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J. Biol. Chem., Vol. 275, Issue 25, 18824-18829, June 23, 2000
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From the
Received for publication, January 5, 2000, and in revised form, February 29, 2000
The small GTP-binding protein ADP-ribosylation
factor (ARF) has been shown to regulate the interaction of actin and
actin-binding proteins with the Golgi apparatus. Here we report that
ARF activation stimulates the assembly of distinct pools of actin on
Golgi membranes. One pool of actin cofractionates with coatomer
(COPI)- coated vesicles and is sensitive to salt extraction and the
plus end actin-binding toxin cytochalasin D. A second
ARF-dependent actin pool remains on the Golgi membranes
following vesicle extraction and is insensitive to cytochalasin D. Isolation of the salt-extractable ARF-dependent actin from
the Golgi reveals that it is bound to a distinct repertoire of
actin-binding proteins. The two abundant actin-binding proteins of the
ARF-dependent actin complex are identified as spectrin and
drebrin. We show that drebrin is a specific component of the
cytochalasin D-sensitive, ARF-dependent actin pool on the
Golgi. Finally, we show that depolymerization of this actin pool with
cytochalasin D increases the extent of the salt-dependent
release of COPI-coated vesicles from the Golgi following cell-free
budding reactions. Together these data suggest that regulation of the
actin-based cytoskeleton may play an important role during ARF-mediated
transport vesicle assembly or release on the Golgi.
Extensive biochemical and genetic studies have allowed a detailed
description of the molecular events surrounding the assembly of coated
transport vesicles (1, 2). In particular, GTPases of the
ARF1 family act as molecular
switches to trigger the assembly of the coat proteins. The
ARF-dependent oligomerization of coat proteins has been
shown to be sufficient to deform the membrane into a vesicle bud (3,
4). Despite this progress, many details have yet to emerge regarding
the mechanisms of cargo packaging, the regulation of vesicle formation,
and the nature of interactions with molecular motors and other
cytoskeletal proteins that allow directed movement.
Although ARF is best characterized for its role in vesicle formation,
recent studies suggest it may serve additional functions in the cell.
One role for ARF may be to regulate the activity of the
phospholipid-modifying enzymes phospholipase D, phosphatidylinositol 4-kinase, and phosphatidylinositol 5-kinase (5-7). This regulation may
play a role in vesicle release and/or be part of signaling pathways not
directly related to transport. A second role for ARF may be in
directing the assembly of the actin-based cytoskeleton. ARF has been
shown to regulate the binding of spectrin, ankyrin, and actin to the
Golgi membranes (8, 9). ARF6 has been implicated in rearranging the
actin cytoskeleton at the plasma membrane (7, 10, 11). ARF isoforms may
also play a similar role in yeast (12).
There is considerable evidence that the actin cytoskeleton and myosin
motors are important for Golgi function in the cell (13, 14). Recent
studies show that actin is likely to be a component of Golgi-derived
transport vesicles (15, 16). In this study centractin, tropomyosin
isoforms, and myosin isoforms were found to be selectively associated
with distinct classes of Golgi-derived vesicles. Some isoforms of
spectrin and ankyrin localize to the Golgi apparatus, suggesting that
this is a site of their action (9, 17, 18). The actin cytoskeleton may also play a role in Golgi morphology and positioning (19).
Here we present evidence that at least two distinct pools of actin are
assembled on the Golgi membrane upon ARF activation. This actin affects
the release of COPI-coated vesicles from the Golgi and regulates the
association of spectrin and the actin-binding protein drebrin with the
membrane. The implications of these findings for the ability of
COPI-coated vesicles to mediate multiple trafficking steps and for the
role of the cytoskeleton in Golgi function will be discussed.
