A Putative Heterotrimeric G Protein Inhibits the Fusion of
COPI-coated Vesicles
SEGREGATION OF HETEROTRIMERIC G PROTEINS FROM COPI-COATED
VESICLES*
J. Bernd
Helms
§,
Désiré
Helms-Brons
,
Britta
Brügger
,
Ioannis
Gkantiragas
,
Heike
Eberle
,
Walter
Nickel
,
Bernd
Nürnberg¶,
Hans-Hermann
Gerdes
, and
Felix T.
Wieland
From the
Biochemie-Zentrum Heidelberg (BZH),
University of Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg,
¶ Freie Universitaet Berlin, Universitätsklinikum Rudolf
Virchow, Institut für Pharmakologie, Thielallee 67-73, 14195 Berlin, and
Department of Neurobiology, University of
Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany
 |
ABSTRACT |
Heterotrimeric G proteins have been implicated in
the regulation of intracellular protein transport, but their mechanism
of action remains unclear. In vivo, secretion of
chromogranin B, tagged with the green fluorescent protein, was
inhibited by the addition of a general activator of trimeric G proteins
(AlF4
) to stably transfected Vero cells and
resulted in an accumulation of the tagged protein in the Golgi
apparatus. In an in vitro assay that reconstitutes
intra-Golgi protein transport, we find that a membrane-bound and
AlF4
-sensitive factor is involved in the
fusion reaction. To determine whether this effect is mediated by a
heterotrimeric G protein localized to COPI-coated transport vesicles,
we determined the presence of G proteins on these vesicles and found
that they were segregated relative to the donor membranes. Because G
proteins do not have an obvious sorting, retention, or retrieval
signal, we considered the possibility that other interactions might be responsible for this segregation. In agreement with this, we found that
trimeric G proteins from isolated Golgi membranes were partially insoluble in Triton X-100. Identification of the proteins that interact
with the heterotrimeric G proteins in the Golgi-derived detergent-insoluble complex might help to reveal the regulation of
protein secretion mediated by heterotrimeric G proteins.
 |
INTRODUCTION |
Vesicular protein transport between intracellular compartments
involves the budding, targeting, and fusion of transport vesicles. These highly specific events are controlled by proteins that belong to
a general protein machinery (1). Among these are various small
molecular weight GTP-binding proteins as well as heterotrimeric GTP-binding (G)1 proteins
that are known to regulate vesicular transport. Of the small molecular
weight GTP-binding proteins, the ADP-ribosylation factor (ARF) is a
stoichiometric component of COP-coated vesicles (2) and is involved in
coatomer recruitment (3, 4) as well as in uncoating of COP-coated
vesicles (5). Another member of the small molecular weight GTP-binding
proteins, the Rab-family of proteins, is thought to act in concert with
v- and t-SNAREs, which regulate the specificity of membrane fusion
(6).
The involvement of heterotrimeric G proteins in intracellular protein
transport was initially suggested by the finding that AlF4
, an activator of heterotrimeric but not
of monomeric G proteins (7), inhibits intra-Golgi transport in a
cell-free system (8). In vivo evidence for the involvement
of heterotrimeric G proteins comes from overexpression of
G
i3, which localizes to the Golgi complex and inhibits
protein transport through the secretory pathway (9).
The mechanisms of the involvement of G proteins in intracellular
protein traffic remains to be established. Probes that interfere with
the classical G protein cycle at the plasma membrane have been used to
study a G protein cycle in reconstituted intracellular vesicular
transport assays, but conflicting results have been obtained (reviewed
in Ref. 10). There seems to be a possibility of alternative G protein
cycles at the early stages of the secretory pathway, whereas a
classical G protein cycle may operate at later stages (10). Alternative
G protein cycles may include interactions with ARF protein, either via
a direct interaction of G
with ARF (reviewed in Ref.
10) or via interaction with an ARF GTPase-activating protein (11).
