The Rab2 protein is a resident of pre-Golgi
intermediates and required for vesicular transport in the early
secretory pathway. We have previously shown that a peptide
corresponding to the amino terminus of Rab2 (residues 2-14) arrests
protein traffic prior to a rate-limiting event in VSV-G movement
through pre-Golgi structures (Tisdale, E. J., and Balch, W. E. (1996) J. Biol. Chem. 271, 29372-29379). To
determine the mechanism by which this peptide inhibits transport, we
investigated the effect of the Rab2 peptide on the distribution of the
-COP subunit of coatomer because COPI partially localizes to
pre-Golgi intermediates. We found that the peptide caused a dramatic
change in the distribution of pre-Golgi intermediates containing
-COP. A quantitative binding assay was employed to measure
recruitment of
-COP to membrane when incubated with the Rab2
(13-mer). Peptide-treated microsomes showed a 25-70% increase in the
level of membrane-associated
-COP. The enhanced recruitment of
coatomer to membrane was specific to the Rab2 (13-mer) and required
guanosine 5'-3-O-(thio)triphosphate, ADP ribosylation factor, and protein kinase C-like activity. The ability to enhance
-COP membrane binding was not limited to the peptide. Similarly, the
addition of recombinant Rab2 protein to the assay promoted
-COP
membrane association. Our results suggest that the Rab2 peptide causes
the persistent recruitment of COPI to pre-Golgi intermediates which
ultimately arrests protein transport due to the inability of membranes
to uncoat.
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INTRODUCTION |
Membrane traffic in the early secretory pathway requires the Rab1
and Rab2 GTPases (1, 2). The Rab1 protein is found in the endoplasmic
reticulum (ER),1 pre-Golgi
intermediates, and early compartments of the Golgi complex (3), whereas
Rab2 has been immunolocalized only to pre-Golgi intermediates (4).
These transport intermediates composed of vesicles and tubular clusters
(VTCs) (5) are distinct from the ER and the Golgi complex (6). VTCs are
morphologically defined by the proteins ERGIC-53/gp58 (p53/gp58) (7, 8) and Rab2 (4), and are the major peripheral site for COPI recruitment (9, 10).
Pre-Golgi intermediates (VTCs) sort and recycle resident proteins from
itinerant proteins destined for secretion (10-12). Although the
mechanism of protein recycling is unknown, it is likely to involve the
COPI coat complex (coatomer and ARF) (10, 13, 14). Coatomer is a
heptameric, soluble complex composed of
,
,
',
,
,
,
and
subunits (15). Perhaps the best characterized component of
coatomer is the
-COP subunit that was first identified as an 110-kDa
peripheral membrane protein associated with pre-Golgi intermediates and
the cis Golgi stack (16, 17). The coat complex is recruited en
bloc (18) or as a subset (19, 20) to membrane after activation of
the small GTPase ADP ribosylation factor (ARF) (21, 22) and
phospholipase D. Phospholipase D hydrolyzes phosphatidylcholine to
phosphatidic acid which in tandem with phosphatidylinositol 4,5-bisphosphate enhances coatomer membrane association (23, 24). The
binding of COP1 deforms the membrane which leads to bud formation and
eventual release of a coated vesicle.
We have reported that a peptide which corresponds to residues 2-14 of
Rab2 arrests protein transport by preventing the flow of cargo through
pre-Golgi intermediates (VTCs) (2). The ability of this peptide to
block transport in a rapid and specific manner makes it a valuable tool
to dissect events in the early secretory pathway. In this study, we
took advantage of the irreversible property of the peptide to determine
the mechanism by which the Rab2 (13-mer) arrests transport. Our
previous observation that peptide affects transport from pre-Golgi
structures prompted us to look at the distribution of transport-related
proteins which co-localize to VTCs. We initially analyzed the
distribution of COPI by focusing on the
-COP subunit. To our
surprise, we found that the peptide had a striking affect on
-COP
recruitment. Membranes incubated with peptide showed a 25-70%
increase in
-COP binding compared with control. This increase in
coatomer binding was specific to the Rab2 (13-mer) and required ARF,
GTP
S, and protein kinase C (PKC) or a PKC-like protein. Similar
results were obtained after addition of recombinant Rab2 protein to the
binding assay. We propose that this enhanced COPI recruitment results
in the inability of vesicles derived from pre-Golgi structures to
uncoat which ultimately leads to the coupled arrest of anterograde and
retrograde traffic. These data suggest that Rab2 plays a role in
protein sorting and recycling from pre-Golgi intermediates.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Polyclonal antiserum made to peptides
corresponding to
-COP (EAGELKPEEEITVGPVQK), N-
-COP (yeast
sequence KMLTKFESKSTRAKGYC), N-
'-COP (PLRLDIKRKLTARSDYC), and
N-
-COP (APPAPGPASGGSGEVYC) were generously provided by Dr. Rohan
Teasdale (R. W. Johnson Pharmaceutical Research Institute, La
Jolla, CA). The serum was applied to cyanogen bromide-activated
Sepharose 4B to which the immunizing peptide was coupled for affinity
purification. The column was washed with 5 bed volumes of PBS and
eluted with 0.1 M glycine, pH 2.8. The eluate was
neutralized to pH 7.2, dialyzed against PBS, then concentrated.