Materials--
Rat liver Golgi membranes and bovine brain
cytosol were isolated as described previously (20). The following
antibodies were used in this study: M3A5 (21), anti-actin (Sigma),
anti-drebrin (Medical & Biological Laboratories Co.), anti Golgi Binding Assays--
The final reaction conditions included
25 mM HEPES (pH 7.2), 2.5 mM magnesium acetate,
15 mM potassium chloride, 0.2 M sucrose, Golgi
membranes (0.2 mg/ml), bovine brain cytosol (1.0 mg/ml), and an
ATP-regenerating system. The incubations were carried out for 20 min at
37 °C, and the final reaction volume was 1 ml. When specified,
brefeldin A, cytochalasin D, or latrunculin A was added at the
indicated concentration. For reactions containing brefeldin A, the
membranes and cytosol were preincubated for 10 min at 37 °C with the
toxin or with a solvent control. For "float-up" binding assays, the
membranes were isolated following the incubation by centrifugation at
15,000 × g for 30 min at 4 °C in a refrigerated microcentrifuge. The membranes were then resuspended in 50 µl of 45%
(w/w) sucrose in 25 mM HEPES (pH 7.2), 25 mM
KCl and placed into a 7 × 20-mm ultracentrifugation tube. The
sample was overlaid with 125 µl of 35% (w/w) sucrose in 25 mM HEPES (pH 7.2), 25 mM KCl and then 25 µl
of 15% (w/w) sucrose in 25 mM HEPES (pH 7.2), 25 mM KCl. The sample was spun at 100,000 rpm for 30 min in a TLA-100 rotor (Beckman Instruments). A 100-µl sample containing the
Golgi membranes was removed from the top of the step gradient, and the
membrane proteins were precipitated by the addition of trichloroacetic
acid to 10%. The pellet from the step gradient and the TCA precipitate
were analyzed by SDS-PAGE and Western blotting. For two-stage
incubations the conditions were just as described above except that
following the first incubation, the membranes were reisolated by
microcentrifugation and incubated with a second reaction mixture prior
to the float-up centrifugation.
COPI-coated Vesicle Budding Reactions--
Unless otherwise
indicated, the final budding reaction conditions were identical to
those used for binding reactions except that the reaction volume was
2.0 ml. Following the incubation, the Golgi membranes were isolated by
centrifugation at 15,000 × g for 15 min and washed one
time with LSSB (25 mM HEPES (pH 7.2), 2.5 mM
magnesium acetate, 50 mM potassium chloride, 0.2 M sucrose). The vesicles were stripped from the membrane by
incubating in HSSB (25 mM HEPES (pH 7.2), 2.5 mM magnesium acetate, 250 mM potassium
chloride, 0.2 M sucrose) for 10 min on ice. Following the
incubation, the membranes were centrifuged at 15,000 × g for 15 min. Where indicated, the pellet containing the
Golgi remnants was resuspended in Laemmli sample buffer for Western
blot analysis. The supernatant was loaded onto a 35% (w/w) sucrose
cushion (25 mM HEPES (pH 7.2), 2.5 mM magnesium
acetate, 250 mM potassium chloride, 35% (w/w) sucrose) and
centrifuged at 350,000 × g for 30 min. The 35%
sucrose pellets were resuspended in Laemmli sample buffer and analyzed
by Western blotting.
Western Blotting--
Proteins were fractionated using SDS-PAGE
and blotted onto polyvinylidene difluoride membranes using standard
protocol for the Bio-Rad minigel and blotting apparatuses. Following
the transfer, the membranes were dried and incubated with appropriate
dilutions of the indicated primary antibodies. The signal was
visualized using horseradish peroxidase-conjugated secondary antibodies
(Bio-Rad) and ECL (Amersham Pharmacia Biotech). Where indicated, the
signals were quantitated using densitometry.
Polypeptide Sequence Determination and Mass
Spectrometry--
Identification by mass spectrometry was done by
cutting protein bands from SDS-PAGE gels and digesting the material
with 0.2 µg of trypsin (24). The resulting peptide mixture was then
loaded onto a 2-µl bed volume of Poros 50 R2 (PerSeptive Biosystems, Foster City, CA) reversed-phase beads (packed into an Eppendorf gel-loading tip) and eluted stepwise with 4 µl of 16% (and then with
4 µl of 30%) acetonitrile, 0.1% formic acid (25). The "16%" and "30%" peptide pools were each analyzed twice by
matrix-assisted laser desorption/ionization time of flight mass
spectrometry in the presence and absence of peptide calibrants (25)
using a REFLEX III (Brüker-Franzen, Bremen, Germany) instrument
equipped with a gridless pulsed extraction ion source and a 2-GHz
digitizer and operated in reflectron mode. Spectra were obtained by
averaging multiple signals. After recalibration with internal
standards, monoisotopic masses were assigned for the most prominent
peaks, and a peptide mass list was generated to search a protein
non-redundant data base (National Center for Biotechnology Information,
Bethesda, MD) using the PeptideSearch (26) algorithm with an accuracy requirement of 40 ppm. For the identification of the 280-kDa band, 35 of 63 experimental masses matched human While characterizing the protein components of Golgi-derived
transport vesicles, we observed that non-muscle actin is a major GTP In the absence of GTP As a more definitive test of the model that ARF activation leads to
changes on Golgi membranes that subsequently allow them to assemble
actin, we used two-stage binding reactions (Fig.
