,
Cla4 is an effector of Cdc42 but not of Rho1 (Park and Bi,
2007). Nevertheless, we could show that Cla4 efficiently
regulates binding between Rdi1 and Rho1 and between Rdi1
and Cdc42. Third, a kinase-dead mutant of Cla4 had almost
no effect on Rdi1 complexes. Because Cla4 kinase activity is
required for its effect on Rdi1, it is tempting to speculate that
complex formation is regulated through phosphorylation of
either Rdi1 or the Rho GTPases by Cla4. However, we could
not demonstrate such a phosphorylation event (data not
shown). Thus, it is conceivable that the regulation of Rdi1 by
Cla4 is mediated via an unknown protein. It is unclear how
Cla4 interferes with Rdi1 function. Cla4 could either inhibit
the formation of a Rho GTPase–Rdi1 complex or alterna-
tively disrupt an already existing complex.
There is some evidence that kinases modulate the inter-
action between RhoGDIs and Rho GTPases (Bourmeyster
and Vignais, 1996; Mehta et al., 2001; Forget et al., 2002;
DerMardirossian et al., 2004). In mammalian cells, Pak1
phosphorylates RhoGDI at Ser101 and Ser174, which results
in the disruption of Rac1–RhoGDI complex (DerMardiros-
sian et al., 2004). Notably, these serine residues are not
conserved in budding yeast Rdi1.
Because Cla4 acts downstream of Cdc42, but also has a
function in the disruption of the Rdi1–Cdc42 complex, Cla4
and Cdc42 may constitute a positive feedback loop involved
in the establishment of cell polarity. According to this
model, GTP-bound Cdc42 might recruit and activate Cla4,
which in turn disrupts binding between Rdi1 and Cdc42. As
a consequence, more Cdc42 is targeted to the plasma mem-
brane at sites of polarized growth, where it is activated by its
GEF Cdc24. Interestingly, Cla4 also phosphorylates Cdc24
(Gulli et al., 2000; Bose et al., 2001), and it was suggested that
both proteins restrict Cdc42 activation via a negative feed-
back loop (Gulli et al., 2000). Together, Cla4 might have a
dual role in the regulation of Cdc42 activity. In the early
stages Cla4 may be involved in the plasma membrane re-
cruitment and subsequent activation of Cdc42, whereas at
later stages Cla4 terminates polarity via Cdc24. Importantly,
Cla4 also has an effect on Rho1. Thus, Rho1 activity is
indirectly regulated by Cdc42. A similar cross talk between
Rho GTPases has been proposed for Pxl1. This paxillin-like
protein may coordinate the function of Rho1 and Cdc42
during bud formation (Gao et al., 2004; Mackin et al., 2004).
We observed that Cla4 has no effect on the interaction be-
tween Rho4 and Rdi1. Thus, Cla4 is highly specific for Cdc42
Degradation of Rho4
Here, we observed that Rho4 protein levels depend on Rdi1.
Lower amounts of Rho4 were found in cells with high levels
of Rdi1 and vice versa. Because Rho4 is a relatively stable
protein and a marked reduction of Rho4 levels took place
after only 2 h of RDI1 overexpression, it is unlikely that this
reduction is due to diminished protein synthesis. Thus, Rdi1
promotes Rho4 proteolysis. Further analysis revealed that
Rho4 degradation was almost completely blocked in the
absence of vacuolar proteases. In addition, using the protea-
some inhibitor MG132, we could demonstrate that Rho4
proteolysis, at least to some extent, also depends on the
proteasome. Finally, we observed that Ygk3, which phos-
phorylates Rho4 (Ptacek et al., 2005), promotes Rho4 degra-
dation. Very little is known about Ygk3. It is one of four
yeast homologues of GSK-3?, and it probably has a role in
ubiquitin-dependent protein degradation (Andoh et al.,
2000; Kassir et al., 2006). GSK-3? has a wide range of func-
tions in higher eukaryotes, including the establishment of
cell polarity (Kim and Kimmel, 2006). Notably, in the
Hedgehog and the canonical Wnt pathway, GSK-3? medi-
ates degradation of a transcription factor through phosphor-
ylation of these proteins (Kim and Kimmel, 2006). Together,
we propose a model, in which Rdi1 and Ygk3 coordinately
regulate Rho4 degradation by vacuolar proteases and the
proteasome (Figure 10).
