T H E J O U R N A L O F C E L L B I O L O G Y
The Journal of Cell Biology, Vol. 169, No. 3, May 9, 2005 383–389
The Rockefeller University Press$8.00
Coatomer-bound Cdc42 regulates dynein recruitment
to COPI vesicles
Raymond V. Fucini,
and Mark Stamnes
Department of Physiology and Biophysics, Roy J. and Lucille A. Carver College of Medicine, The University of Iowa, Iowa City, IA 52242
Molecular Biology Program Memorial Sloan-Kettering Cancer Center, New York, NY 10021
ytoskeletal dynamics at the Golgi apparatus are
regulated in part through a binding interaction
between the Golgi-vesicle coat protein, coatomer,
and the regulatory GTP-binding protein Cdc42 (Wu,
W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000.
Nature . 405:800–804; Fucini, R.V., J.L. Chen, C. Sharma,
M.M. Kessels, and M. Stamnes. 2002.
13:621–631). The precise role of this complex has not been
determined. We have analyzed the protein composition
of Golgi-derived coat protomer I (COPI)–coated vesicles
after activating or inhibiting signaling through coatomer-
bound Cdc42. We show that Cdc42 has profound effects
on the recruitment of dynein to COPI vesicles. Cdc42,
Mol. Biol. Cell.
when bound to coatomer, inhibits dynein binding to
COPI vesicles whereas preventing the coatomer–Cdc42
interaction stimulates dynein binding. Dynein recruitment
was found to involve actin dynamics and dynactin. Reclus-
tering of nocodazole-dispersed Golgi stacks and micro-
tubule/dynein-dependent ER-to-Golgi transport are both
sensitive to disrupting Cdc42 mediated signaling. By
contrast, dynein-independent transport to the Golgi
complex is insensitive to mutant Cdc42. We propose a
model for how proper temporal regulation of motor-
based vesicle translocation could be coupled to the
completion of vesicle formation.
The secretory and endocytic pathways rely on molecular
motors and the cytoskeleton for the directed motility of trans-
port intermediates (Allan et al., 2002; Engqvist-Goldstein and
Drubin, 2003). The efficient use of actively motile vesicles
must involve precise temporal and spatial regulation. For
example, premature action of motor proteins could lead to the
translocation of incompletely assembled transport vesicles.
Delayed action of motors could lead to the accumulation of
nascent vesicles at their sites of assembly. Cytoskeletal proteins
interact with vesicle coat proteins or cargo proteins (Stamnes,
2002). Characterization of these interactions may help explain
how motor proteins and cytoskeletal dynamics are connected
to vesicular transport.
The Golgi complex is typically a compact structure near
the microtubule organizing center (MTOC). Because of its
limited distribution, trafficking to and from the Golgi may be
especially dependent on the cytoskeleton. All three classes of
motor proteins, dynein, kinesin and myosin, have been implicated
in Golgi transport or positioning (Allan et al., 2002). Dynein is
especially important for transport to the Golgi apparatus and for
Golgi positioning at the MTOC (Thyberg and Moskalewski,
1999). Dynein and its adaptor, dynactin, have been found to
associate with Golgi- and ER-derived vesicles (Fath et al.,
1997; Watson et al., 2005).
Actin dynamics are also important for Golgi transport
and morphology (Fucini et al., 2002; Luna et al., 2002; Duran
et al., 2003; Carreno et al., 2004; Chen et al., 2004; Matas et
al., 2004). The GTP-binding protein, Cdc42, regulates actin
dynamics and is linked to several transport steps (Cerione,
2004). Cdc42 function at the Golgi is mediated through a binding
interaction with the coat protomer I (COPI)–vesicle coat
protein, coatomer (Wu et al., 2000; Fucini et al., 2002; Chen et
al., 2004). A COOH-terminal dilysine motif on the putative
cargo receptor, p23, competes with Cdc42 for a binding site on
-COP subunit of coatomer. This suggests a link between
COPI vesicle assembly, cargo packaging, and the regulation of
ADP-ribosylation factor (ARF) stimulates Arp2/3-depen-
dent actin polymerization on Golgi membranes by recruiting
coatomer and Cdc42 (Fucini et al., 2002; Chen et al., 2004).
Cdc42 signaling leads to the assembly of a specific pool of actin
that can be defined by its toxin sensitivity and by its specific
Correspondence to Mark Stamnes: email@example.com
Abbreviations used in this paper: ARF, ADP-ribosylation factor; COPI, coat
protomer I; endoH, endoglycosidase H; IC, intermediate chain; MTOC, micro-
tubule organizing center; VTC, vesiculotubular cluster.
The online version of this article contains supplemental material.