Peptides were synthesized at the R. W. Johnson Pharmaceutical
Research Institute Protein Facility (La Jolla, CA). The monoclonal
antibody to ARF (1D9) and ARF1 cDNA was a gift from Dr. Richard
Kahn (Emory University, Atlanta, GA). Purified coatomer was
kindly provided by Dr. M. Gerard Waters (Princeton
University, Princeton, NJ). Polyclonal antiserum to Rab2 was purchased
from Santa Cruz Biotechnology. Rab2 cDNA was acquired from Dr.
Marino Zerial (EMBL, Heidelberg, Germany). The polyclonal serum to p58
was obtained from Dr. Jaakko Saraste (University of Bergen, Bergen,
Norway). Calphostin C was purchased from Calbiochem (San Diego, CA).
Chelerythrine chloride was obtained from LC Laboratories. Digotonin was
acquired from Boehringer Mannheim. HRP-conjugated antibody was
purchased from Bio-Rad. Monoclonal antibody to
-COP (M3A5) and all
other reagents were purchased from Sigma.
Membrane Binding Reaction--
HeLa cells were washed three
times with ice-cold phosphate-buffered saline (PBS). The cells were
scraped off the dish with a rubber policeman into 10 mM
Hepes, pH 7.2, and 250 mM mannitol, then broken with 15 passes of a 27-gauge syringe. The broken cells were pelleted at
500 × g for 10 min at 4 °C, and the supernatant re-centrifuged at 20,000 × g for 20 min at 4 °C.
The pellet containing ER, pre-Golgi, and Golgi membranes was washed
with 1 M KCl in 10 mM Hepes, pH 7.2, for 15 min
on ice, then centrifuged at 20,000 × g for 20 min at
4 °C. The membranes were resuspended in 10 mM Hepes, pH
7.2, and 250 mM mannitol and employed in the binding reaction as described previously (10, 14). Membranes (30 µg of total
protein) were added to a reaction mixture which contained 27.5 mM Hepes, pH 7.2, 2.75 mM MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM
CaCl2, 1 mM ATP, 5 mM creatine
phosphate, and 0.2 units of rabbit muscle creatine kinase. Peptide or
Rab2 protein were added to obtain the final concentration as indicated
under "Results" and the reaction mixture incubated on ice for 30 min. Rat liver cytosol (50 µg) and 2.0 µM GTP
S were
then added and the reactions shifted to 37 °C and incubated for 10 min. The binding reaction was terminated by transferring the samples to
ice and then centrifuged at 20,000 × g for 10 min at
4 °C. The pellet was resuspended in sample buffer, separated by
SDS-PAGE and transferred to nitrocellulose in 25 mM Tris,
pH 8.3, 192 mM glycine, 20% methanol. The membrane was
blocked in Tris-buffered saline (TBS) which contained 5% nonfat dry
milk and 0.5% Tween 20, incubated with an affinity purified polyclonal
antibody made to the EAGE peptide of
-COP (25) or a polyclonal
antibody to Rab2, or a monoclonal antibody to ARF (1D9) (26), washed,
further incubated with a horseradish peroxidase (HRP)-conjugated
anti-rabbit or anti-mouse antibody, developed with enhanced
chemiluminescence (ECL) (Amersham), then quantitated by
densitometry.
Indirect Immunofluorescence--
NRK cells plated on coverslips
were permeabilized with digitonin (20 µg/ml) as outlined previously
(3). Coverslips with permeabilized cells were inverted and placed in
tissue culture wells that contained the transport mixture described
above, preincubated on ice for 15 min with or without peptide or Rab2
protein and then incubated for 80 min at 15 °C. To terminate
transport, the cells were transferred to ice and fixed in 3%
formaldehyde/PBS for 10 min. Intracellular
-COP was detected by
re-permeabilization of the fixed cells with 0.05% saponin in
PBS/normal goat serum for 10 min, washed with PBS, then co-incubated
for 30 min with a monoclonal antibody to
-COP (M3A5) and a
polyclonal serum to p58, or with polyclonal serum to Rab2. Cells were
then washed with PBS, co-stained for 30 min with Texas Red anti-rabbit
antibody and fluorescein isothiocyanate anti-mouse antibody, washed,
mounted, then viewed under a Zeiss Axiovert fluorescence
microscope.
Purification of Recombinant Rab2 Protein and in Vitro
Prenylation--
Rab2
14 was generated by polymerase chain reaction
using a 5'-oligonucleotide primer which introduced a start codon at the deletion site. The cDNA for Rab2 wild type and Rab2
14 were
cloned into pET3A (Novagen, Milwaukee, WI) and introduced into BL21
(DE3) pLysS (Novagen). A 1-liter culture was grown to an
OD600 of 0.4-0.5 and induced with 0.4 mM
isopropyl-
-thiogalactopyranoside for 3 h at 37 °C. The cells
were centrifuged at 5000 × g for 10 min at 4 °C,
and the cell pellet resuspended in 50 mM Tris, pH 7.4, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl
fluoride, 0.1 mM benzamidine, 1 mM EDTA, and
1% Triton X-100, then homogenized by 20 passes with a Dounce tissue
grinder. Lysozyme (400 µg/ml), DNase I (40 µg/ml), and 25 mM MgCl2 were added to the homogenate and
allowed to digest for 30 min at 4 °C, then centrifuged at 22,000 × g for 30 min at 4 °C. The supernatant was
applied to a 70-ml column containing Q Sepharose Fast Flow (Pharmacia)
equilibrated with Buffer A (50 mM Tris, pH 7.4, 10 mM MgCl2, and 1.0 mM EDTA), washed
with 2 bed volumes of Buffer A, then eluted with a linear NaCl gradient
(0-400 mM) in Buffer A. Three-ml fractions were collected
and an aliquot of each fraction separated by SDS-PAGE and immunoblotted
with a Rab2 polyclonal antibody. Rab2-enriched fractions were pooled,
concentrated, and applied to a 200-ml column containing Sephacryl S-100
(Pharmacia) and eluted with Buffer A. Fractions containing Rab2 or
Rab2
14 proteins were identified by SDS-PAGE and immunoblotting, then
pooled, concentrated, and prenylated in an in vitro
reaction. Briefly, the isoprenylation reaction was performed in a total
volume of 50 µl that contained 5 µg of recombinant Rab2 protein, 10 µg of geranylgeranyl pyrophosphate (Sigma), 1 mM
dithiothreitol, 25 µl of rat liver cytosol, 10 mM MgCl2, 1 mM ATP, 5 mM creatine
phosphate, and 0.2 units of rabbit muscle creatine kinase. The reaction
was incubated for 1 h at 37 °C and then desalted through a 1-ml
column of Sephadex G-25 (Pharmacia) and concentrated. The protein
concentration was determined by Micro BCA Protein Assay Reagent
(Pierce).