2). In the first stage, cytosol, GTP
Activated ADP-ribosylation Factor Assembles Distinct Pools of
Actin on Golgi Membranes*
,,,¶
Department of Physiology and Biophysics,
University of Iowa College of Medicine, Iowa City, Iowa 52242 and the
§ Molecular Biology Program, Memorial Sloan-Kettering Cancer
Center, New York, New York 10021
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-COP
(22), anti-KDEL receptor (23).
-spectrin, and 15 of the
remaining 28 masses matched human 
-spectrin (random matches, 12 of
63). For the identification of the 120-kDa band, 8 of 13 experimental
masses matched human drebrin (random matches, 4 of 13).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
S-dependent protein constituent of both Golgi
membranes and the Golgi-derived vesicles following incubations with
cytosol (data not shown). This finding, together with recent reports
indicating that ARF regulates the levels of actin, spectrin, and
ankyrin on Golgi membranes (8) and that actin-binding proteins
associate with vesicles (15, 16), led us to begin dissecting the nature and role of actin on the Golgi apparatus. As a first step, cell-free Golgi binding assays were used to characterize the conditions for
GTPS/ARF-dependent actin association with the Golgi
membranes. We developed a float-up binding assay that allowed actin
polymerization to be assayed independently of Golgi binding (Fig.
1, see "Experimental Procedures"). In
short, Golgi membranes were incubated with cytosol under conditions
known to promote coated vesicle formation. Following the incubation,
the membranes were resuspended in 45% sucrose and then overlaid with
35% sucrose in a centrifuge tube. Because Golgi membranes have a
buoyant density equivalent to about 30% sucrose, they float to the top
of the gradient during ultracentrifugation (Fig. 1A, G
fraction). By contrast, large cytoskeletal polymers pelleted at
the bottom of the centrifuge tube (Fig. 1A, P
fraction). The presence of the Golgi membranes in the G fraction
is confirmed by blotting for the cis-Golgi marker, KDEL receptor (Fig.
1A).
View larger version (25K):
[in a new window]
Fig. 1.
Actin binding to Golgi membranes.
A, Golgi membranes were incubated with cytosol, and GTP S
and phalloidin were added to the reaction where indicated. Following
the incubation, the reaction was loaded onto a float-up gradient. Shown
is a Western blot of the top Golgi fraction (G) and the
bottom pellet fraction (P) from the gradients. The blot was
probed for actin and the Golgi marker, KDEL receptor. B, a
Western blot probed with the anti-actin antibody of the top Golgi
fractions from float-up Golgi binding. Shown are the results from
duplicate incubations containing brefeldin A (BfA, 200 µM) or the methanol solvent alone as indicated.
C, a Western blot of the top Golgi fraction probed with
antibodies against actin and the coatomer subunit -COP. The
incubations were devoid of cytosol, Golgi membranes, or GTPS as
indicated.
S, the Golgi membranes bound neither to
G-actin, present in the cytosol (Fig. 1A, lanes 1 and 2), nor to F-actin, assembled by the addition of the
actin-polymerizing toxin phalloidin (Fig. 1A, lanes
5 and 6). In the presence of phalloidin, the F-actin
was completely resolved from the Golgi membranes on the step gradient.
By contrast, when GTPS was included in the incubation (Fig.
1A, lanes 3 and 4) 50-80% of the
actin was present in the G fraction. The GTPS-dependent
binding of actin to the Golgi was completely inhibited by the addition
of brefeldin A, indicating that the GTPS effect is mediated by ARF (Fig. 1B). Brefeldin A inhibits nucleotide exchange for ARF1
but not for ARF6 or the rho family of GTPases (27, 28), indicating that
the brefeldin A-sensitive actin binding we observe is distinct from the
modifications of the actin cytoskeleton involving these GTPases. If
either cytosol or the membranes are omitted from the reaction, no actin
appears in the G fraction (Fig. 1C). These findings suggest
that actin is assembled directly by Golgi membranes upon activation of
ARF with GTPS.