JCB • VOLUME 169 • NUMBER 3 • 2005384
association with the actin-binding protein mAbp1 (Fucini et al.,
2002). We report the unexpected finding that Cdc42 regulates
the recruitment of the microtubule-dependent motor protein,
dynein, to coatomer-coated membranes.
Results and discussion
Dynein is a Cdc42-sensitive component
of Golgi vesicles
We analyzed the consequences of blocking Cdc42 recruitment
to budding COPI vesicles using a peptide corresponding to the
coatomer-binding motif of p23 (Wu et al., 2000; Fucini et al.,
2002; Chen et al., 2004). Activating ARF with GTP
cell-free Golgi-vesicle budding assay leads to COPI vesicle
formation as indicated by the presence of coatomer in a vesi-
cle-enriched fraction (Fig. 1, A and B). GTP
Cdc42 causing it to appear in the vesicle fraction (Fig. 1 B). As
expected, the p23 peptide prevented Cdc42 from binding
coatomer and appearing with the vesicles (Fig. 1 B). The levels
of a high molecular weight protein was greatly increased in the
presence of the peptide (Fig. 1 A). Subsequent analysis of tryp-
tic fragments by mass spectrometry led to the identification of
this protein as the heavy chain subunit of dynein.
Dynein is a multimeric microtubule-based motor pro-
tein composed of at least two heavy chains bound to interme-
diate chain (IC) and light chain subunits. Western blots of the
Golgi-vesicle fractions confirmed that the dynein IC levels
are increased when the coatomer–Cdc42 complex is dis-
rupted by the p23 peptide (Fig. 1 B). A control peptide had
no effect on dynein levels (unpublished data). Because both
dynein heavy chain (Fig. 1 A) and IC (Fig. 1 B) behave simi-
larly, it is likely that the entire dynein complex is present in
this vesicle fraction.
Because the p23 peptide might have multiple effects, we
used an independent approach to test the consequences of acti-
vating vesicle formation in the absence of coatomer-bound
Cdc42. Constitutively active recombinant ARF1(Q71L) pro-
moted coatomer assembly on the Golgi membranes, but did not
lead to Cdc42 activation or recruitment (Fig. 1 C). Importantly,
ARF1(Q71L) also stimulated the recruitment of dynein. Thus,
two separate methods for stimulating coatomer assembly with-
out Cdc42 led to increased dynein levels. The results indicate
that coatomer-bound Cdc42 negatively regulates dynein bind-
ing to the vesicles.
S in a
S also activates
Recombinant Cdc42 blocks dynein
Multiple GTP-binding proteins are candidates to function in
the secretory pathway or in motor protein recruitment (Ham-
mer and Wu, 2002; Symons and Rusk, 2003). To test whether
Cdc42 is sufficient to mediate the effects of GTP
nein recruitment, we examined the effects of adding recombi-
nant Cdc42 to the vesicle budding reaction in addition to
ARF1(Q71L) (Fig. 2, A and B). Addition of the constitutively
active Cdc42(Q61L) greatly decreased the amount of dynein in
the vesicle fraction. Cdc42(Q61L) and GTP
reduce dynein levels to the same extent (Fig. 2 A) indicating
S on dy-
S were found to
that Cdc42 is the predominant GTP-binding protein that nega-
tively regulates dynein recruitment in the budding reaction.
We tested whether the binding interaction between co-
atomer and Cdc42 is important by using a mutant form of
Cdc42(Q61Lss) that is constitutively GTP-bound, activates
multiple Cdc42 effectors, but fails to bind coatomer (Wu et al.,
2000). Cdc42(Q61Lss) had no effect on dynein levels (Fig.
2 B), confirming a requirement for the binding interaction
with coatomer. Cdc42(Q61L) is a GTPase-deficient mutant,
whereas a second active form of Cdc42, (F28L), can complete
the GTP binding/hydrolysis cycle. Cdc42(F28L) stimulates ER
to Golgi transport whereas Cdc42(Q61L) blocks it (Wu et al.,
2000). Thus, we anticipated that these two active forms of
Cdc42 might have opposing effects on dynein recruitment to
membranes. Indeed, Cdc42(F28L) did not affect dynein levels
on the vesicles (Fig. 2 B).
We determined whether interfering with Cdc42 signaling
had any effect on dynein distribution in whole cells. Cytoplas-
mic dynein is normally found in dispersed punctate structures
that are concentrated in the perinuclear region (Fig. 2 C). In
cells expressing Cdc42(Q61L), the perinuclear dynein becomes
more diffuse or reduced and there is an increase in cell-surface
dynein (Fig. 2 C). The ability of recombinant Cdc42(Q61L) to
inhibit dynein binding and alter dynein distribution in cells
(A) Shown is a Coomassie blue–stained gel of Golgi vesicle-enriched fractions
obtained from budding incubations performed in the presence of 20 ?M
GTP?S with or without 250 ?M p23 peptide as indicated. The identities of
the coatomer subunits, ?-COP, ?-COP, ??-COP, and ?-COP were confirmed
by Western blotting (not depicted). (B) Immunoblots of the vesicle-enriched
fractions were probed with the indicated antibodies. (C) Shown is a blot of
Golgi-binding assays performed in the presence of 20 ?M GTP?S or 25
?g/ml of recombinant ARF1(Q71L) and probed as indicated.
