The EMBO Journal Vol. 20 No. 20 pp. 5657±5665, 2001
Cdc42p functions at the docking stage of yeast
vacuole membrane fusion
Oliver MuÈller, Douglas I.Johnson1 and
Andreas Mayer2
Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft,
Spemannstrasse 37±39, 72076 TuÈbingen, Germany and 1Department of
Microbiology and Molecular Genetics, Markey Center for Molecular
Genetics, University of Vermont, Burlington, VT 05405, USA
2
Corresponding author
e-mail: [email protected]
Membrane fusion reactions have been considered to
be primarily regulated by Rab GTPases. In the model
system of homotypic vacuole fusion in the yeast
Saccharomyces cerevisiae, we show that Cdc42p, a
member of the Rho family of GTPases, has a direct
role in membrane fusion. Genetic evidence suggested
a relationship between Cdc42p and Vtc1p/Nrf1p, a
central part of the vacuolar membrane fusion
machinery. Vacuoles from cdc42 temperature-sensitive mutants are de®cient for fusion at the restrictive
temperature. Speci®c amino acid changes on the
Cdc42p protein surface in these mutants de®ne the
putative interaction domain that is crucial for its function in membrane fusion. Af®nity-puri®ed antibodies
to this domain inhibited the in vitro fusion reaction.
Using these antibodies in kinetic analyses and assays
for subreactions of the priming, docking and postdocking phase of the reaction, we show that Cdc42p
action follows Ypt7p-dependent tethering, but precedes the formation of trans-SNARE complexes. Thus,
our data de®ne an effector binding domain of Cdc42p
by which it regulates the docking reaction of vacuole
fusion.
Keywords: actin/Rho GTPase/Saccharomyces cerevisiae/
SNARE/tethering
Introduction
Small GTPases are key regulatory proteins in many
cellular processes (Lazar et al., 1997; Matozaki et al.,
2000; Zerial and McBride, 2001). They can bind and
hydrolyze GTP and act as molecular switches by cycling
between an active, GTP-bound state and an inactive, GDPbound state. Depending on the activation state they can
expose a lipid anchor and insert into membranes. In the
active state, they associate with other proteins that mediate
downstream effects. Three other factors regulate the
different states of small GTPases. GTP hydrolysis is
accelerated by GTPase-activating proteins (GAPs), exchange of GDP for GTP is regulated by guaninenucleotide exchange factors (GEFs), and membrane
association is controlled by guanine-dissociation inhibitors
(GDIs), which extract the protein from the membrane in its
ã European Molecular Biology Organization
GDP-bound state. This extraction is thought to help in
recycling the proteins after a signaling event.
Small GTPases can be grouped into several families.
Original classi®cations suggested that Ras proteins in¯uence cell growth, Rho proteins control the actin cytoskeleton architecture, Rab and Arf proteins participate in
intracellular traf®cking and Ran directs nucleocytoplasmic
transport. However, it has become increasingly apparent
that a given GTPase can interact with a variety of targets
and in¯uence a diverse range of cellular responses. An
example of this is Cdc42, a member of the Rho subfamily
(Johnson, 1999; Erickson and Cerione, 2001).
The gene encoding Cdc42 was ®rst identi®ed in
Saccharomyces cerevisiae as an essential element in the
assembly of the bud site (Adams et al., 1990; Johnson and
Pringle, 1990). Further studies in yeast suggested a role for
Cdc42 in the establishment of cell polarity through effects
on the actin cytoskeleton (Ziman et al., 1991, 1993; Li
et al., 1995). Studies in mammalian cells revealed that
Cdc42 in¯uences cell shape and structure by initiating
actin cytoskeleton remodeling (Kozma et al., 1995; Nobes
and Hall, 1995). Recent work has shown a direct role of
Cdc42 in stimulating actin polymerization (Ma et al.,
1998a,b; Rohatgi et al., 1999, 2000). Cdc42 relieves the
autoinhibition of a C-terminal region of Wiscott±Aldrich
syndrome protein (WASP), which can then couple to the
Arp2/3 complex (Kim et al., 2000; Prehoda et al., 2000).
This complex promotes the incorporation of actin monomers into F-actin polymers. Cdc42 also appears to play a
role in reactions of vesicular traf®c. Cdc42 interacts with
Golgi proteins involved in vesicle budding such as ARF
(ADP ribosylation factor) (Erickson et al., 1996) and the
g-subunit of the COP1 coatomer complex (Wu et al.,
2000). Other targets for activated Cdc42 are the ACKs
(activated Cdc42-associated tyrosine kinases) (Manser
et al., 1993; Yang and Cerione, 1997; Yang et al., 1999).
One of these non-receptor tyrosine kinases, ACK2,
competes with AP-2 (adaptor protein-2) for binding to
clathrin, leading to an inhibition of AP-2-mediated
transferrin receptor endocytosis (Erickson and Cerione,
2001). Previous studies in MDCK cells, dendritic cells and
Schizosaccharomyces pombe support an involvement of
Cdc42 in endocytosis (Kroschewski et al., 1999; Garrett
et al., 2000; Murray and Johnson, 2001). A role for Cdc42
in exocytosis has been suggested based on studies on
secretion in mast cells (Brown et al., 1998; Hong-Geller
and Cerione, 2000). Cdc42 has also been implicated in the
maintenace of tight junctions, as well as in the regulation
of RNA processing (Erickson and Cerione, 2001). Thus,
Cdc42 acts on a number of different targets in a variety of
cellular processes.