Recently, G proteins have been implicated in the maintenance of the
Golgi structure (12, 13). It is not clear, however, whether this
signaling pathway is the same as the one involved in protein
secretion.
To investigate the role of G proteins in early stages of the secretory
pathway, we expanded initial experiments that showed an inhibitory
effect of AlF4
in intra-Golgi transport (8).
We show that the action of AlF4
is distinct
from the action of GTP
S. Heterotrimeric G proteins are excluded from
COPI-coated transport vesicles, possibly by interaction with other
proteins in a detergent-insoluble complex.
 |
EXPERIMENTAL PROCEDURES |
Materials--
L-1-Tosylamido-2-phenylethyl
chloromethyl ketone-treated trypsin was obtained from Worthington
Biochemical Corp., Freehold, NJ. Soybean trypsin inhibitor was
purchased from Sigma. CHO Golgi membranes were isolated as described
(14). Bovine brain cytosol was prepared according to Malhotra et
al. (15). A total membrane fraction from normal rat kidney cells
was prepared by passing a 2-ml cell pellet, resuspended in a total
volume of 15 ml of 20 mM Tris/HCl, pH 7.4, 1 mM
EDTA, 50 mM NaCl (TEN buffer) several times through a G25
needle until more than 90% of the cells were broken as judged by
trypan blue permeability. Whole cells and nuclei were spun down by
centrifugation for 5 min at 500 × g. The postnuclear
supernatant was centrifuged 45 min at 100,000× g,
and the membrane pellet was resuspended in 8 ml of TEN
buffer and stored at
80 °C. Antibodies used against the G
protein-specific subunits are AS 6 (G
oc), AS 232 (G
12), AS 266 (G
ic), AS 348 (G
s), AS 369 (G
q/11) and are described in
(16). AS398 (G
c) is described in (17). In some
experiments a G
i3- and a G
i1/2-specific antibody from NEN Life Science Products was used (EC/2 and AS/7, respectively). Polyclonal rabbit antibodies against p23 (#1327) and
'-COP (#891) were used as described (18, 19).
AlF4
was freshly prepared as a 10× stock
solution by premixing equal volumes of 1 mM
AlCl3 with 600 mM NaF.
Cell-free Intra-Golgi Transport Assay--
The standard
incubation conditions were as described previously (14, 15).
In Vivo Secretion of Green Fluorescent Protein-tagged Human
Chromogranin B (hCgB-GFP)--
Vero cells (clone V7) stably
transfected with a hCgB-GFP fusion protein were grown on glass
coverslips to ~40% confluency, and expression of the GFP construct
was as described (20). Cells were mounted in Fluoromount G (Biozol) and
analyzed using a Zeiss Axiovert 35 microscope equipped with the
appropriate filters for fluorescein isothiocyanate-derived
fluorescence.
Protease Digest--
10 µg of isolated CHO Golgi membranes was
pre-incubated in 25 mM Hepes/KOH, pH 7.2, 20 mM
KCl, 2.5 mM magnesium acetate, and 10 µM GDP
to analyze the G protein in its inactive conformation, which is more
susceptible to protease digest (21). The membranes were then incubated
with 2.5 µg of trypsin and/or 25 µg of soybean trypsin inhibitor
for 15 min at 30 °C. The Golgi membranes were pelleted through a
sucrose cushion (15% sucrose w/v) at 14,000 × g for
30 min. The proteins were analyzed by SDS-PAGE and subsequent Western
blot analysis (ECL) using a G
i3-specific antibody (EC/2) and a p23-specific antibody raised against a luminal part of the protein (#1327) (18). Protein bands were quantified by densitometry measurements using a flatbed scanner and NIH image software (National Institutes of Health, U. S. A.).
Isolation of COPI-coated Vesicles--
COPI-coated vesicles were
isolated from large scale incubation of CHO Golgi membranes with bovine
brain cytosol under standard cell free assay conditions in the presence
of 20 µM GTP
S or 20 µM GTP
S + AlF4
(50 µM AlCl3 + 30 mM NaF) (15, 22).