Dynein is present on Golgi vesicles assembled without Cdc42.
CDC42 REGULATES DYNEIN ON GOLGI VESICLES • CHEN ET AL. 385
supports the model that the coatomer–Cdc42 complex regu-
lates dynein binding to membranes.
Dynein recruitment is sensitive to actin
Cdc42 could affect dynein recruitment by regulating Arp2/3-
dependent actin polymerization or alternatively through a par-
allel effector that is independent of actin dynamics. We used
the actin toxin, cytochalasin D, to test whether disrupting actin
dynamics affected dynein binding to the membrane. We found
that cytochalasin D stimulated dynein binding but only at con-
centrations around 10–20
g/ml (Fig. 2 D). In this range, over-
all levels of bound actin were only moderately affected (Fig. 2
D, inset). The sensitivity of dynein recruitment to low levels of
actin toxins suggests that Cdc42 regulates dynein through a
specific toxin-sensitive actin-based structure.
Because a binding interaction between spectrin and the
dynactin complex has been implicated in dynein association
with membranes (De Matteis and Morrow, 2000; Holleran et
al., 2001), we hypothesized that high levels of cytochalasin D
caused a more general disruption of actin and spectrin on the
Golgi membrane that precluded dynein binding (Fig. 2 D). Dy-
nactin has also been shown to associate with ER-derived
COPII-coated vesicles (Watson et al., 2005). Thus, we investi-
gated whether dynactin is involved in dynein binding to the
Golgi membranes. The levels of the dynactin subunit, p150
also increased when ARF(Q71L) is added to the Golgi-binding
assay (Fig. 2 E). Tubulin levels on the Golgi are not affected by
ARF1(Q71L) suggesting that dynein is not recruited through
changes in microtubule dynamics (Fig. 2 E). Together, our re-
sults indicate that Cdc42-mediated changes in actin dynamics
regulate dynactin-based recruitment of dynein motors to the
Dynein associates with COPI vesicles in
vitro and in whole cells
Although dynein associates with budding vesicles on the Golgi
(Fath et al., 1997), it has not been implicated directly in COPI-
vesicle–mediated trafficking. Hence, we tested whether COPI
vesicles cofractionate with dynein upon flotation through an
isopycnic sucrose gradient (Fig. 3 A). COPI vesicles fractionate
with a buoyant density equivalent to
5–8). Addition of either GTP
S or ARF1(Q71L) activates COPI
vesicle formation as indicated by the presence of coatomer in the
center of the gradient (Fig. 3 A and Fig. S1, available at http://
www.jcb.org/cgi/content/full/jcb.200501157/DC1). Dynein co-
fractionates with COPI vesicles when vesicle formation is acti-
vated by ARF1(Q71L). However, when ARF and Cdc42 are ac-
tivated simultaneously with GTP
bottom load fractions.
As an additional test that dynein is associated with COPI
vesicles, as opposed to another vesicle type with a similar den-
sity, we performed immuno-isolation experiments with either
anti-dynein (Fig. 3 B) or anti-coatomer (
(Fig. 3 C). Coprecipitation was observed when ARF1(Q71L)
was used to activate vesicle budding but not with GTP
imentation of vesicles at high speed (Fig. 3 B) or precipitation
-COP (Fig. 3 C) confirmed that vesicle recovery was simi-
lar whether ARF1(Q71L) or GTP
provide strong evidence that COPI vesicles associate with dy-
42% sucrose (fractions
S, dynein remains in the
S was included. The results
ization. (A) A Western blot of COPI-vesicle–
enriched fractions isolated by flotation from
Golgi-budding reactions was probed with
the indicated antibodies. Incubations were
performed in the presence of 25 ?g/ml
ARF1(Q71L), 20 ?g/ml Cdc42(Q61L), and
GTP?S as indicated. (B) Plotted are the aver-
age levels of dynein and coatomer found in
the COPI-vesicle enriched fraction isolated as
in A. 20 ?g/ml of recombinant mutant Cdc42
proteins were added as indicated. The error
bars represent the SEM (n ? 3). (C) NRK cells
that had been transfected (asterisk) with GFP-
Cdc42(Q61L) (inset image) were labeled with
an antibody against the dynein light chain
(red). Bars, 20 ?m. (D) Golgi-binding assays
were used to determine the levels of bound
dynein and actin (inset, graph) at various con-
centrations of cytochalasin D. The error bars
represent the SEM (n ? 3). (E) A Golgi-binding
assay were performed adding ARF1(Q71L)
when indicated and probed with the indicated
Cdc42 and actin affect dynein local-
JCB • VOLUME 169 • NUMBER 3 • 2005386
nein predominantly when vesicle formation is activated by
To determine whether COPI vesicles associate with dy-
nein in cells, we characterized the localization of coatomer and
dynein within Vero cells using immunoelectron microscopy.