We have investigated the role of Cdc42 in membrane
fusion using the model system of homotypic vacuole
fusion in the yeast S.cerevisiae (Wickner and Haas, 2000).
5657
O.MuÈller, D.I.Johnson and A.Mayer
This reaction occurs in sequential phases of priming,
tethering, docking and membrane fusion. It can be
reconstituted in vitro and the different kinetic stages of
the reaction can be analyzed (Conradt et al., 1994; Mayer
et al., 1996; Wickner and Haas, 2000). The priming event
activates the machinery required for recognition and
membrane attachment, including the SNARE proteins
Vam3p, Nyv1p, Vam7p, Vti1p and Ykt6p, the Rab
GTPase Ypt7p, and the HOPS complex of tethering
factors (Vps11, 16, 18, 33, 39, 41) (Ungermann et al.,
1999; Price et al., 2000a,b; Sato et al., 2000; Wurmser
et al., 2000). Tethering factors and Rab GTPases can
establish an initial SNARE-independent interaction of the
membranes (Cao et al., 1998; Ungermann et al., 1998a;
Waters and Pfeffer, 1999). The ATPase Sec18p/NSF and
its cofactor Sec17p/a-SNAP disassemble cis-SNARE
complexes, i.e. complexes of SNARE proteins on the
same membrane (Ungermann et al., 1998b), and Sec17p/
a-SNAP is released from the membrane (Mayer et al.,
1996). LMA1 stabilizes the primed state of SNAREs (Xu
et al., 1998), which is a prerequisite for the formation
of trans-SNARE complexes, i.e. complexes between
SNAREs on apposed membranes. Trans-SNARE pairing
is believed to establish a very close attachment (docking)
of the membranes, which activates a calcium ef¯ux from
the vacuolar lumen (Peters and Mayer, 1998). Calcium
binds to calmodulin and enhances its association with the
vacuolar membrane probably through its interaction with
the V0 sector of the vacuolar H+-ATPase (Peters et al.,
2001). V0 sectors from apposed membranes form transcomplexes that might result in a proteinaceous fusion pore.
The maintenance of V0 trans-complexes is independent of
trans-SNARE complexes (Peters et al., 2001), as is the
completion of fusion (Ungermann et al., 1998a).
The proteins involved in this sequence of events are
connected by physical interactions. Tethering factors
regulate the nucleotide exchange on Ypt7p and, together
with Ypt7p, are required for the formation of transSNARE complexes (Eitzen et al., 2000; Price et al.,
2000a; Sato et al., 2000; Seals et al., 2000). Different
subunits of the Vtc complex regulate priming and a postdocking event and link Sec18p/NSF activity to V0 transcomplex formation (O.MuÈller, M.Bayer, J.Anderson,
M.Mann and A.Mayer, submitted). Genetic evidence
suggested an interaction between Cdc42p and Vtc1p/
Nrf1p (Murray and Johnson, 2000) and led us to investigate the involvement of Cdc42p in membrane fusion.
Results
Cdc42p is present on vacuoles
In a genetic screen, Vtc1p/Nrf1p, a protein of the Vtc
complex (Nelson et al., 2000) that has recently been
shown to link different steps of vacuole fusion (O.MuÈller,
M.Bayer, J.Anderson, M.Mann and A.Mayer, submitted),
was isolated as a negative regulator of Cdc42p in S.pombe
(Murray and Johnson, 2000). Therefore, we asked whether
Cdc42p is involved in the fusion reaction. As a tool for the
analysis of Cdc42p, we used an antibody raised against a
Cdc42p-speci®c peptide (Ziman et al., 1991). The antibodies were af®nity puri®ed on recombinantly expressed
glutathione S-transferase (GST)±Cdc42p coupled to a
Sepharose matrix. Western analysis showed that Cdc42p is
5658
Fig. 1. Cdc42p is associated with the vacuolar membrane. (A) 70 mg of
isolated vacuoles (Vac), cytosol (Cyt) or a whole-cell extract (Cell)
from strain BJ3505 were analyzed by SDS±PAGE and western blotting
for their content of Cdc42p, Ypt7p and Pgk1p. (B) Longer exposure of
the blots for Cdc42p and Ypt7p in (A) with the bands visible in the
whole-cell extract (lane 3).
present on isolated vacuoles (Figure 1, lane 1). The
antibody recognized a single band at the expected
molecular weight of ~21 kDa. Previous studies showed
that Cdc42p is a geranylgeranylated peripheral membrane
protein mainly localized to sites of bud emergence where
transport vesicles fuse with the plasma membrane (Ohya
et al., 1993; Ziman et al., 1993). We show that, like the
Rab GTPase Ypt7p, Cdc42p is also highly enriched on
vacuolar membranes when compared with the the same
protein amounts of cytosol (Figure 1A and B, lane 2) or
whole-cell extract (Figure 1A and B, lane 3). As a control
we also decorated for the cytosolic marker Pgk1p, which is
highly enriched in the cytosolic fraction (Figure 1A).