Lipid Analysis--
Lipids were extracted according to Bligh and
Dyer (23) in the presence of 14:0/14:0 phosphatidylcholine (PC) as a
nonnatural standard for quantitation. Lipids were analyzed and
quantified by nano-electrospray ionization tandem mass spectrometry
(24). PC was measured by detection of (M + H)+ ions by
precursor scanning for m/z 184 (collision cell
offset,
35 V).
Isolation of a Detergent-insoluble Fraction from Golgi
Membranes--
Golgi membranes (5 µg) were pelleted at 14,000 × × g at 4 °C for 30 min and resuspended in 50 µl of
25 mM Pipes, pH 6.5, 2 mM EDTA, 150 mM NaCl (PEN buffer, or as described in the legends) containing 1% detergent, as indicated in the figure legend. After a
30-min incubation on ice, the detergent-insoluble fraction was isolated
by centrifugation at 14,000 × g at 4 °C for 30 min.
The pellet was resuspended in SDS-PAGE sample mixture and analyzed by
SDS-PAGE and subsequent Western blot analysis (ECL) using antibodies as
indicated in the figure legends.
 |
RESULTS |
In Vivo Inhibition of Secretion by an
AlF4
-sensitive Factor--
Heterotrimeric G
proteins have been localized to intracellular membranes along the
secretory pathway including the endoplasmic reticulum and the Golgi
complex (reviewed in Ref. 10). To determine at what stage G proteins
might be involved in the regulation of intracellular protein transport,
we used hCgB-GFP (20, 25), a marker of the secretory pathway. The
fusion protein was stably transfected in Vero cells and is expressed
upon induction with butyrate (20). hCgB-GFP is secreted from the cells
at 37 °C, resulting in the absence of a fluorescent signal in these
cells as determined by fluorescent microscopy (Fig.
1, bottom left panel). When,
however, AlF4
is added to these cells at
37 °C, a perinuclear staining is observed (Fig. 1, bottom
right panel). This indicates that the secretory pathway is
inhibited at the stage of the fluorescent signal. A similar fluorescent
signal was observed when cells were incubated at 15 and 20 °C,
conditions known to block protein transport through the Golgi complex
or at the trans-Golgi network (Fig. 1, top
panels) and which is in agreement with previous experiments (20,
25). These data indicate that the hCgB-GFP accumulates in the Golgi complex upon treatment of the cells with
AlF4
. No inhibition of protein export from
the endoplasmic reticulum is observed. This is a surprising observation
as G proteins have been implicated in the export of proteins from the
endoplasmic reticulum (26). Alternatively, trimeric G proteins at
the Golgi complex might be more intimately involved in regulation
of protein secretion than at the endoplasmic reticulum.

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Fig. 1.
AlF4 inhibits
anterograde transport through the Golgi of a hCgB-GFP fusion
protein. Vero cells stably transfected with a hCgB fusion protein
were incubated in the presence of 2 mM butyrate to
stimulate protein expression. After 20 h at 37 °C, cells were
incubated with AlF4 for 1 h at
37 °C, incubated at 15 °C for 1 h, incubated at 20 °C for
1 h, or incubated at 37 °C for 1 h as a control. Cells
were fixed, permeabilized, and analyzed as described under
"Experimental Procedures."
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|
Inhibition of COPI-mediated Transport by an
AlF4
-sensitive Factor--
To further define
an AlF4
-sensitive factor responsible for
inhibition of protein transport through the Golgi, we employed a
cell-free system that reconstitutes COPI-coated vesicular transport between Golgi cisternae (14, 15). This assay previously revealed the
involvement of a GTP
S- and an
AlF4
-sensitive factor (8). In subsequent
publications, the GTP
S-sensitive factor was identified as a
member(s) of the ARF family (2, 27). Here, we determined whether
inhibition of COPI-mediated transport by AlF4
is dependent on the cytosol (high speed supernatant) concentration. In
agreement with previous studies, COPI-mediated transport of the VSV G
protein is dependent on the presence of cytosolic factors (14), and
only at high concentrations of cytosol, GTP
S inhibits transport of
the VSV G protein (Fig. 2 and Ref. 8).