Purification of Recombinant ARF and Preparation of ARF-depleted
Cytosol--
Recombinant myristoylated ARF was prepared from the BL21
bacterial strain that had been co-transformed with a plasmid encoding N-myristoyltransferase and wild type ARF1 (27). The
transformed cells were induced with 1.0 mM
isopropyl-
-thiogalactopyranoside for 3 h at 25 °C in the
presence of 50 µM myristate. The cells were lysed and the
expressed ARF1 purified on a DEAE-Sephacel and AcA54 Ultrogel columns
as described by Weiss et al. (28). For ARF depletion, rat
liver cytosol was fractionation on a Superose 6 column equilibrated in
25 mM Hepes, pH 7.2, 125 mM KOAc, 2.5 mM MgOAc, and 1 mM dithiothreitol (28).
ARF-enriched and ARF-depleted fractions were identified by Western
blotting. Those fractions lacking ARF were pooled, and concentrated to
the original volume.
 |
RESULTS |
N-terminal Peptide to Rab2 Alters
-COP Distribution--
We
analyzed the distribution of
-COP in permeabilized NRK cells
incubated with or without peptide for 80 min at 15 °C by indirect
immunofluorescence. Cells were incubated at this reduced temperature to
accumulate and enhance visualization of pre-Golgi intermediates (VTCs).
In control cells, anti-
-COP antibody labeled the juxtanuclear Golgi
complex and vesicular structures scattered throughout the cytoplasm and
in proximity to the Golgi complex (Fig.
1A). This distribution of
-COP is in agreement with that found in other cell types (6, 9, 10).
In contrast, cells incubated in the presence of the Rab2 peptide
displayed prominent
-COP-labeled structures that ringed the nucleus
in a collar-like manner (Fig. 1B). These cells did not
appear to contain peripherally-located
-COP-labeled vesicles. The
-COP containing ring-like structure partially overlapped with VTCs
located near the cis Golgi region that stained with the antibody to p58
(29) (Fig. 1D) and with antibody to Rab2 (Fig.
1H). Both of these polypeptides are considered marker
proteins for VTCs (4, 7, 8). As we observed with antibody to
-COP,
p58 (Fig. 1D) and Rab2 (Fig. 1H)-labeled vesicles were not found in peripheral locations. In control cells, p58 (Fig.
1C) and Rab2 (Fig. 1G) were localized in
vesicular elements that were concentrated in the Golgi region and
dispersed throughout the cytoplasm. This staining pattern is similar to
that observed with antibody to
-COP. This striking change in VTC
distribution prompted us to further explore the relationship of Rab2 to
COPI recruitment.

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Fig. 1.
Rab2 (13-mer) causes redistribution of
-COP-labeled structures. NRK cells grown on coverslips were
permeabilized with digotonin, and then incubated in a complete
transport mixture in the absence (A, C, E, and G)
or presence (B, D, F, and H) of 50 µM Rab2 peptide for 80 min at 15 °C. The distribution
of -COP (A and B) and p58 (C and
D), or -COP (E and F) and Rab2
(G and H) was determined by indirect
immunofluorescence as described under "Experimental Procedures." In
control cells, antibody to -COP labeled the Golgi region and
peripherally located pre-Golgi intermediates or VTCs. These structures
overlap with vesicles that stain with antibody to p58 (panel
C) and with antibody to Rab2 (panel G). Peptide-treated
cells displayed punctate -COP-labeled vesicles that ringed the
nucleus. This ring-like structure co-distributed with vesicles that
stained with antibody to p58 (panel D) and with antibody to
Rab2 (panel H).
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|
Rab2 (13-mer) Increases Membrane-associated
-COP--
A
quantitative binding assay was used to measure
-COP recruitment to
membranes in the presence of the Rab2 peptide (10, 21). For this assay,
microsomes were prepared from whole cell homogenates and washed with 1 M KCl to remove pre-bound COPI. These membranes were
preincubated in buffer for 30 min on ice with or without peptide, and
then supplemented with GTP
S and rat liver cytosol and incubated at
37 °C for 10 min. The peptide concentrations employed in the binding
assay were equivalent to those which inhibit protein traffic in an
in vitro transport assay (2). To terminate the reaction,
membranes were collected by centrifugation, separated by SDS-PAGE,
transferred to nitrocellulose, then probed with an affinity purified
antibody to
-COP. We observed in peptide-treated membranes a marked
increase in
-COP recruitment that occurred in a dose dependent
manner and required GTP
S (Fig. 2). The
amount of membrane-associated
-COP increased ~50% when incubated
with 25 µM peptide. In our previous studies (2), this
peptide concentration reduced ER to Golgi transport by 50% in an
in vitro transport assay. To learn if recruitment was
specific to the Rab2 (13-mer), peptides were synthesized to the amino
terminus of other Rab proteins and evaluated in the binding assay (see Table I). Stimulation of
-COP membrane
binding was specific to the Rab2 (13-mer). As shown in Fig.