Fig. 3 (D and E) shows examples of vesicle profiles that are
positively labeled for both coatomer and dynein. 61% (48/79)
of small 50–150-nm circular or pleiomorphic profiles that were
decorated with two or more large gold particles (coatomer) also
contained at least two small gold particles (dynein). Many
structures were observed that were decorated only with the
anti-dynein antibody (Fig. 3, D and E), which is consistent with
dynein’s role in the motility of a variety of structures. The re-
sults provide strong evidence that COPI vesicles interact with
dynein both in vitro and in whole cells.
Golgi clustering is disrupted by mutant
Microtubules play an important role in Golgi localization
within cells (Thyberg and Moskalewski, 1999). Depolymeriz-
ing microtubules with nocodazole leads to a dramatic dispersal
of Golgi membranes. When nocodazole is removed, microtu-
bules repolymerize and the dispersed Golgi recluster at the jux-
tanuclear MTOC via dynein-based translocation (Ho et al.,
1989; Corthesy-Theulaz et al., 1992; Hafezparast et al., 2003).
Because this processes involves dynein-based motility of co-
atomer-coated structures, we tested whether mutant Cdc42 af-
fected it (Fig. 4).
budding reactions performed with GTP?S or ARF1(Q71L) were fractionated
on a sucrose gradient. Shown are immunoblots of the fractions probed as
indicated. (B) Shown is a blot of proteins precipitated with the anti-dynein IC
antibody from a vesicle extract. Coatomer levels were determined using
anti–?-COP and anti–?-COP. Dynein levels were inferred using anti-
p150glued. The blot on the left indicates the total amount of COPI vesicles in
the extract isolated by sedimentation. (C) Vesicles were precipitated with
the anti–?-COP antibody as in B. The amounts of coatomer and dynein
were determined by probing immunoblots with the appropriate antibodies.
(D and E) Cryosections were taken from Vero cells and decorated with anti–
?-COP, large gold particles, and anti-dynein IC, small gold particles. The
large arrows indicate structures labeled with both antibodies and the small
arrows indicate structures labeled only with anti-dynein. Bar, 300 nm.
Dynein is recruited to COPI vesicles. (A) Vesicle extracts from
(A) NRK cells were transfected with a plasmid for the expression of myc-
Cdc42(Q61L). The cells were treated with nocodazole and washed for
the indicated times. The Golgi were labeled using an anti-GM130 anti-
body (red). Transfected cells (asterisks) were identified using an anti-myc
antibody (green). (B) Before nocodazole treatment, NRK cells were trans-
fected (asterisks) with HA–wild-type Cdc42 (WT), myc-Cdc42(Q61L), or
HA-Cdc42(F28L) as indicated (green). The cells were allowed to recover
for 60 min after the nocodazole washout as in A. The Golgi apparatus
was labeled with anti-GM130 (red). Bar, 10 ?m.
Reclustering of Golgi membranes is sensitive to Cdc42 function.
CDC42 REGULATES DYNEIN ON GOLGI VESICLES • CHEN ET AL. 387
We confirmed that Golgi membranes dispersed in NRK
cells after a 2-h incubation with nocodazole (Fig. 4 A). In non-
transfected cells, normal Golgi morphology was largely re-
stored within 1 h after washing out the nocodazole (Fig. 4, A
and B). In the presence of Cdc42(Q61L), the dispersed Golgi
membranes coalesced into larger punctate structures, but they
were not translocated back toward the nucleus (Fig. 4, A and
B). Neither wild-type Cdc42 nor Cdc42(F28L) expression had
any detectable effect on the ability of Golgi stacks to recluster
in a juxtanuclear region (Fig. 4 B). When scored blindly, 79%
(49/62) of Cdc42(Q61L)-transfected cells had dispersed Golgi
membranes after the 1-h washout. By contrast, only 14% (9/66)
of wild-type Cdc42-transfected cells, 16% (10/61) of the
Cdc42(F28L)-transfected cells, and 9% (8/88) of nontrans-
fected cells displayed dispersed Golgi. This is consistent with
the results presented above indicating the Cdc42(Q61L) specif-
ically disrupts dynein recruitment.