This indicates that the GTP
S-sensitive factor is recruited from the
cytosol (8). In contrast, inhibition of transport by
AlF4
does not show any cytosol dependence,
indicating that this factor resides on the Golgi membranes (Fig. 2).
This is in agreement with the membranous localization of intracellular
heterotrimeric G proteins (28), although it cannot be excluded that
AlF4
acts on some other proteins as well (see
"Discussion").

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Fig. 2.
The
AlF4 -sensitive factor resides on Golgi
membranes. Transport assays were performed under standard
conditions (see "Experimental Procedures") with increasing
concentrations of bovine brain cytosol either in the absence ( ) or
in the presence of GTP S ( ) or AlF4
( ). After incubation, incorporation of [3H]UDP-GlcNac
into VSV G protein was measured as a marker for transport.
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|
To determine the topological orientation of heterotrimeric G proteins
on Golgi membranes, we took the G
i3 subunit of G
proteins as a representative of the G
class of proteins. The
G
i3 subunit of heterotrimeric G proteins is localized to
the cis side of Golgi stacks (9, 29). As shown in Fig.
3, more than 80% of membrane-bound G
i3 was digested by trypsin in the absence of detergent.
In contrast, only the carboxyl-terminal fragment (~1 kDa) was removed
from p23, a Golgi-localized type I membrane protein with a short
cytoplasmic tail, showing that the large luminal domain of p23 was not
accessible to trypsin and that the membranes are sealed under these
conditions. In a control experiment it was shown that the luminal
domain can in principle be digested by trypsin in the presence of
detergent (Fig. 3, lane 4). Thus, with intact (sealed)
membranes, G
i3 is accessible to trypsin and therefore
must have a cytosolic orientation.

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Fig. 3.
G proteins are localized to the cytosolic
face of Golgi membranes. Isolated Golgi membranes (lane
1, Input) were digested with trypsin (lane
2) or mock-digested with trypsin in the presence of soybean
trypsin inhibitor (STI) (lane 3). As a control,
Golgi membranes were digested with trypsin in the presence of 1%
Triton X-100 (TX) (lane 4). Incubations and
analysis were performed as described under "Experimental
Procedures."
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The inhibitory effect of AlF4
in the
cell-free system was further investigated, and the formation of
transport intermediates resistant to GTP
S and NEM (8) was compared
with those formed in the presence of AlF4
. To
this end, incubation of the cell-free system was started at
t = 0 min, the transport inhibitors were added at
t = 20 min, and the reaction was continued to
completeness (60 min). Thus, if the inhibitor acts at an early step in
the vesicle transport route (e.g. budding), then it will not
act on the vesicles that have already formed (but not fused yet) during
the 20-min incubation. These vesicles can then still fuse during the
remaining time of incubation and result in a signal. On the other hand,
if the inhibitor acts late (e.g. in a fusion process), most
of the vesicles that have already formed after 20 min can still be
prevented from fusing with the acceptor membrane. An incubation without
inhibitors represents the maximal possible signal (Fig.
4, first lane). When samples are put on ice after 20 min, this represents the minimal signal i.e. a complete and instant block after 20 min of incubation
(Fig. 4, second lane). Under these conditions, vesicles that
have already formed are not able to fuse with the acceptor membrane.
Any signal above the minimal signal represents the presence of a
transport intermediate, resistant to the inhibitor. When NEM or GTP
S
were added at t = 20 min, most of the vesicles that had
formed during this time were still inhibited with NEM and to a
significantly lower extent with GTP
S, indicating a late and an early
inhibition step, respectively (Ref. 8 and Fig. 4, third and
fourth lanes). A similar experiment with
AlF4
(Fig. 4, fifth lane) resulted
in a transport intermediate resistant to
AlF4
, which is intermediate with respect to
GTP
S and NEM, indicating that a G protein acts after binding of ARF
to Golgi membranes (GTP
S effect) but before the
NSF-dependent fusion step of COPI-coated vesicles with the
acceptor membranes (NEM effect).