Very few examples for the regulation of small GTPases
through degradation have been reported. In fibroblasts, the
HECT domain E3 ubiquitin ligase Smurf1 promotes the local
degradation of RhoA and regulates cell polarity and protru-
by Rdi1. Rdi1 selectively extracts Cdc42, Rho1, and
Rho4 from membranes and forms a complex with
these proteins. Rho2 and Rho3 not only attach to the
membrane through a prenyl group but also are
palmitoylated. Cla4 disrupts binding between Rdi1
and Cdc42 and between Rdi1 and Rho1. Because
Cla4 acts as a downstream effector of Cdc42, both
proteins may constitute a positive feedback loop
involved in the establishment of cell polarity. Fur-
thermore, Cdc42 may regulate Rho1 activity via
Cla4. The GSK-3? homologue Ygk3 and Rdi1 target
Rho4 for degradation by vacuolar proteases and the
proteasome after membrane extraction.
Model for the regulation of Rho GTPases
C. Tiedje et al.
Molecular Biology of the Cell2894
sion (Wang et al., 2003). Similarly, the related Smurf2 trig-
gers Rap1B proteolysis during neuronal differentiation
(Schwamborn et al., 2007). There are several important dif-
ferences between these examples and our observations.
RhoA and Rap1B degradation depends on HECT domain E3
ubiquitin ligases. In contrast, Rsp5, the sole yeast ubiquitin
ligase with a HECT domain (Rotin et al., 2000), is not in-
volved in Rho4 degradation. Furthermore, Rho4 is degraded
by vacuolar proteases, which has not been reported for
RhoA and Rap1B. Finally, we demonstrate the involvement
of the Rho GDI Rdi1 and the GSK-3? homologue Ygk3 in
Rho4 degradation. Thus, Rho4 is likely to be degraded by a
It seems unusual that Rho4 degradation depends on the
proteasome and the vacuolar system, but this has been re-
ported for other proteins as well. For example, a mutated
version of the plasma membrane H?-ATPase Pma1 mis-
folds, and it is subsequently degraded by the proteasome
and the autophagy pathway (Mazo ´n et al., 2007). However,
it remains unclear why some proteins are degraded by both
The physiological relevance of Rho4 proteolysis is not
clear. RhoA and Rap1B are both degraded to restrict these
proteins to a defined membrane region (Wang et al., 2003;
Schwamborn et al., 2007). This does not seem to be the case
for Rho4, which is localized to the entire plasma membrane
and enriched at the presumptive bud site. If this polarized
localization was achieved by Rho4 degradation, major
changes in Rho4 protein levels during the cell cycle would
be expected, but they were not observed in this study.
Alternatively, it is conceivable that Rdi1 removes only active
Rho4 from the membrane and targets it for destruction to
terminate Rho4 activity. Thus, only a relatively small portion
of Rho4 would be degraded; consequently, cell cycle-depen-
dent changes may not be detectable by immunoblotting. In
support of this model, we could also demonstrate that over-
expression of constitutively active RHO4 is deleterious for
Importantly, Rho GTPases, Rho GDIs and GSK-3? can be
found in higher eukaryotes, where they also play a pivotal
role in cell polarity. Therefore, the proposed regulatory link
may be conserved from yeast to mammalian cells.
We are grateful to Paul Cullen (State University of New York, Buffalo, Buffalo,
NY), Linda Hicke (Northwestern University, Evanston, IL), Peter Pryciak
(University of Massachusetts Medical School, Worcester, MA), Yoshimi Takai
(Osaka University Graduate School of Medicine, Osaka, Japan), Kazuma
Tanaka (Hokkaido University, Sapporo, Japan), and Jeremy Thorner (Univer-
sity of California, Berkeley, Berkeley, CA) for providing constructs and
strains. We thank Melanie Boß for excellent technical support and Gunnar
Dittmar for strains and helpful discussions. This work is part of the doctoral
thesis of C.T. and the diploma thesis of I.S. The project was supported by the
Deutsche Forschungsgemeinschaft HO 2098/2-1.
Ammerer, G., Hunter, C. P., Rothman, J. H., Saari, G. C., Valls, L. A., and
Stevens, T. H. (1986). PEP4 gene of Saccharomyces cerevisiae encodes proteinase
A, a vacuolar enzyme required for processing of vacuolar precursors. Mol.
Cell Biol. 6, 2490–2499.
Andoh, T., Hirata, Y., and Kikuchi, A. (2000). Yeast glycogen synthase kinase
3 is involved in protein degradation in cooperation with Bul1, Bul2, and Rsp5.
Mol. Cell Biol. 20, 6712–6720.
Barale, S., McCusker, D., and Arkowitz, R. A. (2006). Cdc42p GDP/GTP
cycling is necessary for efficient cell fusion during yeast mating. Mol. Biol.
Cell 17, 2824–2838.