Vacuoles from a speci®c cdc42ts mutant are
thermolabile for fusion
Deletion of the CDC42 gene is lethal (Johnson and
Pringle, 1990). Therefore, we tested the involvement of
Cdc42p in vacuole fusion using different cdc42ts mutants
(Kozminski et al., 2000). We also deleted either PEP4 or
PHO8 in the mutants and the corresponding wild-type
strain to make them suitable for the in vitro fusion assay.
In the in vitro fusion assay, two populations of vacuoles
are used. One is lacking the proteinase Pep4p and therefore
only bears the inactive pro-alkaline phosphatase (Pho8p),
because Pep4p is needed for the maturation of pro-Pho8p
to the active enzyme. The other population has Pep4p, but
is lacking Pho8p. Upon fusion and contents mixing, proPho8p is activated. It can be assayed colorimetrically as a
quantitative readout of fusion (Haas, 1995).
Vacuoles from a cdc42ts-123 mutant were thermolabile
for fusion (Figure 2). At 23°C they fused even slightly
better than wild-type vacuoles, but they showed a dramatic
decrease in fusion activity compared with wild type at
30°C. The wild-type fusion activity was also lower at
30°C due to the narrow temperature optimum of the
in vitro fusion reaction (T.Sattler and A.Mayer, unpublished observation). In contrast, vacuoles from the cdc42ts124 mutant behaved like wild type (Figure 2). These cdc42
alleles have mutations in different functional domains
(Kozminski et al., 2000). The cdc42ts-123 allele contains
the R163A and K166A substitutions and the cdc42ts-124
allele has the K183A, K184A, K186A and K187A
mutations. Both mutants are temperature sensitive for
growth, but only cdc42ts-123 is temperature sensitive for
membrane fusion. Since both sets of mutations map to
different areas on the surface of Cdc42p (Kozminski et al.,
2000), this suggests that Cdc42p uses a speci®c surface
region to regulate membrane fusion.
Cdc42p in yeast vacuole membrane fusion
Fig. 2. In vitro fusion of vacuoles from cdc42ts mutants. Standard
fusion reactions with cytosol were incubated for 70 min at 23 or
30°C. The indicated tester strain combinations were used: OMY14/
OMY15(wt), OMY16/OMY17 (cdc42ts-123) and OMY18/OMY19
(cdc42ts-124). Growth of cells and preparation of vacuoles was at 23°C
instead of the 30°C used in the standard protocol. Four experiments
were averaged. The 100% fusion activities of the wild type at 23°C
were between 2.15 and 2.38 U.
In agreement with our ®ndings, another temperaturesensitive mutant, namely cdc42ts-1 (Adams et al., 1990),
also shows thermolability of vacuole fusion in vitro (see
Eitzen et al., 2001). Furthermore, vacuoles in these
mutants rapidly fragment into smaller vesicles upon shift
to non-permissive temperature. These data provide in vivo
evidence for an involvement of Cdc42p in vacuole fusion
(see Eitzen et al., 2001).
Cdc42p functions up to the docking stage of
membrane fusion
For kinetic analyses we used a Cdc42p-speci®c antibody
directed against a peptide (residues 167±183; Ziman et al.,
1991) encompassing a putative interaction domain with no
known function (Kozminski et al., 2000). The af®nitypuri®ed antibody inhibited fusion in a concentrationdependent manner (Figure 3A; IC50 » 2.4 mM). In a kinetic
experiment, we monitored when the reaction became
resistant to anti-Cdc42p (Figure 3B). The bulk of vacuoles
complete distinct reaction steps within de®ned intervals
and become resistant to inhibitors of these steps (Conradt
et al., 1994; Mayer et al., 1996), revealing the sequence of
events in vacuole fusion. Inhibitors or control buffer were
added into an ongoing fusion reaction at different times
and the samples were incubated further until the end of a
standard fusion period (70 min). Another aliquot was
chilled to stop fusion at that time and monitor progression
of the reaction. All inhibitors abolished fusion when added
at the start of the reaction (Figure 3B). After 15 min the
reaction was resistant to anti-Sec18p, i.e. priming was
completed. After 35 min, the reaction was resistant to
Gdi1p, the GDI that extracts Ypt7p from the vacuolar
membrane (Wickner and Haas, 2000), and to antibodies to
the SNARE Vam3p. Resistance to these two reagents
indicates the completion of docking (Mayer and Wickner,
1997; Nichols et al., 1997; Ungermann et al., 1998a). The
inhibition curve for anti-Cdc42p overlapped with those of
Fig. 3. Cdc42p functions up to the docking stage. (A) Antibody
inhibition. The indicated amounts of af®nity-puri®ed antibodies to
Cdc42p were added to standard fusion reactions without cytosol. After
pre-incubation for 10 min on ice, ATP was added and fusion was
measured after 70 min at 27°C. (B) Kinetic analysis. Standard fusion
reactions without cytosol were started. After the indicated times at
27°C inhibitors or control buffer were added. The samples were left on
ice for 10 min. Then they were transferred to 27°C or left on ice for
the remainder of the 70 min reaction period. Finally, fusion activity
was assayed. Inhibitors used were: anti-Sec18p, 2 mM; Gdi1p, 5 mM;
anti-Vam3p, 2 mM; Mastoparan, 20 mM; anti-Cdc42p, 8 mM.