S,
and membranes were incubated either with or without the actin
monomer-binding toxin latrunculin A to block actin assembly. Fig. 2
shows that upon reisolation of the membranes following the first stage,
actin bound in a GTPS-dependent manner and that this
binding was completely blocked by the presence of latrunculin A. If the
reisolated Golgi membranes were then incubated with cytosol in a second
stage without GTPS and latrunculin A, actin was found to be present
on the membranes provided that GTPS had been included in stage 1. Actin assembled on the Golgi without GTPS in stage 2 even though
actin assembly had been blocked by latrunculin A in stage 1. This
result supports the model that actin assembles directly on the Golgi in
the presence of membrane-bound activated ARF, as opposed to a model
whereby the Golgi membranes associate with actin that had been
previously assembled through a membrane-independent but
GTPS-dependent mechanism. Because the 21-kDa ARF
proteins are the major small GTPases to associate with the Golgi
membranes when incubated with GTPS (29) and no additional G proteins
are activated in stage 2, this result also provides strong additional
evidence that the observed GTPS effects on actin assembly are
ARF-mediated.
View larger version (16K):
[in a new window]
Fig. 2.
ARF-bound Golgi assemble actin independently
of additional G protein activation. Shown is a Western blot probed
with anti-actin for isolated Golgi membranes after stage 1 and stage 2 of two-stage binding assays. Following stage 1 half of the membranes
were loaded onto SDS-PAGE for analysis, and half were used for the
stage 2 incubation. GTP S was included only in stage 1 for
reactions 2 and 3. Latrunculin A (Lat
A) was included only in stage 1 for reaction 3.
Because ARF triggers coated vesicle assembly and the conditions used
above for ARF-dependent actin binding are known to bud COPI-coated vesicles from the Golgi, we wished to examine whether the
ARF-dependent actin pool was involved in vesicle assembly or release. Following cell-free budding reactions, COPI-coated vesicles
are typically stripped from the membrane with 250 mM KCl
and then purified using an isopycnic sucrose gradient (20) or by
pelleting through a sucrose cushion (30). We find that much of the
ARF-dependent actin is extracted from the Golgi with 250 mM KCl (as are COPI-coated vesicles) and pellets through a 35% cushion during ultracentrifugation (Fig.
3). This finding could indicate that the
ARF-dependent actin is directly associated with the
vesicles.
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In addition to the actin that cofractionated with the vesicles, we
observed that some actin is resistant to the salt extraction and
remained on the Golgi membranes left behind after removing the vesicles
(Fig. 3, Golgi remnants). Surprisingly, although the
appearance of actin in the vesicle-enriched 35% sucrose pellet was
completely blocked by the addition of the plus-end-binding toxin
cytochalasin D, the levels of actin on the Golgi remnants were largely
unaffected by the presence of this toxin (Fig. 3). Blotting for the
vesicle marker -COP revealed that the levels of Golgi membrane and
vesicles were unaffected by the toxin treatment. Tubulin levels, both
on the Golgi remnants and in the 35% sucrose pellet, were also
unaffected by GTPS or cytochalasin D, indicating that these reagents
did not have effects on microtubule assembly or disassembly. The actin
monomer-binding toxin latrunculin A reduced the levels of actin in both
the 35% sucrose pellet and the Golgi remnants (Fig. 3B).
These results indicate the presence of distinct pools of actin with
unique toxin sensitivities on the Golgi membranes following incubation
with ARF.
In an effort to identify factors that may confer these distinct
properties to Golgi-associated actin, we examined the protein composition of the ARF-dependent salt-extractable actin
pool. Fig. 4 shows that when
GTPS-assembled actin is extracted from the Golgi and pelleted
through a cushion, a remarkably simple set of abundant proteins is
present (Fig. 4, lane 2). Besides actin, two proteins of 120 and 220 kDa disappeared when cytochalasin D was added to the reaction
(lane 1), indicating that these proteins are
actin-associated. This was an unexpectedly small number of actin-bound
proteins, given that these incubations were carried out with a whole
cytosol preparation. To rule out that our cytosol was devoid of
additional actin-binding proteins, we incubated cytosol alone under the
identical conditions used in the budding reaction in the presence
(lane 3) or absence (lane 4) of phalloidin to
polymerize the actin. The phalloidin-polymerized actin was extracted
with the 250 mM high salt stripping buffer using the same
conditions as for the standard budding reaction. The
phalloidin-polymerized actin contained several additional major
actin-binding proteins not found on the ARF-dependent
actin. Whether or not the 250 mM KCl wash was carried out
on the phalloidin-polymerized actin had no effect on the levels of
actin-binding proteins, ruling out that actin-binding proteins are lost
during the vesicle/actin extractions (data not shown). Thus, based on
the salt extractability, the toxin sensitivity, and the protein
composition, the ARF-dependent actin in the 35% sucrose
pellet is distinct.