Bose, I., Irazoqui, J. E., Moskow, J. J., Bardes, E. S., Zyla, T. R., and Lew, D. J.
(2001). Assembly of scaffold-mediated complexes containing Cdc42p, the
exchange factor Cdc24p, and the effector Cla4p required for cell cycle-regu-
lated phosphorylation of Cdc24p. J. Biol. Chem. 276, 7176–7186.
Bosl, W. J., and Li, R. (2005). Mitotic-exit control as an evolved complex
system. Cell 121, 325–333.
Bourmeyster, N., and Vignais, P. V. (1996). Phosphorylation of Rho GDI
stabilizes the Rho A-Rho GDI complex in neutrophil cytosol. Biochem. Bio-
phys. Res. Commun. 218, 54–60.
Caviston, J. P., Longtine, M., Pringle, J. R., and Bi, E. (2003). The role of
Cdc42p GTPase-activating proteins in assembly of the septin ring in yeast.
Mol. Biol. Cell 14, 4051–4066.
Chuang, T. H., Bohl, B. P., and Bokoch, G. M. (1993). Biologically active lipids
are regulators of Rac.GDI complexation. J. Biol. Chem. 268, 26206–26211.
Cole, K. C., McLaughlin, H. W., and Johnson, D. I. (2007). Use of bimolecular
fluorescence complementation to study in vivo interactions between Cdc42p
and Rdi1p of Saccharomyces cerevisiae. Eukaryot. Cell 6, 378–387.
Court, H., and Sudbery, P. (2007). Regulation of Cdc42 GTPase activity in the
formation of hyphae in Candida albicans. Mol. Biol. Cell 18, 265–281.
DerMardirossian, C., and Bokoch, G. M. (2005). GDIs: central regulatory
molecules in Rho GTPase activation. Trends Cell Biol. 15, 356–363.
DerMardirossian, C., Schnelzer, A., and Bokoch, G. M. (2004). Phosphoryla-
tion of RhoGDI by Pak1 mediates dissociation of Rac GTPase. Mol. Cell 15,
Dovas, A., and Couchman, J. R. (2005). RhoGDI: multiple functions in the
regulation of Rho family GTPase activities. Biochem. J. 390, 1–9.
Dransart, E., Olofsson, B., and Cherfils, J. (2005). RhoGDIs revisited: novel
roles in Rho regulation. Traffic 6, 957–966.
Eitzen, G., Thorngren, N., and Wickner, W. (2001). Rho1p and Cdc42p act
after Ypt7p to regulate vacuole docking. EMBO J. 20, 5650–5656.
Forget, M. A., Desrosiers, R. R., Gingras, D., and Beliveau, R. (2002). Phos-
phorylation states of Cdc42 and RhoA regulate their interactions with Rho
GDP dissociation inhibitor and their extraction from biological membranes.
Biochem. J. 361, 243–254.
Gao, X. D., Caviston, J. P., Tcheperegine, S. E., and Bi, E. (2004). Pxl1p, a
paxillin-like protein in Saccharomyces cerevisiae, may coordinate Cdc42p and
Rho1p functions during polarized growth. Mol. Biol. Cell 15, 3977–3985.
Gladfelter, A. S., Bose, I., Zyla, T. R., Bardes, E. S., and Lew, D. J. (2002). Septin
ring assembly involves cycles of GTP loading and hydrolysis by Cdc42p.
J. Cell Biol. 156, 315–326.
Gulli, M. P., Jaquenoud, M., Shimada, Y., Niederhauser, G., Wiget, P., and
Peter, M. (2000). Phosphorylation of the Cdc42 exchange factor Cdc24 by the
PAK-like kinase Cla4 may regulate polarized growth in yeast. Mol. Cell 6,
Hoffman, G. R., Nassar, N., and Cerione, R. A. (2000). Structure of the Rho
family GTP-binding protein Cdc42 in complex with the multifunctional reg-
ulator RhoGDI. Cell 100, 345–356.
Ho ¨fken, T., and Schiebel, E. (2002). A role for cell polarity proteins in mitotic
exit. EMBO J. 21, 4851–4862.
Ho ¨fken, T., and Schiebel, E. (2004). Novel regulation of mitotic exit by the
Cdc42 effectors Gic1 and Gic2. J. Cell Biol. 164, 219–231.
Irazoqui, J. E., Gladfelter, A. S., and Lew, D. J. (2003). Scaffold-mediated
symmetry breaking by Cdc42p. Nat. Cell Biol. 5, 1062–1070.
Irazoqui, J. E., Gladfelter, A. S., and Lew, D. J. (2004). Cdc42p, GTP hydrolysis,
and the cell’s sense of direction. Cell Cycle 3, 861–864.