Gdi1p and anti-Vam3p, indicating that Cdc42p acts at
least up to the docking stage of vacuole fusion.
An independent approach by Eitzen et al. (2001) further
supports this conclusion. In that study, Rdi1p, the RhoGDI
that speci®cally extracts Cdc42p from the membrane, was
used as a tool to investigate Cdc42p function in vacuole
fusion. Like the speci®c antibodies to Cdc42p, Rdi1p
inhibits the fusion reaction in a concentration-dependent
manner and shows inhibition up to the docking stage in a
kinetic experiment.
Cdc42p is not required for the priming reaction
Sensitivity of the reaction to anti-Cdc42p in a kinetic
experiment up to the docking stage does not exclude a
further involvement of Cdc42p in the preceding priming
phase. For example, Vtc4p, a component of the Vtc
complex involved in vacuole fusion (O.MuÈller et al.,
submitted), controls priming, but has further activities
required up to the docking stage. Therefore, we asked
whether priming is affected by anti-Cdc42p. We tested this
by monitoring Sec17p/a-SNAP release from the vacuolar
membrane, which is a molecularly de®ned subreaction
occurring as a consequence of priming (Mayer et al.,
5659
O.MuÈller, D.I.Johnson and A.Mayer
Fig. 4. Sec17p/a-SNAP release is not compromised by anti-Cdc42p.
Standard fusion reactions (3-fold volume) without cytosol were
incubated for 70 min without ATP on ice (0 min) or with ATP at 27°C
for 15 or 70 min. Where indicated, 6 mM anti-Cdc42p was added.
Reactions were chilled on ice and centrifuged (10 000 g, 10 min, 4°C).
The supernatants (S) were recovered and the pellets (P) resuspended in
90 ml of PS buffer. All samples were trichloroacetic acid precipitated
and processed for non-reducing SDS±PAGE and western blotting with
the indicated antibodies. Alkaline phosphatase (Pho8p) serves as a
membrane integral vacuolar marker.
1996). At concentrations suf®cient to block overall fusion,
anti-Cdc42p did not compromise Sec17p/a-SNAP release
(Figure 4), indicating that Cdc42p is not required for
priming.
Cdc42p acts after Ypt7p but before trans-SNARE
pairing
Several molecular events kinetically map to the docking
step, such as Ypt7p action, trans-SNARE pairing and the
triggering of a calcium ef¯ux from the lumen of the
organelles. In order to determine more precisely when
Cdc42p acts within the sequence of these different
subreactions of the docking stage, we used anti-Cdc42p
in speci®c assays for these subreactions.
The in vitro reaction can be blocked at distinct stages by
the use of reversible inhibitors. GTPgS, a poorly hydrolyzable analog of GTP, locks Ypt7p in its GTP-bound state
(Eitzen et al., 2000). A GTPgS block can be released by reisolating the vacuoles and resuspending them in fresh
fusion buffer without GTPgS (Mayer et al., 1996). After
release of the GTPgS block, different inhibitors worked
with different ef®ciencies, indicating the sequence of
events (Figure 5A). BAPTA, a calcium chelator that
inhibits fusion by quenching the calcium ef¯ux triggered
by docking (Peters and Mayer, 1998), inhibited the
released reaction completely, indicating that calcium
ef¯ux from the vacuolar lumen coincides with or follows
a GTPgS-sensitive step (cf. Eitzen et al., 2000). Antibodies
to Sec18p/NSF, which is involved in priming, i.e. in a
subreaction preceding docking, did not inhibit the released
reaction. Gdi1p did not inhibit the released reaction
(Figure 5A; cf. Eitzen et al., 2001), probably because the
GTPgS bound state of Ypt7p is resistant to GDI extraction.
Anti-Cdc42p still inhibited the released reaction to ~50%
(Figure 5A), as did antibodies to the SNARE Vam3p. This
is consistent with the fact that formation of trans-SNARE
complexes requires Ypt7p (Sato et al., 2000) and that
Ypt7p function is necessary to pass the Cdc42p-dependent
step. Further data by Eitzen et al. (2001) show that
Rdi1pÐby extracting Cdc42pÐstill inhibits after a
reversible block by Ypt7p antibodies. Collectively, these
data strongly suggest that Cdc42p and Vam3p act after
Ypt7p.
After a BAPTA block, the reaction was resistant to antiCdc42p (Figure 5B), as it was to anti-Sec18p, Gdi1p and
5660
Fig. 5. Kinetic analysis of Cdc42p action. (A and B) Standard fusion
reactions (10-fold volume) without cytosol were incubated at 27°C in
the presence of (A) 2 mM GTPgS or (B) 2 mM BAPTA. After 30 min,
reactions were chilled on ice and vacuoles were re-isolated (6800 g,
2 min, 4°C). Vacuoles were resuspended in fresh fusion buffer with
cytosol. In the case of the BAPTA block, 200 mM CaCl2 was added.
Aliquots were supplemented with the indicated inhibitors and incubated
for another 60 min at 27°C. Finally, fusion activity was measured.