Cdc42 affects ER-Golgi transport only in
cells using dynein-mediated motility
Molecular motor proteins and actin dynamics may play multi-
ple roles in trafficking between the Golgi and the ER (Allan et
al., 2002; Luna et al., 2002; Matanis et al., 2002; Short et al.,
2002; Stamnes, 2002; Duran et al., 2003). The translocation of
vesiculotubular clusters (VTCs) from ER exit sites to the juxta-
nuclear Golgi complex is a trafficking step where coatomer
(Aridor et al., 1995; Scales et al., 1997; Stephens et al., 2000;
Presley et al., 2002), Cdc42 (Wu et al., 2000; Fucini et al.,
2002), and dynein (Burkhardt et al., 1997; Presley et al., 1997)
have each been implicated. As with Golgi positioning (Fig. 4),
we anticipated that Cdc42 might regulate the dynein-based mo-
tility of coatomer-coated VTCs. Cdc42(Q61L) expression led
to an increase in non-Golgi processed (endoglycosidase H [en-
doH]-sensitive) VSVG(ts045) after release from the ER (Fig. 5
A and Fig. S2, available at http://www.jcb.org/cgi/content/full/
jcb.200501157/DC1) confirming the previous studies (Wu et
al., 2000; Fucini et al., 2002). In Cdc42(Q61L)-transfected
cells, VSVG appeared to be adjacent to SEC23-positive ER
exit sites (Fig. S3 A, available at http://www.jcb.org/cgi/
content/full/jcb.200501157/DC1). The results indicate that
Cdc42(Q61L) expression leads to immobile VTCs.
Although translocation of VTCs to the Golgi normally re-
lies on dynein and microtubules, cells that have adapted to no-
codazole resume ER to Golgi transport in a microtubule-inde-
pendent manner (Thyberg and Moskalewski, 1999). In this
case, the Golgi membranes are found dispersed throughout the
cell near ER-exit sites (Fig. S3 B). We found that endoH-sensi-
tive VSVG was not observed in nocodazole-adapted cells ex-
pressing Cdc42(Q61L) indicating that ER-to-Golgi transport
was no longer compromised (Fig. 5 A and Fig. S2). VSVG
colocalized with the dispersed Golgi compartments in the
Cdc42(Q61L)-transfected nocodazole-adapted cells (Fig. 5 B).
VSVG and Golgi markers did not colocalize in Cdc42(Q61L)
expressing cells without nocodazole. When another inhibitor,
BAPTA-AM, is used to block ER-to-Golgi transport (Ahluwa-
lia et al., 2001; Chen et al., 2002), VSVG is not transported to
the Golgi even after adaptation to nocodazole (Fig. 5 C). These
results suggest that Cdc42 signaling in the early secretory path-
way is especially important during dynein-mediated transloca-
tion of the VTCs.
The regulation of dynein-based vesicle
Our data suggest a model through which temporal regulation of
dynein motors could be connected to transport vesicle coat as-
sembly and cargo packaging. Early in vesicle formation, when
cargo proteins are not yet concentrated, coatomer would be as-
sociated with Cdc42 thereby stimulating actin assembly but in-
hibiting dynein binding. As coatomer binds cargo proteins such
as p23 and vesicle formation is completed, Cdc42 would disso-
ciate from coatomer, halting actin polymerization and allowing
dynein recruitment and motility. Given the emerging con-
nections between vesicle formation and cytoskeletal function
(Stamnes, 2002), it seems likely that related regulatory pro-
cesses may be used by various trafficking steps within the cell.
Materials and methods
Rat liver Golgi membranes and bovine-brain cytosol were isolated as de-
scribed previously (Malhotra et al., 1989). Recombinant myristoylated
ARF1(Q71L) was obtained by expression in
2001). Nocodazole and anti–
-tubulin were obtained from Sigma-Aldrich.
The following antibodies were used: anti-dynein IC 74.1 (Covance), anti-
dynein LC E16 (Santa Cruz Biotechnology, Inc.), anti-p150
sciences), and anti-GM130 (BD Biosciences). All other antibodies and ma-
terials were obtained as described previously (Fucini et al., 2000, 2002).
E. coli (Ahluwalia et al.,
Golgi membrane binding and budding reactions
Golgi membrane binding and budding reactions were performed as de-
scribed previously (Fucini et al., 2000, 2002; Chen et al., 2004). For the
binding assays, the Golgi membranes were reisolated from the reaction by
flotation (Fucini et al., 2000). In Fig. 1 (A and B), vesicles were enriched
from the high salt supernatant by sedimentation through a sucrose cushion
(Fucini et al., 2000) in order to facilitate large scale reactions needed for
cells were cotransfected with vectors expressing GFP-VSVG(ts045) and
myc-tagged Cdc42(Q61L). VSVG was accumulated in the ER. Where indi-
cated, 20 ?M nocodazole was added for 6 h before incubating at 32?C
for 15 min. The cells were lysed and digested with endoglycosidase H (En-
doH) where indicated. VSVG levels were determined by Western blotting.