Jaffe, A. B., and Hall, A. (2005). Rho GTPases: biochemistry and biology.
Annu. Rev. Cell Dev. Biol. 21, 247–269.
Janke, C. et al. (2004). A versatile toolbox for PCR-based tagging of yeast
genes: new fluorescent proteins, more markers and promoter substitution
cassettes. Yeast 21, 947–962.
Jensen, S., Geymonat, M., Johnson, A. L., Segal, M., and Johnston, L. H. (2002).
Spatial regulation of the guanine nucleotide exchange factor Lte1 in Saccha-
romyces cerevisiae. J. Cell Sci. 115, 4977–4991.
Kassir, Y., Rubin-Bejerano, I., and Mandel-Gutfreund, Y. (2006). The Saccha-
romyces cerevisiae GSK-3 beta homologs. Curr. Drug Targets 7, 1455–1465.
Kim, L., and Kimmel, A. R. (2006). GSK3 at the edge: regulation of develop-
mental specification and cell polarization. Curr. Drug Targets 7, 1411–1419.
Knop, M., Siegers, K., Pereira, G., Zachariae, W., Winsor, B., Nasmyth, K., and
Schiebel, E. (1999). Epitope tagging of yeast genes using a PCR-based strategy:
more tags and improved practical routines. Yeast 15, 963–972.
Koch, G., Tanaka, K., Masuda, T., Yamochi, W., Nonaka, H., and Takai, Y.
(1997). Association of the Rho family small GTP-binding proteins with Rho
Regulation of Rho GTPases by Rdi1
Vol. 19, July 2008 2895
GDP dissociation inhibitor (Rho GDI) in Saccharomyces cerevisiae. Oncogene
Lee, D. H., and Goldberg, A. L. (1996). Selective inhibitors of the proteasome-
dependent and vacuolar pathways of protein degradation in Saccharomyces
cerevisiae. J. Biol. Chem. 271, 27280–27284.
Longtine, M. S., McKenzie, A., Demarini, D. J., Shah, N. G., Wach, A., Brachat,
A., Philippsen, P., and Pringle, J. R. (1998). Additional modules for versatile
and economical PCR-based gene deletion and modification in Saccharomyces
cerevisiae. Yeast 14, 953–961.
Mackin, N. A., Sousou, T. J., and Erdman, S. E. (2004). The PXL1 gene of
Saccharomyces cerevisiae encodes a paxillin-like protein functioning in polar-
ized cell growth. Mol. Biol. Cell 15, 1904–1917.
Marco, E., Wedlich-Soldner, R., Li, R., Altschuler, S. J., and Wu, L. F. (2007).
Endocytosis optimizes the dynamic localization of membrane proteins that
regulate cortical polarity. Cell 129, 411–422.
Masuda, T., Tanaka, K., Nonaka, H., Yamochi, W., Maeda, A., and Takai, Y.
(1994). Molecular cloning and characterization of yeast rho GDP dissociation
inhibitor. J. Biol. Chem. 269, 19713–19718.
Mazo ´n, M. J., Eraso, P., and Portillo, F. (2007). Efficient degradation of mis-
folded mutant Pma1 by endoplasmic reticulum-associated degradation re-
quires Atg19 and the Cvt/autophagy pathway. Mol. Microbiol. 63, 1069–
Mehta, D., Rahman, A., and Malik, A. B. (2001). Protein kinase C-alpha signals
rho-guanine nucleotide dissociation inhibitor phosphorylation and rho acti-
vation and regulates the endothelial cell barrier function. J. Biol. Chem. 276,
Mo ¨sch, H. U., Roberts, R. L., and Fink, G. R. (1996). Ras2 signals via the
Cdc42/Ste20/mitogen-activated protein kinase module to induce filamentous
growth in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA 93, 5352–5356.
Nomanbhoy, T. K., and Cerione, R. (1996). Characterization of the interaction
between RhoGDI and Cdc42Hs using fluorescence spectroscopy. J. Biol.
Chem. 271, 10004–10009.
Palecek, S. P., Parikh, A. S., and Kron, S. J. (2002). Sensing, signalling and
integrating physical processes during Saccharomyces cerevisiae invasive and
filamentous growth. Microbiology 148, 893–907.
Park, H. O., and Bi, E. (2007). Central roles of small GTPases in the develop-
ment of cell polarity in yeast and beyond. Microbiol. Mol. Biol. Rev. 71, 48–96.
Ptacek, J. et al. (2005). Global analysis of protein phosphorylation in yeast.
Nature 438, 679–684.