3, a randomized form of Rab2 (13-mer) had
no effect on
-COP binding even at a concentration that results in
maximum recruitment by the wild-type peptide. Likewise, a peptide that
corresponds to the first 7 amino acids of Rab2 had no influence on
-COP membrane binding. The amount of membrane-associated
-COP was
not affected by control peptides made to the analogous N-terminal
domains of Rab5 and Rab3A. We also noted that a peptide to the amino
terminus of Rab1B, a protein essential for ER to Golgi transport failed
to recruit coatomer above the control level. These results show that
the enhanced recruitment of
-COP to membrane was specific to the
Rab2 (13-mer).

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Fig. 2.
Rab2 peptide stimulates -COP membrane
recruitment. Microsomes were prepared from HeLa cell homogenates
as described under "Experimental Procedures," then preincubated
with or without increasing concentrations of the Rab2 peptide for 30 min on ice. Cytosol and GTP S were added to one set of membranes
(closed bar), whereas the other set received only cytosol
(striped bar) and then all membranes incubated for 10 min at
37 °C to promote COPI binding. Microsomes were collected by
centrifugation, separated by SDS-PAGE, and immunoblotted with
affinity-purified antibody to -COP. Rab2 peptide treatment resulted
in a marked increase in membrane-associated -COP that required
GTP S.
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Table I
Peptides tested in membrane binding assay
Membrane binding assay was performed as described under "Experimental
Procedures."
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Fig. 3.
Enhanced -COP membrane binding is specific
to the Rab2 (13-mer). Microsomes were prepared from HeLa cell
homogenates as described under "Experimental Procedures," and then
preincubated for 30 min on ice in 50 µM peptide
corresponding to the amino terminus of the peptides indicated (see
Table I). Cytosol and GTP S were added and the membranes incubated
for 10 min at 37 °C. Membranes were pelleted and then subjected to
SDS-PAGE and Western blotting. The blot was incubated with a polyclonal
antibody to -COP, then further incubated with HRP-conjugated
secondary antibody and developed with ECL. The amount of recruited
-COP was quantitated by densitometry.
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|
The availability of affinity-purified antibodies that recognize the
,
', and
subunits of coatomer allowed us to test for co-recruitment after peptide treatment. In all cases, we observed a
linear increase in membrane binding of these subunits comparable to
-COP (data not shown). It appears that the peptide stimulates, at
the very least, recruitment of a subcomplex if not all coatomer subunits.
The Rab2 Peptide Increases the Rate of
-COP Recruitment--
We
performed a time course in which membrane was incubated with or without
peptide to determine whether the Rab2 (13-mer) affects the rate of
coatomer recruitment to membrane. As shown in Fig.
4A, the Rab2 peptide
accelerated coatomer binding that resulted in a t1/2
of ~5 min compared to t1/2 of ~7.5 min for the control. No increase in
-COP recruitment was observed after ~10 min of incubation for both control and peptide-treated membranes. The
apparent saturability was not due to the rate of COPI binding, since
longer incubations times did not result in an increased level of
membrane-bound
-COP (data not shown). To further learn if the
membranes were saturated with
-COP or if cytosol was limiting, microsomes were preincubated with or without the Rab2 peptide and then
GTP
S and increasing amounts of cytosol added. Fig. 4B shows that control and peptide-treated membranes were saturated with
-COP when incubated with 0.1 mg of cytosol (total protein). Incubation of membranes with higher cytosol concentrations did not
result in additional
-COP recruitment. Peptide-treated membranes showed an ~ 30% increase in membrane-bound
-COP compared
with control membrane. These combined results suggest that the Rab2 (13-mer) not only affects the extent of
-COP recruitment but also
causes a rapid recruitment of COPI to membrane.

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Fig. 4.
The Rab2 peptide accelerates the rate of
-COP recruitment to membrane. Panel A, HeLa cell
membranes were incubated for increasing time in the absence
(closed circle) or presence (open square) of 50 µM Rab2 peptide. At the time indicated, membranes were
shifted to ice, pelleted, separated by SDS-PAGE, and transferred to
nitrocellulose. The blot was probed with antibody to -COP and the
amount of -COP quantitated by densitometry. Panel B,
binding reactions were preincubated for 30 min at 0 °C in the
absence or presence of 50 µM Rab2 peptide. Increasing
amounts of cytosol and 2.0 µM GTP S were then added and
the reaction incubated for 20 min at 37 °C. Membranes were pelleted,
subjected to SDS-PAGE and Western blotting. The amount of
membrane-associated -COP was determined as described under
"Experimental Procedures." The Rab2 peptide increased the rate and
extent of -COP membrane binding.
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|
Rab2 Protein Increases Membrane-associated
-COP--
The Rab2
peptide inhibits protein traffic in an irreversible manner (2) which
may result from the inability of the Rab2 protein to bind to pre-Golgi
elements. We pursued this question by first establishing a
dose-response curve for the Rab2 protein. The addition of 200 ng of
Rab2 protein saturated the membranes (Fig.