(B) Cells expressing Cdc42(Q61L) and GFP-VSVG(ts045) were treated
as in A then decorated with antiGM130. (C) Cells expressing GFP-
VSVG(ts045) were treated with nocodazole (for 6 h) and either BAPTA-AM
or DMSO (for 1 h) before the shift to 32?C for 15 min. The amount of
endoH-sensitive VSVG was determined by Western blotting.
Translocation of VTCs is sensitive to Cdc42 function. (A) Vero
JCB • VOLUME 169 • NUMBER 3 • 2005388
identification. In all other cases, vesicles were purified by fractionating the
high salt supernatant on a 5-ml 30–50% sucrose isopycnic gradient (Mal-
hotra et al., 1989; Serafini et al., 1991; Ostermann et al., 1993). Frac-
tions were recovered from the top and analyzed by Western blotting.
Where indicated Western blot signals were quantified by densitometry.
Immuno-isolation of COPI vesicles
Budding incubations were performed as described above. The high salt
supernatant containing vesicles was diluted twofold in 25 mM Hepes, pH
7.4, 2.5 mM magnesium acetate. Anti-dynein IC 74.1 or anti–
body was used to saturate protein G agarose (Invitrogen). 20
were added to 200
l of supernatant and incubated for 1 h at 4
mixing. After washing three times in 25 mM Hepes, pH 7.4, 0.5 mM
EDTA, 50 mM KCl, the beads were collected and the bound proteins were
analyzed by Western blot.
l of beads
Vero cells were fixed with 3% PFA plus 0.1% glutaraldehyde in 0.1 M
PBS, pH 7.4. The cells were washed, pelleted in 9% gelatin in PBS, and
infiltrated with 2.3 M sucrose in PBS. The pellet was frozen in liquid nitro-
gen and sectioned with a cryomicrotome. Thin sections, collected on
grids, were blocked in 5% goat serum and incubated at RT for 60 min
with a mixture of the primary antibodies (anti-dynein and anti–
grids were washed in PBS and immunolabeled with anti–mouse and anti–
rabbit IgG coupled to gold (Electron Microscopy Sciences). Grids were
washed, embedded in 0.3% uranyl acetate in 2% methyl cellulose, and
examined using a microscope (model H-7000; Hitachi).
Transfection and Golgi clustering assay
NRK cells were transfected using lipofectamine (Invitrogen) with plasmids
expressing wild-type Cdc42, Cdc42(Q61L), or Cdc42(F28L). For the
Golgi clustering assay, NRK cells were exposed to nocodazole (20
C for at least 2 h to scatter the Golgi. The cells were then washed
-MEM media and incubated without nocodazole for the indicated
times. Immunofluorescence was performed as described previously (Fucini
et al., 2002). Images were acquired using a confocal microscope (model
LSM-510; Carl Zeiss MicroImaging, Inc.) and a 63
MicroImaging, Inc.) with an NA of 1.40.
objective (Carl Zeiss
GFP-VSVG(ts045) transport assay
Vero cells were cotransfected using lipofectamine (Invitrogen) with GFP-
ts045-VSVG (Presley et al., 1997) and the Cdc42(Q61L) plasmid. Trans-
fected cells were incubated for 14–16 h at the restrictive temperature
C) to accumulate VSVG protein in the ER. VSVG protein was re-
leased from the temperature block by switching the cells to 32
g/ml cycloheximide. The cells were incubated for 15 min
C and lysed for Western blot analysis or processed for immunofluo-
rescence. Nocodazole or BAPTA-AM was added at the indicated times by
replacing the medium with medium containing 20
M BAPTA-AM plus 10
g/ml cycloheximide. The treatment of VSVG pro-
tein with endoH (Calbiochem) was done according to the manufacturer’s
M nocodazole or 50
A vesicle-enriched pellet was obtained as described above from budding
reactions performed in the presence of cytochalasin D. The gel-resolved
high molecular weight band was digested with trypsin and the mixture
was fractionated on a Poros 50 R2 RP micro-tip (Erdjument-Bromage et al.,
1998). Resulting peptide pools were then analyzed by matrix-assisted la-
ser desorption/ionization reflectron time-of-flight mass spectrometry using
a Reflex III instrument obtained from Bruker Daltonics. Selected mass val-
ues were taken to search the protein nonredundant database (National
Center for Biotechnology Information, Bethesda, MD) using the Pep-
tideSearch (Mann et al., 1993) algorithm.