Fusion without re-isolation was 3.94 U. Inhibitors used were: antiSec18p, 2 mM; Gdi1p, 3.7 mM; anti-Cdc42p, 6 mM; anti-Vam3p, 2 mM;
BAPTA, 2 mM; GTPgS, 2 mM. (C) Ca2+ release from vacuoles in the
course of the fusion reaction was assayed as described in Materials and
methods. Inhibitors used were: Gdi1p, 4 mM; anti-Sec18p, 1 mM;
anti-Cdc42p, 6 mM.
anti-Vam3p. Only BAPTA itself and GTPgS still inhibited
fusion. The same effect as with anti-Cdc42p could be
observed with Rdi1p (Eitzen et al., 2001). These results
show that Cdc42p and Vam3p act before the BAPTAsensitive step, i.e. before calcium ef¯ux from the lumen of
the vacuole. This could be tested by direct measurements
of calcium ef¯ux (Peters and Mayer, 1998). Anti-Cdc42p
inhibited this calcium ef¯ux, as did anti-Sec18p and Gdi1p
(Figure 5C).
Trans-SNARE pairing is thought to establish the docked
state of the membranes that precedes the calcium ef¯ux.
Cdc42p in yeast vacuole membrane fusion
Fig. 6. Cdc42p acts before trans-SNARE pairing. Standard fusion
reactions (5-fold volume) with cytosol containing vacuoles from strains
BJ3505Dvam3 and BJ3505Dnyv1 were incubated without ATP on ice
or with ATP at 27°C. One of the samples at 27°C received buffer only
(None) and another one 6 mM af®nity-puri®ed antibodies to Cdc42p
(Ab). After 45 min, trans-SNAREs were assayed as described in
Materials and methods by the amount of Nyv1p that co-immunoprecipitates with Vam3p. As can be seen by comparison of coprecipitated Nyv1p with the corresponding input, formation of
trans-SNARE pairs in the control reaction is in the range of literature
values (Ungermann et al., 1998a).
Since trans-SNARE complex formation was also inhibited
by anti-Cdc42p (Figure 6), Cdc42p must act after Ypt7p
has ful®lled its function, but before trans-SNARE pairing
occurs, and before the ef¯ux of lumenal calcium is
triggered.
Discussion
To date, mainly Rab GTPases have been implicated as
direct players in membrane fusion reactions. In this study
we identify another class of GTPase as a new factor in
vacuole fusion. The Rho GTPase Cdc42p functions at the
docking stage of vacuole fusion. Our kinetic analysis
places Cdc42p between Rab (Ypt7p) action and transSNARE pairing. This is surprising because Rab and
SNARE action are intimately connected, as illustrated by
the fact that Rab and SNAREs are part of a large complex
(McBride et al., 1999). Furthermore, Rab activity is
required for trans-SNARE pairing (Eitzen et al., 2000;
Sato et al., 2000; Seals et al., 2000; Wurmser et al., 2000),
and SNARE overexpression can compensate for loss of
Rab function (Dascher et al., 1991). The observation that
Cdc42p acts between Rab and SNAREs suggests that Rab
proteins and associated tethering factors may not be the
sole controllers of trans-SNARE pairing, but require other
GTPases as mediators. In agreement with this view,
Cdc42p and Rho1p, but not Rho2p, Rho3p or Rho4p, are
required in vacuole fusion (cf. Eitzen et al., 2001). Rho1 is
also required for spatial regulation of the exocyst, a
complex of tethering factors involved in exocytosis in
yeast (Guo et al., 2001). Rho3p has a direct role in
exocytosis that is distinct from its role in actin polarity
(Adamo et al., 1999). It was suggested that it may
coordinate several different events necessary for delivery
of proteins to speci®c sites on the cell surface. In addition,
Ran, a small GTPase well known for its role in nuclear
protein import and RNA export, has been implicated in a
fusion process, the reassembly of the nuclear envelope
from mitotic vesicles (Hetzer et al., 2000).
Our ®ndings and those of Eitzen et al. (2001) now
present a detailed kinetic analysis of Cdc42p function in
vacuole fusion, which allows us to map the requirement
for this protein within the reaction sequence leading to
fusion. To investigate Cdc42p function, two speci®c and
independent reagents were used. We used an af®nitypuri®ed antibody to Cdc42p, while Eitzen et al. (2001)
used Rdi1p, the RhoGDI that speci®cally extracts Cdc42p
from the membrane. The biochemical data for Cdc42p
involvement in membrane fusion were reinforced by data
on vacuole fusion in cdc42 temperature-sensitive mutants.
Eitzen et al. (2001) show in vitro and in vivo defects in
vacuole fusion in a cdc42ts-1 mutant and we demonstrate
fusion defects in a cdc42ts-123 mutant. This mutant
originated from a site-directed mutagenesis approach to
explore the functions and interactions of Cdc42p in
S.cerevisiae (Kozminski et al., 2000). Groups of charged
residues expected to be at the protein surface were mutated
and the mutants were tested for functional consequences.
This approach not only con®rmed the importance of
domains that were already predicted to be interaction sites,
but also de®ned a new putative protein interaction domain.