5). The microsomal binding assay was then
performed with increasing peptide concentrations in the presence of 200 ng of recombinant Rab2 protein. The amount of membrane-bound Rab2
protein did not change when co-incubated with 10-75 µM
peptide (Fig. 5, inset). These results suggest that the Rab2
(13-mer) does not compete with Rab2 protein binding to membrane.

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Fig. 5.
Rab2 amino-terminal peptide does not compete
with Rab2 protein membrane binding. HeLa cell microsomes were
incubated with increasing concentrations of purified recombinant Rab2
protein in an assay buffer supplemented with GTP S for 15 min at
37 °C. The membranes were pelleted, separated by SDS-PAGE,
transferred to nitrocellulose, and the blot probed with a polyclonal
antibody to Rab2. After incubation with an HRP-conjugated secondary
antibody, the blot was developed with ECL and the amount of recruited
Rab2 quantitated by densitometry. Membranes are saturated with 200 ng
of Rab2 protein. Inset, HeLa cell membranes were
preincubated with 200 ng of recombinant Rab2 protein in the presence or
absence of increasing Rab2 peptide concentrations for 30 min on ice.
GTP S was then added and the reactions shifted to 37 °C for 15 min. Microsomes were collected by centrifugation, separated by
SDS-PAGE, and immunoblotted with polyclonal antibody to Rab2, and
quantitated by densitometry. The amount of membrane-bound Rab2 did not
change with increasing peptide concentration which suggests that the
Rab2 (13-mer) does not interfere with Rab2 protein binding to
membrane.
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We reasoned that if there was no competition between the peptide and
the Rab2 protein for binding, then the Rab2 (13-mer) might mimic the
function of the intact protein and therefore potentiate Rab2 activity.
To address this possibility, the microsomal binding assay was performed
by incubating membranes with three concentrations of recombinant Rab2
protein with GTP
S and cytosol for 15 min at 37 °C. As we observed
with the Rab2 (13-mer), Rab2 protein membrane binding resulted in a
dose-dependent increase in membrane-associated
-COP
(Fig. 6A). This result was
further investigated in the morphological assay which showed that the
Rab2 protein changed the intracellular distribution of
-COP. In
these cells we observed large
-COP containing structures located in
the perinuclear region (Fig. 7C). These structures appeared
to overlap with juxtanuclear structures that labeled with antibody to
p58 (Fig. 7D) and with antibody to Rab2 (Fig. 7D,
inset). This accumulation of COPI containing intracellular
structures in response to the Rab2 protein is consistent with the
enhanced
-COP recruitment observed in the microsomal binding assay.

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Fig. 6.
Recombinant Rab2 protein promotes -COP
membrane association. Recombinant Rab2 wild-type protein was
purified and isoprenylated in vitro, as described under
"Experimental Procedures." HeLa cell microsomes were incubated with
0, 50, 100, or 200 ng of recombinant Rab2 protein (panel A)
or recombinant Rab2 14 protein (panel B) in assay buffer
supplemented with GTP S and cytosol for 15 min at 37 °C. Membranes
were pelleted, separated by SDS-PAGE, transferred to nitrocellulose,
and the blot probed with a monoclonal antibody to -COP and a
polyclonal antibody to Rab2. After incubation with HRP-conjugated
secondary antibodies, the blot was developed with ECL and the amount of
recruited Rab2 and -COP was quantitated by densitometry. A co-linear
increase in Rab2 and -COP was noted with increasing protein
concentration. The amino-terminal truncated form of Rab2 had no effect
on -COP recruitment.
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Fig. 7.
Rab2 protein alters -COP distribution in
permealized cells. NRK cells grown on coverslips were
permeabilized with digotonin, and then incubated in a complete
transport mixture (panels A and B), or in a
complete transport mixture supplemented with 200 ng of Rab2 protein
(panels C and D) or with 200 ng of Rab2 14
(panels E and F) for 80 min at 15 °C. The
distribution of -COP (all panels), the distribution of
p58 (inset panels A, C, and E), and the
distribution of Rab2 (inset panel B, D, and F)
was determined by indirect immunofluorescence as described under
"Experimental Procedures." In control cells, antibody to -COP
predominantly labeled the Golgi region. Rab2 protein-treated cells
displayed enhanced perinuclear -COP staining that overlapped with
structures stained for p58 and Rab2. Arrows denote alignment
of co-stained cells.
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From our earlier work we know that overexpression of Rab2 wild-type
caused a decrease (25%) in the processing of VSV-G to endo H-resistant
forms (2). Deletion of the first 14 amino-terminal residues from Rab2
wild-type did not affect prenylation, but restored transport to control
levels. To determine if a similar truncation of Rab2 wild-type could
modify the ability of the protein to recruit
-COP, we generated
recombinant Rab2
14 protein and introduced the mutant protein into
the binding assay. Fig. 6B shows that membranes treated with
Rab2
14 did not recruit
-COP to the level obtained by incubation
with the wild-type protein. Furthermore, the truncated protein did not
alter
-COP distribution when introduced into the morphological assay
(Fig. 7E). These cells displayed a similar distribution of
VTCs labeled with p58 (Fig. 7F) and Rab2 (Fig. 7F,
set) compared with control (Fig. 7, A and
B). These combined results support our contention that the
peptide functions as a bona fide domain of the Rab2 protein and more
importantly, demonstrates that the amino-terminal residues of Rab2 are
necessary to promote COPI binding.