Richman, T. J., Sawyer, M. M., and Johnson, D. I. (2002). Saccharomyces
cerevisiae Cdc42p localizes to cellular membranes and clusters at sites of
polarized growth. Eukaryot. Cell 1, 458–468.
Richman, T. J., Toenjes, K. A., Morales, S. E., Cole, K. C., Wasserman, B. T.,
Taylor, C. M., Koster, J. A., Whelihan, M. F., and Johnson, D. I. (2004).
Analysis of cell-cycle specific localization of the Rdi1p RhoGDI and the
structural determinants required for Cdc42p membrane localization and clus-
tering at sites of polarized growth. Curr. Genet. 45, 339–349.
Roberts, R. L., Mo ¨sch, H. U., and Fink, G. R. (1997). 14–3-3 proteins are
essential for RAS/MAPK cascade signaling during pseudohyphal develop-
ment in S. cerevisiae. Cell 89, 1055–1065.
Roth, A. F., Wan, J., Bailey, A. O., Sun, B., Kuchar, J. A., Green, W. N.,
Phinney, B. S., Yates, J. R. 3rd, and Davis, N. G. (2006). Global analysis of
protein palmitoylation in yeast. Cell 125, 1003–1013.
Rotin, D., Staub, O., and Haguenauer-Tsapis, R. (2000). Ubiquitination and
endocytosis of plasma membrane proteins: role of Nedd4/Rsp5p family of
ubiquitin-protein ligases. J. Membr. Biol. 176, 1–17.
Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger, D., and Gierschik, P.
(2000). The Rac-RhoGDI complex and the structural basis for the regulation of
Rho proteins by RhoGDI. Nat. Struct. Biol. 7, 122–126.
Schwamborn, J. C., Muller, M., Becker, A. H., and Puschel, A. W. (2007).
Ubiquitination of the GTPase Rap1B by the ubiquitin ligase Smurf2 is re-
quired for the establishment of neuronal polarity. EMBO J. 26, 1410–1422.
Seshan, A., Bardin, A. J., and Amon, A. (2002). Control of Lte1 localization by
cell polarity determinants and Cdc14. Curr. Biol. 12, 2098–2110.
Takahashi, K., Sasaki, T., Mammoto, A., Takaishi, K., Kameyama, T., Tsukita,
S., and Takai, Y. (1997). Direct interaction of the Rho GDP dissociation
inhibitor with ezrin/radixin/moesin initiates the activation of the Rho small
G protein. J. Biol. Chem. 272, 23371–23375.
Tcheperegine, S. E., Gao, X. D., and Bi, E. (2005). Regulation of cell polarity by
interactions of Msb3 and Msb4 with Cdc42 and polarisome components. Mol.
Cell Biol. 25, 8567–8580.
Valdez-Taubas, J., and Pelham, H. R. (2003). Slow diffusion of proteins in the
yeast plasma membrane allows polarity to be maintained by endocytic cy-
cling. Curr. Biol. 13, 1636–1640.
Wang, H. R., Zhang, Y., Ozdamar, B., Ogunjimi, A. A., Alexandrova, E.,
Thomsen, G. H., and Wrana, J. L. (2003). Regulation of cell polarity and
protrusion formation by targeting RhoA for degradation. Science 302, 1775–
Wedlich-Soldner, R., Wai, S. C., Schmidt, T., and Li, R. (2004). Robust cell
polarity is a dynamic state established by coupling transport and GTPase
signaling. J. Cell Biol. 166, 889–900.
Yamashita, T., and Tohyama, M. (2003). The p75 receptor acts as a displace-
ment factor that releases Rho from Rho-GDI. Nat. Neurosci. 6, 461–467.
Yamochi, W., Tanaka, K., Nonaka, H, Maeda, A., Musha, T., and Takai, Y.
(1994). Growth site localization of Rho1 small GTP-binding protein and its
involvement in bud formation in Saccharomyces cerevisiae. J. Cell Biol. 125,
Ziman, M., O’Brien, J. M., Ouellette, L. A., Church, W. R., and Johnson, D. I.
(1991). Mutational analysis of CDC42Sc, a Saccharomyces cerevisiae gene that
encodes a putative GTP-binding protein involved in the control of cell polar-
ity. Mol. Cell. Biol. 11, 3537–3544.
Ziman, M., Preuss, D., Mulholland, J., O’Brien, J. M., Botstein, D., and Johnson,
D. I. (1993). Subcellular localization of Cdc42p, a Saccharomyces cerevisiae
GTP-binding protein involved in the control of cell polarity. Mol. Biol. Cell 4,
C. Tiedje et al.
Molecular Biology of the Cell2896