The R163A and K166A mutations in the cdc42ts-123
mutant, for which we now show defects in membrane
fusion, are in this new putative interaction domain. They
are in a region (a5-helix, residues 166±177) predicted by
structural models to be a speci®city determinant for
Cdc42p±protein interactions (Guo et al., 1998; AbdulManan et al., 1999; Mott et al., 1999; Kozminski et al.,
2000). The cdc42ts-124 mutant, which has mutations in
another interaction domain, is also temperature sensitive
for growth, but behaves like wild type in vacuole fusion.
The inhibitory antibody we used was raised against
residues 167±183 of Cdc42p, which contains the a5helix region. Thus, two independent approaches pointed to
the same domain. This strongly suggests that the new
putative interaction domain altered in the cdc42ts-123
mutant is responsible for Cdc42p action in membrane
fusion. This domain binds proteins with a CRIB (Cdc42/
Rac interactive binding) motif (Burbelo et al., 1995; Guo
et al., 1998; Abdul-Manan et al., 1999; Mott et al., 1999;
Kozminski et al., 2000). Yeast proteins with such a CRIB
motif are Cla4p, Gic1p, Gic2p and Ste20p (Burbelo et al.,
1995). Cla4p is a Cdc42p-regulated serine/threonine
kinase involved in the control of the actin cytoskeleton
(Holly and Blumer, 1999). A direct interaction of Cla4p
with the GTP-bound form of Cdc42p was shown by
overlay and two-hybrid assays (Cvrckova et al., 1995;
Richman et al., 1999). Strikingly, cla4 deletion mutants
have a phenotype of fragmented vacuoles (S.Seeley,
M.Kato, G.Eitzen and W.Wickner, personal communication), suggesting an involvement of Cla4p in vacuole
fusion.
Could the actin cytoskeleton play a role in vacuole
fusion and explain the function of Rho GTPases like
Cdc42p and Rho1p in this process? This is likely for
several reasons. First, actin is involved in the process of
vacuole fusion, as drugs that affect actin inhibit the in vitro
reaction (see Eitzen et al., 2001). Secondly, phosphatidylinositol 4,5-bisphosphate (PIP2) is required at two steps in
vacuole fusion, in priming and in the subsequent docking
reaction (Mayer et al., 2000). PIP2 is involved in the
regulation of Rho GTPase targets and in actin dynamics
(Higgs and Pollard, 2000; Kim et al., 2000; Prehoda et al.,
2000; Rohatgi et al., 2000). Thirdly, vacuole inheritance,
i.e. transmission of vacuolar fragments into the nascent
daughter cells of yeast, requires myosin-dependent transport of vacuoles along actin ®laments (Hill et al., 1996)
5661
O.MuÈller, D.I.Johnson and A.Mayer
and illustrates the capacity of vacuoles to bind actin. Actin
binding may involve Vac8p, an armadillo repeat protein
required for vacuole inheritance and fusion (Wang et al.,
1998, 2001; Veit et al., 2001). In other traf®cking
pathways Rho GTPases are believed to act by regulating
actin remodeling too. One possibility is that a layer of
F-actin might shield the membrane of organelles, making
it necessary to locally remove it by depolymerization
before a vesicle can dock or fuse (Orci et al., 1972; Trifaro
and Vitale, 1993; Vitale et al., 1995). In line with this
possibility, actin depolymerization was shown to stimulate
exocytosis in pancreatic acinar cells (Muallem et al.,
1995). However, the polymerization of actin was also
shown to facilitate phagosome fusion in vitro (Jahraus
et al., 2001). Thus, proper dynamics of dis- and reassembly of F-actin may be needed for fusion on intracellular compartments as well as for endo- (Ayscough,
2000) and exocytosis (Bernstein et al., 1998). F-actin may
also provide tracks for vesicles in endo- and exocytosis,
facilitating mechanical movement of the vesicles inside
cells (Govindan et al., 1995; Murphy et al., 1996; Lamaze
et al., 1997; Lang et al., 2000).
The role of Cdc42p in vacuole fusion, which we de®ne
here, might involve actin regulation, e.g. to support actindependent interaction of vesicles over longer distances.
This could facilitate clustering of vacuoles by transport
along actin ®laments and promote membrane tethering.
But this can not be the sole function of Cdc42p in fusion
because Cdc42p acts after Ypt7p, which mediates vacuole
tethering (Mayer and Wickner, 1997; Ungermann et al.,
1998a). Furthermore, priming, i.e. Sec18p/NSF-dependent
SNARE activation, must occur before the Cdc42p requirement can be satis®ed (see Eitzen et al., 2001). If Cdc42p
were just needed to facilitate non-speci®c actin-dependent
vacuole clustering by vesicle movement along polymerizing actin ®laments, it would be hard to understand why
Cdc42p is so tightly integrated into the reaction sequence.
In this case we would expect that the Cdc42p requirement
should precede Ypt7p-mediated tethering and be independent of Sec18p/NSF and priming. A further argument
against a pure clustering function is the fact that vacuoles
fuse well without the addition of cytosol (Mayer et al.,
1996). Therefore, an external reservoir of G-actin is not
required and a function for actin cables as vesicle tracks is
unlikely in our in vitro reaction.