Online supplemental material
Fig. S1 shows the quantification of coatomer and dynein levels in vesicle
fractions isolated by flotation as in Fig. 3 A. Fig. S2 shows the time course
of ER-to-Golgi VSVG transport in the presence of nocodazole and/or
Cdc42(Q61L). Fig. S3 shows the localization of VSVG and Golgi mem-
branes relative to ER exit sites. Online supplemental material is available
We thank J. Topp, C. Sharma, A. Navarrete, J. Shao, and R. Nessler for con-
tributions to this work. We are grateful to J. Ahluwalia, R. Piper, L. Weisman,
and M. Anderson for helpful discussions.
Support was received from the National Institutes of Health
(GM068674), American Cancer Society, and the Carver Charitable Trust (to
M. Stamnes), the American Heart Association Heartland Affiliate (to J.-L. Chen),
and a National Cancer Institute core grant P30 CA08748 (to P. Tempst).
Submitted: 31 January 2005
Accepted: 31 March 2005
Ahluwalia, J.P., J.D. Topp, K. Weirather, M. Zimmerman, and M. Stamnes.
2001. A role for calcium in stabilizing transport vesicle coats.
Allan, V.J., H.M. Thompson, and M.A. McNiven. 2002. Motoring around the
Nat. Cell Biol.
Aridor, M., S.I. Bannykh, T. Rowe, and W.E. Balch. 1995. Sequential coupling
between COPII and COPI vesicle coats in endoplasmic reticulum to
J. Cell Biol.
Burkhardt, J.K., C.J. Echeverri, T. Nilsson, and R.B. Vallee. 1997. Overexpres-
sion of the dynamitin (p50) subunit of the dynactin complex disrupts dy-
nein-dependent maintenance of membrane organelle distribution.
Carreno, S., A.E. Engqvist-Goldstein, C.X. Zhang, K.L. McDonald, and D.G.
Drubin. 2004. Actin dynamics coupled to clathrin-coated vesicle forma-
tion at the trans-Golgi network.
J. Cell Biol.
Cerione, R.A. 2004. Cdc42: new roads to travel.
Chen, J.L., J.P. Ahluwalia, and M. Stamnes. 2002. Selective effects of calcium
chelators on anterograde and retrograde protein transport in the cell.
Chen, J.L., L. Lacomis, H. Erdjument-Bromage, P. Tempst, and M. Stamnes.
2004. Cytosol-derived proteins are sufficient for Arp2/3 recruitment and
ARF/coatomer-dependent actin polymerization on Golgi membranes.
Corthesy-Theulaz, I., A. Pauloin, and S.R. Pfeffer. 1992. Cytoplasmic dynein
participates in the centrosomal localization of the Golgi complex.
De Matteis, M.A., and J.S. Morrow. 2000. Spectrin tethers and mesh in the bio-
J. Cell Sci.
Duran, J.M., F. Valderrama, S. Castel, J. Magdalena, M. Tomas, H. Hosoya, J.
Renau-Piqueras, V. Malhotra, and G. Egea. 2003. Myosin motors and
not actin comets are mediators of the actin-based Golgi-to-endoplasmic
reticulum protein transport.
Mol. Biol. Cell.
Engqvist-Goldstein, A.E., and D.G. Drubin. 2003. Actin assembly and endocy-
tosis: from yeast to mammals.
Annu. Rev. Cell Dev. Biol.
Erdjument-Bromage, H., M. Lui, L. Lacomis, A. Grewal, R.S. Annan, D.E. Mc-
Nulty, S.A. Carr, and P. Tempst. 1998. Examination of micro-tip re-
versed-phase liquid chromatographic extraction of peptide pools for
mass spectrometric analysis.
J. Chromatogr. A.
Fath, K.R., G.M. Trimbur, and D.R. Burgess. 1997. Molecular motors and a
spectrin matrix associate with Golgi membranes in vitro.
Fucini, R.V., A. Navarrete, C. Vadakkan, L. Lacomis, H. Erdjument-Bromage,
P. Tempst, and M. Stamnes. 2000. Activated ADP-ribosylation factor as-
sembles distinct pools of actin on Golgi membranes.
Fucini, R.V., J.L. Chen, C. Sharma, M.M. Kessels, and M. Stamnes. 2002.
Golgi vesicle proteins are linked to the assembly of an actin complex de-
fined by mAbp1.
Mol. Biol. Cell.
Hafezparast, M., R. Klocke, C. Ruhrberg, A. Marquardt, A. Ahmad-Annuar, S.
Bowen, G. Lalli, A.S. Witherden, H. Hummerich, S. Nicholson, et al.
2003. Mutations in dynein link motor neuron degeneration to defects in
retrograde transport. Science. 300:808–812.
Hammer, J.A., III, and X.S. Wu. 2002. Rabs grab motors: defining the connec-
tions between Rab GTPases and motor proteins. Curr. Opin. Cell Biol.