ARF Enhances
-COP Binding in Rab2 Peptide and Rab2
Protein-treated Microsomes--
Numerous studies show that the small
GTPase ARF facilitates COPI binding to pre-Golgi elements and the Golgi
complex (10, 21, 22). However, it has also been reported that membranes prepared from PtK1 cells can form COPI-coated vesicles in the absence
of exogenous ARF (24). This ARF independent COPI recruitment requires
phospholipase D activity to produce phosphatidic acid which forms
stable binding sites for coatomer. On the basis of these results, we
wanted to determine whether peptide recruitment of
-COP required
pre-bound ARF. Microsomes were incubated with increasing concentrations
of the Rab2 (13-mer) for 10 min at 37 °C. The membranes were
collected, subjected to SDS-PAGE and Western blotting, then probed with
a monoclonal antibody to ARF (1D9) (26) and
-COP. As shown in Fig.
8A, peptide stimulated a
modest increase in membrane-bound ARF (see bar graph). This
finding contrasts with the peptide induced linear increase of
-COP
and most likely reflects ARF-saturated membrane. In support of this
interpretation, dose-response curves generated with increasing cytosol
showed that the concentration used in the binding assay contains
near-saturating amounts of ARF (data not shown).

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Fig. 8.
Rab2 amino-terminal peptide and Rab2 protein
require ARF to stimulate -COP membrane binding. Panel A,
HeLa cell microsomes prepared as described under "Experimental
Procedures" were preincubated with increasing concentrations of the
Rab2 (13-mer) for 30 min on ice. Cytosol and GTP S were then added
and the membranes incubated for 10 min at 37 °C. After separation by
SDS-PAGE and Western blotting, the transfer was probed with antibody to
-COP and to ARF, washed, further incubated with HRP-conjugated
secondary antibody, and developed with ECL. The amount of -COP and
ARF was quantitated by densitometry. Panel B, salt-washed
microsomes were incubated with purified coatomer (75 ng) and GTP S in
the absence or presence of 50 µM Rab2 peptide or 100 ng
of Rab2 protein for 10 min at 37 °C. To one set of binding reactions
was added 75 ng of purified ARF (+). The amount of membrane-associated
-COP was determined by densitometry. Panel C, membrane
was incubated with ARF-depleted cytosol ( ARF) in the
absence or presence of 100 ng of Rab2 protein or 50 µM
Rab2 peptide supplemented with GTP S and incubated for 10 min at
37 °C. One set of reactions was supplemented with 75 ng of ARF1
(+ARF) and incubated as above. Membranes were pelleted,
separated by SDS-PAGE, transferred to nitrocellulose, and the blot
probed with a monoclonal antibody to -COP (anti- -COP) and a
monoclonal antibody to ARF (anti-ARF) and developed by ECL.
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To further study the role of ARF in coatomer recruitment, purified
components were evaluated in the binding assay. We used coatomer and
ARF concentrations that were comparable to the cytosolic concentrations
of these components employed in the assay. Fig. 8B shows
control membranes bound a small amount of coatomer. The addition of
exogenous ARF stimulated
-COP binding. Similarly, coatomer
recruitment was significantly enhanced when ARF was added to microsomes
treated with the Rab2 (13-mer) or the Rab2 protein. Both Rab2 peptide-
and Rab2 protein-incubated membranes recruited more
-COP than
control. As further proof for an ARF requirement in the assay, we
depleted ARF from cytosol by gel filtration on a Superose 6 FPLC column
(28). The binding reaction was then performed with the ARF-deficient,
coatomer-containing cytosol in the presence or absence of the Rab2
(13-mer) or Rab2 protein (Fig. 8C). In these reactions, the
amount of membrane-associated
-COP was negligible. However, the
addition of purified recombinant ARF to the assay restored
-COP
binding. As we observed with the purified components, the level of
membrane-bound COPI is greater in reactions containing Rab2 peptide and
Rab2 protein. These membranes also recruit more
-COP than microsomes
incubated with purified components suggesting that an additional
cytosolic factor may promote membrane binding. Interestingly, the
amount of bound ARF is comparable between control and treated membranes
(Fig. 8C, anti-ARF). Most likely ARF acts catalytically to
facilitate coatomer recruitment. These combined results suggest that
the Rab2 peptide and protein do not "by-pass" ARF but require
ARF-dependent assembly of
-COP containing coat complex
onto membrane.
Calphostin C Inhibits Rab2 Peptide and Rab2 Protein Recruitment of
-COP--
It has been reported that ARF/coat assembly increases in
response to protein kinase C (PKC) (30). We know from our previous studies that the specific PKC inhibitor calphostin C, arrests protein
traffic prior to the block by the Rab2 peptide (2) and therefore PKC or
a PKC-like protein functions upstream of Rab2. To determine whether PKC
influenced Rab2 peptide and Rab2 protein activity, coatomer binding was
assessed in the presence of calphostin C. This molecule recognizes the
highly conserved cysteine-rich motif present in the PKC regulatory
domain and competes for binding with diacylglycerol and phorbol esters
(31, 32). Membranes preincubated with 2.5 µM calphostin C
did not recruit ARF or
-COP (Fig. 9).
We observed a similar result with peptide-treated membrane. The ability
of the Rab2 peptide to increase COPI binding was completely lost when
membrane was co-incubated with calphostin C (Fig. 9). These membranes
contained barely detectable ARF. This effect was not limited to the
peptide. Membranes incubated with Rab2 protein failed to bind
-COP
when treated with calphostin C. However, calphostin C does not
interfere with Rab2 protein membrane association. As we observed with
the peptide, membrane-bound ARF was negligible. These data provide
evidence that PKC or a PKC-like protein is an upstream effector and
required for the Rab2 (13-mer) and the Rab2 protein to stimulate COPI
recruitment.