The view of actin as a barrier to fusion, however, is
consistent with the data available so far. There is actin on
the surface of isolated vacuoles that polymerizes under
conditions of the in vitro reaction (P.Sluszarewicz, A.Merz
and W.Wickner, personal communication). Local actin
disassembly may be required to facilitate docking, i.e. a
close and tight apposition of the membranes involving
trans-SNARE pairing. An actin meshwork on the membranes would be strongly expected to interfere with such
close apposition. Due to the topology and dimensions of
the SNARE complex, its formation can be expected to
bring the membranes into a distance of ~2±3 nm (Sutton
et al., 1998), leaving barely any space for actin ®laments,
which have diameters of 5±9 nm (Holmes et al., 1990).
Finally, the available data still leave the possibility of actin
being an intrinsic component of the fusion machinery. The
exact role of actin in the fusion process remains to be
elucidated.
5662
Cdc42p could also be connected to the fusion machinery
in a more direct way. Its function in vacuole fusion could
be related to the Vtc complex. This complex regulates the
capacity of Sec18p/NSF to prime SNAREs and links it to
the late reaction step of trans-complex formation of V0
sectors of the vacuolar H+-ATPase (O.MuÈller, M.Bayer,
J.Anderson, M.Mann and A.Mayer, submitted). It is
striking that Vtc1p/Nrf1p, a subunit of the Vtc complex,
was isolated in a genetic screen as a regulator of Cdc42p
and co-localizes with Cdc42p to vacuolar membranes in
S.pombe (Murray and Johnson, 2000, 2001). In view of
these connections, it may also be relevant that mutations in
VMA5, the gene encoding subunit C of the vacuolar
H+-ATPase, show synthetic lethality with mutations in
CDC24 (White and Johnson, 1997), the GEF for Cdc42p.
The same study also suggested a role for Cdc24p in
vacuole function because one of the csl (CDC24 syntheticlethal) mutants displayed phenotypes similar to those
observed with calcium-sensitive, Pet± V-ATPase mutants
defective in vacuole function.
Thus, Cdc42p has several relations to proteins involved
in vacuole fusion, supporting our conclusion of a direct
role of Cdc42p in this reaction. New assays will have to be
developed to monitor actin dynamics in the fusion reaction
and explore the function of Rho proteins in detail.
Materials and methods
Strains
Standard fusion strains were Saccharomyces cerevisiae BJ3505 and
DKY6281 (Mayer et al., 1996). BJ3505Dvam3 and BJ3505Dnyv1 were
described previously (Nichols et al., 1997).
For the analysis of cdc42 ts mutants, strains DDY1300 (CDC42, wt),
DDY1336 (cdc42ts-123) and DDY1338 (cdc42ts-124) were used, which
were described previously (Kozminski et al., 2000). To make these strains
suitable for the in vitro fusion assay, either PEP4 or PHO8 was deleted by
replacing the gene with a loxP-kanMX-loxP cassette from plasmid pUG6
(GuÈldener et al., 1996). The resulting strains were OMY14 (CDC42
Dpep4), OMY15 (CDC42 Dpho8), OMY16 (cdc42ts-123 Dpep4), OMY17
(cdc42ts-123 Dpho8), OMY18 (cdc42ts-124 Dpep4) and OMY19 (cdc42ts124 Dpho8). The oligonucleotides used for generation of deletion
cassettes by PCR were: pep4.5¢.Del.kan, 5¢-TGGTCAGCGCCAACCAAGTTGCTGCAAAAGTCCACAAGGCCAGCTGAAGCTTCGTACGC-3¢; pep4.3¢.Del.kan, 5¢-AATCGTAAATAGAATAGTATTTACGCAAGAAGGCATCACCGCATAGGCCACTAGTGGATCTG-3¢; pho8.5¢.Del.kan, 5¢-GGCTACATAAACATTTACATATCAGCATACGGGACATTATCAGCTGAAGCTTCGTACGC-3¢; and pho8.3¢.Del.kan,
5¢-CGTATTAAATAATATGTGAAAAAAGAGGGAGAGTTAGATAGCATAGGCCACTAGTGGATCTG-3¢. Deletions were veri®ed by
western blotting.
General procedures
Vacuole isolation, preparation of cytosol, Gdi1p, inhibitory antibodies
and other reagents, as well as fusion assays, were essentially performed as
described (Peters et al., 1999). PS buffer is 10 mM PIPES±KOH pH 6.8,
200 mM sorbitol. 13 PIC is a protease inhibitor cocktail: 100 mM
pefabloc SC, 100 ng/ml leupeptin, 50 mM o-phenanthroline, 500 ng/ml
pepstatin A.
GST±Cdc42p and af®nity puri®cation of antibodies
The plasmid pGEX-Cdc42 (M.Ziman and D.I.Johnson, unpublished
results), which encodes a GST±Cdc42p fusion protein for expression in
Escherichia coli, was generated by inserting the CDC42 coding region on
a blunt-ended NdeI fragment into HindIII-cleaved, blunt-ended pGEXKG (Guan and Dixon, 1991). Escherichia coli BL21 cells containing
pGEX-Cdc42 were grown in LB/ampicillin medium (37°C, 225 r.p.m.) to
an OD600 of ~0.6. Expression was induced with 1 mM isopropylb-D-thiogalactopyranoside at 27°C for 4 h. Cells were broken by a
freeze±thaw cycle and soni®cation in buffer A [20 mM Tris±HCl pH 6.8,
150 mM KCl, 1 mM phenylmethylsulfonyl ¯uoride (PMSF), 13 PIC].