Ho, W.C., V.J. Allan, G. van Meer, E.G. Berger, and T.E. Kreis. 1989. Reclus-
tering of scattered Golgi elements occurs along microtubules. Eur. J.
Cell Biol. 48:250–263.
Holleran, E.A., L.A. Ligon, M. Tokito, M.C. Stankewich, J.S. Morrow, and E.L.
Holzbaur. 2001. beta III spectrin binds to the Arp1 subunit of dynactin.
J. Biol. Chem. 276:36598–36605.
Luna, A., O.B. Matas, J.A. Martinez-Menarguez, E. Mato, J.M. Duran, J. Bal-
lesta, M. Way, and G. Egea. 2002. Regulation of protein transport from the
Golgi complex to the endoplasmic reticulum by CDC42 and N-WASP.
Mol. Biol. Cell. 13:866–879.
Trends Cell Biol.
J. Cell Biol.
J. Biol. Chem.
CDC42 REGULATES DYNEIN ON GOLGI VESICLES • CHEN ET AL.389 Download full-text
Malhotra, V., T. Serafini, L. Orci, J.C. Shepherd, and J.E. Rothman. 1989. Puri-
fication of a novel class of coated vesicles mediating biosynthetic protein
transport through the Golgi stack. Cell. 58:329–336.
Mann, M., P. Hojrup, and P. Roepstorff. 1993. Use of mass spectrometric mo-
lecular weight information to identify proteins in sequence databases.
Biol. Mass Spectrom. 22:338–345.
Matanis, T., A. Akhmanova, P. Wulf, E. Del Nery, T. Weide, T. Stepanova, N.
Galjart, F. Grosveld, B. Goud, C.I. De Zeeuw, et al. 2002. Bicaudal-D
regulates COPI-independent Golgi-ER transport by recruiting the dy-
nein-dynactin motor complex. Nat. Cell Biol. 4:986–992.
Matas, O.B., J. Martinez-Menarguez, and G. Egea. 2004. Association of Cdc42/
N-WASP/Arp2/3 Signaling pathway with Golgi membranes. Traffic.
Ostermann, J., L. Orci, K. Tani, M. Amherdt, M. Ravazzola, Z. Elazar, and J.E.
Rothman. 1993. Stepwise assembly of functionally active transport vesi-
cles. Cell. 75:1015–1025.
Presley, J.F., N.B. Cole, T.A. Schroer, K. Hirschberg, K.J. Zaal, and J. Lippin-
cott-Schwartz. 1997. ER-to-Golgi transport visualized in living cells.
Presley, J.F., T.H. Ward, A.C. Pfeifer, E.D. Siggia, R.D. Phair, and J. Lippin-
cott-Schwartz. 2002. Dissection of COPI and Arf1 dynamics in vivo and
role in Golgi membrane transport. Nature. 417:187–193.
Scales, S.J., R. Pepperkok, and T.E. Kreis. 1997. Visualization of ER-to-Golgi
transport in living cells reveals a sequential mode of action for COPII
and COPI. Cell. 90:1137–1148.
Serafini, T., L. Orci, M. Amherdt, M. Brunner, R.A. Kahn, and J.E. Rothman.
1991. ADP-ribosylation factor is a subunit of the coat of Golgi-derived
COP-coated vesicles: a novel role for a GTP-binding protein. Cell. 67:
Short, B., C. Preisinger, J. Schaletzky, R. Kopajtich, and F.A. Barr. 2002. The
Rab6 GTPase regulates recruitment of the dynactin complex to Golgi
membranes. Curr. Biol. 12:1792–1795.
Stamnes, M. 2002. Regulating the actin cytoskeleton during vesicular transport.
Curr. Opin. Cell Biol. 14:428–433.
Stephens, D.J., N. Lin-Marq, A. Pagano, R. Pepperkok, and J.P. Paccaud. 2000.
COPI-coated ER-to-Golgi transport complexes segregate from COPII in
close proximity to ER exit sites. J. Cell Sci. 113:2177–2185.
Symons, M., and N. Rusk. 2003. Control of vesicular trafficking by Rho GTP-
ases. Curr. Biol. 13:R409–R418.
Thyberg, J., and S. Moskalewski. 1999. Role of microtubules in the organiza-
tion of the Golgi complex. Exp. Cell Res. 246:263–279.
Watson, P., R. Forster, K.J. Palmer, R. Pepperkok, and D.J. Stephens. 2005.
Coupling of ER exit to microtubules through direct interaction of COPII
with dynactin. Nat. Cell Biol. 7:48–55.
Wu, W.J., J.W. Erickson, R. Lin, and R.A. Cerione. 2000. The gamma-subunit
of the coatomer complex binds Cdc42 to mediate transformation. Na-