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Fig. 9.
Calphostin C blocks the ability of the Rab2
peptide and Rab2 protein to stimulate -COP recruitment.
Microsomes prepared as described under "Experimental Procedures"
were preincubated with or without 2.5 µM calphostin C in
the presence of 50 µM Rab2 (13-mer) or 100 ng of
recombinant Rab2 protein for 30 min on ice. The membranes were then
supplemented with cytosol and GTP S and incubated at 37 °C for 10 min. The amount of membrane-bound -COP and ARF was determined as
described under "Experimental Procedures." Calphostin C inhibited
binding of ARF and coatomer to membranes incubated with the Rab2
(13-mer) and Rab2 protein.
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To further study the role of PKC in mediating Rab2 (13-mer) and Rab2
protein-activated COPI recruitment, we performed the binding reaction
with chelerythrine chloride. This molecule acts on the catalytic domain
of PKC and specifically inhibits PKC kinase activity (33). Fig.
10 (lane D) shows membranes
treated with 2.0 µM chelerythrine chloride bound
-COP
and ARF at a level equal to control membranes supplemented with GTP
S
(Fig. 10, lane B). The reagent is active because control
kinase reactions using bovine PKC were inhibited (data not shown). We
co-incubated chelerythrine chloride and the Rab2 peptide in the binding
assay. The kinase inhibitor had no influence on the ability of the Rab2
peptide to stimulate COPI binding nor did the reagent affect ARF
membrane association (Fig. 10, lane E). Similarly,
chelerythrine chloride did not influence
-COP or ARF binding to Rab2
protein-treated membranes (Fig. 10, lane G). These results
indicate that PKC phosphorylating activity is not required.

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Fig. 10.
PKC kinase activity is not required for COPI
binding. HeLa cell membranes were preincubated with the following
reagents in the absence of cytosol and GTP S for 30 min on ice, then
cytosol and GTP S were added and incubated at 37 °C for 10 min to
promote coatomer (closed bar) and ARF recruitment
(open bar); lane A, control membranes, no
GTP S; lane B, control membranes supplemented with
GTP S; lane C, 50 µM Rab2 peptide and
GTP S; lane D, 2.0 µM chelerythrine chloride
and GTP S; lane E, 2.0 µM chelerythrine
chloride, 50 µM Rab2 peptide and GTP S; lane
F, 100 ng of Rab2 protein and GTP S; lane G, 2.0 µM chelerythrine chloride, 100 ng of Rab2 protein, and
GTP S. The amount of -COP and ARF recruited to membrane was
quantitated as described under "Experimental Procedures."
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DISCUSSION |
We previously reported that a peptide corresponding to the amino
terminus of Rab2 was a potent and irreversible inhibitor of ER to Golgi
transport. The Rab2 peptide did not interfere with vesicle budding from
the ER nor did it prevent intra-Golgi trafficking (2). These results
suggested that the Rab2 (13-mer) specifically arrested transport from
VTCs. Since Rab2 immunolocalizes to pre-Golgi intermediates and the
Rab2 peptide interferes with traffic from these intermediates, we
wished to learn if the peptide influenced other transported-related
proteins which also target to these structures. Our thought was that
this information might shed light on the mechanism by which the Rab2
peptide inhibits protein trafficking. To address this question, we
analyzed the distribution of COPI by focusing on the
-COP subunit.
We observed a dramatic change in
-COP distribution after peptide
treatment. Vesicular structures containing
-COP accumulated near the
nucleus and in proximity to the Golgi complex. Similar results have
been reported for anoxic pancreatic acinar cells (34). In those
studies, ATP-depletion did not alter the Golgi stack but greatly
reduced the number of ER to Golgi transport vesicles that were replaced
by cytoplasmic, fibrillar aggregates containing
-COP. These
aggregates represent cytoplasmic pools of coatomer that self-associate
after disruption of ER to Golgi traffic. Although like the anoxic
condition the organization of the Golgi complex and the endoplasmic
reticulum did not change after Rab2 peptide treatment, we observed a
different
-COP phenotype. Our studies show structures that
overlapped when labeled with antibody to
-COP, antibody to p58, and
antibody to Rab2, indicating that the Rab2 peptide did not cause
-COP membrane dissociation to the cytosol. On the contrary, we
believe that VTCs are perpetually coated with COPI which interferes
with subsequent trafficking through the Golgi complex. This result is
similar to the effect produced by GTP
S when added to the intra-Golgi transport assay (35). In that case, coated vesicles and coated membranes form which cannot uncoat and inhibit subsequent fusion events
necessary for intercisternal transport. Since VTCs in peptide-treated cells are "stalled," components are not retrieved from these
structures that are necessary for cargo transport from the ER. Our
previous observation that the Rab2 (13-mer) caused ts045 VSV-G to
accumulate in punctate structures which were smaller and less abundant
than VTCS that accumulate at 15 °C, supports this interpretation
(2). We predict that prolonged incubation with the Rab2 peptide would ultimately result in complete inhibition of transport from the ER as
well as recycling from pre-Golgi and Golgi compartments.