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To identify the putative actin-binding proteins, the tryptic fragments
of the 120- and 220-kDa polypeptides were analyzed by matrix-assisted
laser desorption/ionization time-of-flight mass spectrometry. Using
mass fingerprint analysis, the identity of these proteins was
determined to be drebrin and a mixture of - and -spectrin,
respectively. Spectrin has been previously identified as part of an
ARF-regulated cytoskeleton on the Golgi and Golgi-derived vesicles (8,
9). Drebrin has mostly been characterized as a protein involved in
neuronal differentiation (31, 32). Interestingly, the yeast homolog of
drebrin, ABP1, has been implicated in endocytosis, suggesting that this
family of actin-binding proteins could play a role in trafficking
(33).
We confirmed that drebrin binding to the Golgi membranes is GTPS-
and ARF-dependent using a Western blot analysis of the vesicle-budding reaction. Drebrin appeared in the 35% sucrose pellet
and on Golgi remnants in a GTPS-dependent manner (Fig. 5). The addition of brefeldin A
completely blocked the appearance of drebrin in both fractions,
indicating that drebrin is ARF-dependent. When cytochalasin
D was included in the incubation, actin and drebrin completely
disappeared from the 35% sucrose pellet. The cytochalasin D resistant
pool of actin is observed on the Golgi remnants (lane 7) and
is found to be ARF-dependent. Interestingly, drebrin is not
associated with this pool of actin. Together these data suggest that
ARF activation leads to the assembly of at least two pools of actin on
the Golgi. The first pool is cytochalasin D-sensitive,
salt-extractable, and contains drebrin. The second pool is less
extractable with salt, is cytochalasin D-resistant, and does not
contain drebrin.
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Because the same small GTPase, ARF, triggered vesicle formation and the
assembly of actin on the Golgi and because actin may be directly
associated with the Golgi-derived vesicles (Fig. 1) (15, 16), we
reasoned that the ARF-dependent actin assembly could play a
role in vesicle assembly or release. Therefore, we examined the effects
of adding the actin-binding toxin cytochalasin D on COPI vesicle
formation using the cell-free budding reaction. Addition of the toxin
had no effect on the binding of ARF and coatomer to Golgi membranes,
suggesting that actin does not play a role in these early events of
COPI-coated vesicle assembly (data not shown). Effects were observed,
however, on the release of vesicles following the incubation.
Typically, vesicles are extracted from the Golgi membranes following
cell-free budding reactions by a high salt wash (20). At the standard
salt concentration (250 mM KCl), cytochalasin D had little
effect on the release of COPI-coated vesicles (Fig.
6). Studies on the
giantin/GM130/p115-mediated docking of COPI-coated vesicles revealed
that the effects of disruption of this complex on vesicle release could
be observed when lower salt concentrations were used for extracting
vesicles (30). Because some aspects of vesicle docking and release are
salt-sensitive, we examined the effects of cytochalasin D on
COPI-coated vesicle release at lower salt concentrations. Fig. 6 shows
that when vesicles were stripped at the lower salt concentrations, 150 and 75 mM KCl, significantly more -COP and KDEL receptor
were found in the 35% sucrose pellet when cytochalasin D was included
in the incubations. Therefore, the salt-dependent vesicle
release appears to be facilitated by interfering with actin assembly on
the Golgi membranes. These results indicate that ARF-mediated changes
in the cytoskeleton could play an important role in regulating
vesicular trafficking through the Golgi.
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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We describe an ARF-dependent salt extractable actin
pool assembled from whole cytosol with a remarkably simple protein
composition. One of the two major actin-binding proteins of this
complex was identified as a mixture of - and -spectrin,
confirming previous studies showing an ARF-mediated interaction between
spectrin and the Golgi (8). The second protein was identified as
drebrin. Previously, drebrin had been characterized as a
neuron-specific protein that may play a role in neuronal development.