Cdc42p in yeast vacuole membrane fusion
Triton X-100 was added to a ®nal concentration of 1% (w/v) and the
lysate was mixed for 30 min at 4°C to complete solubilization. The lysate
was cleared (100 000 g, 20 min, 4°C) and the supernatant was repeatedly
passed over 2 ml of GSH±Sepharose (Pharmacia) in a column for 4 h at
4°C. The column was washed with 20 column vols of buffer B (20 mM
Tris±HCl pH 6.8, 150 mM NaCl). The fusion protein was cleaved on the
column by adding thrombin (Pharmacia; 25 U in 1 ml of buffer B) at 4°C
for 4 h. The eluate was diluted with 20 mM Tris±HCl pH 6.8 to a ®nal KCl
concentration of 50 mM and run over a MiniQ column (PE 4.6/50;
Pharmacia; 0.5 ml/min) pre-equilibrated with buffer C (20 mM Tris±HCl
pH 6.8, 50 mM KCl). Under these conditions, all contaminating proteins
bound to the column while the pure, cleaved Cdc42p was in the ¯ow
through. The protein was dialyzed into PS buffer (10 mM PIPES±KOH
pH 6.8, 200 mM sorbitol) containing 150 mM KCl, concentrated by
ultra®ltration in centricon-10 (Amicon), and stored at ±80°C.
Antibodies were af®nity puri®ed with the recombinant protein
immobilized on activated CH-Sepharose 4B (Pharmacia) in coupling
buffer (0.1 M NaHCO3 pH 8.0, 0.5 M NaCl). Remaining active groups
were blocked with 0.1 M Tris±HCl pH 8.0. Antibodies were eluted with
0.1 M glycine pH 2.75/150 mM NaCl and the pH was immediately
neutralized by addition of 1 M Tris±HCl pH 8.8. The antibodies were
dialyzed into PS buffer (10 mM PIPES±KOH pH 6.8, 200 mM sorbitol)
containing 150 mM KCl, concentrated by ultra®ltration in centricon-30
(Amicon), and stored at ±20°C.
Assay for trans-SNARE pairing
Trans-SNARE pairing was analyzed with a modi®ed method according to
Ungermann et al. (1998a). Five standard fusion reactions with cytosol
containing 20 mg of vacuoles from strain BJ3505Dvam3 and 20 mg of
vacuoles from BJ3505Dnyv1 and 10 mM microcystin LR, were incubated
without ATP-regenerating system on ice or with ATP at 27°C. After
45 min, the reactions were chilled on ice and centrifuged (20 000 g, 5 min,
4°C). The vacuole pellet was solubilized in 300 ml of buffer A (0.5%
Triton X-100, 2 mM EDTA, 1 mM PMSF in phosphate-buffered saline)
and incubated for 10 min at 4°C on a mixer. The lysate was cleared
(100 000 g, 10 min, 4°C) and 5% of the lysate was extracted with
chloroform/methanol (Wessel and FluÈgge, 1984) to serve as an input
control. The rest of the lysate was rotated for 45 min at 4°C with 10 mg of
af®nity-puri®ed antibodies to Vam3p and 20 ml of protein A±Sepharose
(Pharmacia). The beads were washed four times with 1 ml of buffer A,
transferred to a fresh tube and eluted with non-reducing sample buffer.
Proteins were resolved by SDS±PAGE and analyzed by western blotting
for immunoprecipitated Vam3p and co-immunoprecipitated Nyv1p.
Measurement of Ca2+ ef¯ux
Ca2+ release from vacuoles in the course of the fusion reaction was
assayed with a modi®ed method according to Peters and Mayer (1998).
Ca2+ was directly measured with aequorin in ongoing fusion reactions
running in parallel in a microtiter plate luminometer (EG&G Berthold,
Germany). Reactions (30 ml) contained 10 mg of vacuoles from strain
BJ3505 in PS buffer with 150 mM KCl and 0.5 ml of cytosol (30 mg/ml).
Respective inhibitors were included. After 2 min of pre-incubation on ice,
2 ml of ATP-regenerating system (same composition as in the fusion
assay) and 3 ml of a freshly prepared 2:2:1 mix of the following stock
solutions were added: 2.5 mM aequorin (Aqualite, Molecular Probes),
1 mM EGTA±KOH pH 7.5 and 500 mM native coelenterazine (Molecular
Probes) in EtOH. Samples were pipetted into a microtiter plate and light
emission of aequorin was measured with the luminometer for 60 min at
27°C. The assay was calibrated with solutions of different calcium
concentrations as described (Peters and Mayer, 1998).
Acknowledgements
We thank Susanne BuÈhler, Tanja Baader, Christa Baradoy and Kurt
Toenjes for assistance, and David Drubin for providing strains. This work
was supported by the Deutsche Forschungsgemeinschaft (SFB446,
A.M.), the Boehringer-Ingelheim-Foundation (A.M.), the BoehringerIngelheim-Fonds (O.M.) and National Science Foundation grants
MCB-9728218 and MCB-0076826 (D.I.J.).
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Received July 12, 2001; revised and accepted August 20, 2001
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