The results of our microsomal binding assay show that the Rab2 (13-mer)
increased the rate and extent of COPI membrane association and that
this enhanced recruitment is specific to the Rab2 peptide. We did not
observe a similar level of
-COP binding after incubation with a
scrambled peptide or with amino-terminal peptides made to other Rab
proteins. Interestingly, the Rab1B peptide and Rab2 peptide share 8 amino acids, yet the Rab1B peptide did not interrupt protein traffic
(2) nor affect COPI recruitment. The ability of the Rab2 protein to
promote
-COP membrane association and the lack of recruitment by the
Rab2
14 protein supports our contention that the peptide functions as
a bona fide Rab2 domain.
Combined biochemical and morphological data suggests that the peptide
acts at a post-ER budding stage but before VSV-G exit from pre-Golgi
structures. These structures segregate anterograde from
retrograde-transported proteins, an event that requires the participation of ARF and coatomer (10). Our results indicate that ARF
is necessary for Rab2 peptide and Rab2 protein-stimulated COPI binding.
We suggest that ARF functions catalytically to facilitate Rab2 peptide
and Rab2 protein enhancement of COPI binding. This requirement for ARF
is consistent with the numerous publications showing ARF involvement in
COPI recruitment (10, 21, 22). In particular, addition of an activated
form of ARF (ARF1 Q71L) causes VSV-G to accumulate in pre-Golgi
structures that contain
-COP and p53/p58 (10). Furthermore, ARF1 can
mediate the in vitro recruitment of the COPI coat to
subcellular fractions enriched in cis Golgi/intermediate compartment
membranes (26).
The Rab2 (13-mer) binds to membrane. However, the peptide did not
interfere with Rab2 protein membrane association. Although it appears
that the peptide is not a competive inhibitor for Rab2 protein
membrane-binding sites, the Rab2 (13-mer) might influence Rab2 function
by interaction with a downstream Rab2 effector. This effector could
require interaction with the Rab2 amino terminus to regulate coat
recruitment. The Rab2 peptide would therefore block the functional
interaction of Rab2 with the downstream effector. This inhibition would
lead to uncontrolled coatomer binding ultimately inhibiting vesicular
traffic in the early secretory pathway. We have observed enhanced
-COP recruitment when membranes are incubated with Rab2 protein and
GTP (data not shown). However, a significant increase in
-COP
binding was obtained when GTP
S was included in the assay. The
irreversible binding of the Rab2 protein to membrane mimics the
irreversible nature of the Rab2 (13-mer). We propose that in steady
state, Rab2 plays a role in coatomer recruitment to pre-Golgi
intermediates (VTCs). This recruitment process requires PKC or a
PKC-like protein.
Protein kinase C activity has been found in regulated (36) and
constitutive exocytosis (31) and is required for receptor traffic
through the endocytic path (37). All members of this family of
isoenzymes have an amino-terminal regulatory domain that contains
binding sites for phospholipid/diacylglycerol and calcium, and a
carboxyl-terminal catalytic domain that binds ATP and has kinase
activity (38). A variety of compounds are available which specifically
bind to these domains and either activate or inhibit. These compounds
allow one to determine if PKC exerts an effect independent of kinase
activity. We evaluated the effect of calphostin C in the binding assay
because PKC regulates ARF and COPI (30). Additionally, PKC inactivation
by calphostin C arrests transport in the early secretory pathway before
the site of Rab2 peptide inhibition (2, 39). Calphostin C binds to the
PKC regulatory domain and blocks binding of diacylglycerol. In this
study, calphostin C inhibited Rab2 (13-mer) and Rab2 protein stimulation of
-COP membrane binding which indicates that PKC or a
PKC-like protein functions upstream of the Rab2 protein. The kinase
activity of this enzyme is not required based on the result that
chelerythrine chloride which binds to the catalytic domain had no
influence on
-COP recruitment.
The role of PKC in mediating intracellular trafficking independent of
phosphorylation has been reported for ER to Golgi transport (39) and in
the production of post-Golgi vesicles (40). In both cases, a
phorbol-ester binding protein was proposed to participate in the
respective transport event. However, unlike the soluble phorbol
ester-sensitive protein required for ER to Golgi transport, we believe
that the PKC-like molecule required in Rab2 activity is membrane
associated. Membranes preincubated with calphostin C, then pelleted and
resuspended in cytosol and GTP
S do not bind
-COP (data not
shown). PKC isozymes are differentially expressed in tissue and
cell-type and each translocate to a specific intracellular site upon
activation by phorbol esters (41, 42). It is possible that the PKC-like
molecule required for Rab2 activated
-COP recruitment differs from
the phorbol ester-binding protein that regulates exit from the ER.
The Proposed Role of Rab2 in the Secretory Pathway--
The
phylogenetic tree of the Ras superfamily shows that Rab2 is most
closely related to Rab4 which is interpreted to mean that these
proteins are similar in function (43). Therefore, it is conceivable
that the role of Rab2 in exocytosis is analogous to the role of Rab4 in
endocytosis. In the endocytic pathway, Rab4 regulates the early
recycling step from the "sorting endosome" to the plasma membrane
(44-46). Since COPI-coated vesicles are involved in retrograde
transport (13), the Rab2 protein may function in protein sorting and
recycling events from pre-Golgi intermediates or "sorting exosomes"
(7, 10). In this case, Rab2 would regulate vesicular traffic through a
subcompartment within pre-Golgi elements. Such a subcompartment might
function to sort and recycle escaped ER proteins that possess retrieval sequences. The recruitment of COPI to this subcompartment may result in
vesicles that contain recycling proteins. The Rab4 protein plays a
similar role in endocytosis by controlling a rate-limiting step in
receptor transport from early endosomes to recycling endosomes (44).
We thank Dr. Nick Davis for criticial reading
of the manuscript.