In particular, it has been localized to dendrites and may play a role
in dendrite outgrowth. If drebrin were restricted to neuronal
dendrites, it would be unlikely that it plays a general role in Golgi
transport or other vesicular trafficking steps. Several findings
indicate that this is not the case. First, we find by Western blot
analysis that a protein identical in size to brain cytosol drebrin is
recognized by the drebrin antibody in Chinese hamster ovary cells (data
not shown), indicating that drebrin or a closely related isoform is present in non-neuronal cells. Second, other recent studies have found
drebrin to be present in several non-neuronal tissues (34, 35).
Finally, a homolog of drebrin, ABP1, is present in the yeast
Saccharomyces cerivisiae. ABP1 interacts
genetically with genes encoding proteins required for endocytosis (33).
This finding, together with the results we report here, raises the possibility that drebrin is involved in vesicular trafficking. Additional studies will be required to determine the precise role of
drebrin or drebrin-related proteins in these processes.
The effects of cytochalasin D on the salt-dependent extraction of COPI-coated vesicles could indicate a role for actin in the fission reaction or vesicle release. It is, however, equally likely that actin plays a role in a vesicle tethering or docking event that is not directly related to the fission reaction. In this respect, it is interesting that similar results have been obtained when the function of the GM130/giantin complex is compromised. This complex is thought to tether vesicles to the Golgi after they are formed (36) or to dock vesicles to a target organelle prior to SNARE-mediated fusion (37, 38). Although additional studies will be required to establish the precise role of actin in these trafficking steps, the cytochalasin D effects indicate that the ARF-dependent actin plays a role in the interaction of COPI vesicles with the Golgi and therefore is likely to function during COPI-mediated trafficking to, from, or within the Golgi.
It is of interest to speculate why pools of actin with distinct properties are assembled on the Golgi. One possibility is that the different pools of actin reflect different classes of assembling vesicles. ARF triggers the assembly of both COPI-coated and AP1/clathrin-coated vesicles on the Golgi apparatus (39, 40). It is possible that ARF directs the assembly of distinct actin pools for different classes of vesicles. Additional evidence supporting this model comes from the recent finding that different classes of Golgi-derived vesicles associate with distinct actin-binding proteins (15). A second possibility is that ARF directs the assembly of different actin pools on different types of membranes. For example, actin assembled on cis-Golgi membranes may have different properties from actin assembled on trans-Golgi membranes. Assembling actin with specific properties on each type of membrane or at each class of vesicle assembly site could direct the overall organization, positioning, or morphology of these organelles. Alternatively or in addition, it could be involved in conferring cytoskeleton-dependent directionality and targeting to the transport process.
Targeting proteins in the cell almost certainly requires specific
interactions with molecular motor proteins and cytoskeletal elements to
move them in the proper direction to the correct location in the cell.
In some instances, the same class of vesicle coat can be used for
multiple transport steps; for example, COPI-coated vesicles appear to
be used for anterograde and retrograde trafficking from Golgi cisternae
as well as in the endocytic pathway (41-45). Therefore, it is highly
probable that there is cellular machinery in addition to the coat
proteins that specify interactions with the cytoskeleton. The
ARF-mediated changes in the actin cytoskeleton may be part of this
machinery. Thus ARF-regulated vesicle assembly may not only involve the
directed oligomerization of coat proteins but also additional complex
regulation of cytoskeletal components to ensure that the assembled
vesicle moves along a specific cytoskeletal filament with the correct
molecular motor and thus arrives at the proper destination in the cell.
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FOOTNOTES |
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* This work was supported by the Roy J. Carver Charitable Trust, a March of Dimes Basil O'Connor starter scholar award, and the University of Iowa Diabetes and Endocrinology Research Center (to M. S.) and by an NCI core grant (to P. T.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed. E-mail: mark-stamnes@uiowa.edu.
Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M000024200
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ABBREVIATIONS |
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The abbreviations used are:
ARF, ADP-ribosylation factor;
SDS-PAGE, SDS-polyacrylamide gel
electrophoresis;
GTPS, guanosine
5'-3-O-(thio)triphosphate.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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F. D. Brown, A. L |