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Fig. 4.
AlF4 acts between
the GTP s- and NEM-sensitive transport steps. Transport assays
were performed under standard conditions (see "Experimental
Procedures"). As a control, transport of VSV G protein was measured
after a 1-h incubation at 37 °C (1st lane). Parallel
incubations were initiated at 37 °C, and after 20 min, the samples
were put on ice (2nd lane) or inhibitors were added (as
indicated in the 3rd-5th lanes), and the
reaction was continued at 37 °C for 40 min (1 h total incubation).
The error bars represent the mean average of 5 incubations.
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These results raised the possibility that heterotrimeric G proteins are
localized to COPI-coated vesicles and from there exert their inhibitory
action. To determine the presence of G proteins on COPI-coated
vesicles, we isolated these vesicles from a large scale incubation of
Golgi membranes in the cell-free system in the presence of GTP
S. The
total protein content of the vesicles cannot be used for quantitation
because this also includes contamination of the vesicle preparation
with relatively high amounts of actin and tubulin, resulting in an
overestimation of the amount of vesicles. Therefore the vesicles were
quantified based on their lipid (PC) composition. This lipid is present
in relatively high amounts in membranes and is not expected to be
sorted within the Golgi stack (30). Fig.
5 shows a comparison of equal amounts of
donor Golgi and COPI-coated vesicles, based on their
phosphatidylcholine content. p23, a Golgi-localized protein, is
enriched in Golgi membranes and becomes more concentrated in
COPI-coated vesicles, in agreement with previous observations (18). In
addition,
'-COP, a subunit of the coatomer complex, is enriched in
the vesicle fraction, as expected (Fig. 5). Antibodies against various
sub-classes of heterotrimeric G proteins were then used to identify the
presence of these G proteins on the vesicles. Most of the antibodies do detect the presence of G proteins on the donor Golgi membranes, except
for antibodies raised against the G
o subclass. In
contrast, these antibodies could not detect the presence of G proteins
in the purified COPI-vesicle fraction derived from the donor Golgi membranes, including antibodies against the G
s-,
G
i-, and G
o- subclass. Antibodies against
the G
q/11 and G
12 subclass of G proteins
detected small amounts of their antigens in COPI-coated vesicles but
clearly in much smaller amounts as compared with the donor membranes.
Likewise, an antibody against a common motif of various G
isoforms
failed to reveal any of the Golgi-localized isoforms in COPI-coated
vesicles. Also under conditions where vesicles were generated in the
presence of both GTP
S and AlF4
(to
activate G proteins during the generation of COPI-coated vesicles) are
G proteins strongly segregated from COPI-coated vesicles (Fig. 5,
lane 4).

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Fig. 5.
Heterotrimeric G proteins are excluded from
COPI-coated vesicles. CHO cells were homogenized, and Golgi
membranes were isolated from these homogenates as described under
"Experimental Procedures." COPI-coated vesicles were generated by
incubation in the cell-free system of the Golgi membranes (see
"Experimental Procedures") in the presence of GTP S or GTP S
and AlF4 . Golgi membranes and COPI-coated
vesicles were quantified by electrospray ionization tandem mass
spectrometry (see "Experimental Procedures"). The samples were
analyzed by SDS-PAGE (16) and subsequent Western blotting. Lane
1, homogenate (Hom) (10 µg of total protein);
lane 2, CHO Golgi (10 µg of total protein, corresponding
to 3.0 µg of PC); lane 3, COPI-coated vesicles
(Ves) generated in the presence of GTP S (containing 3.0 µg of PC); lane 4, COPI-coated vesicles generated in the
presence of GTP S and AlF4 (containing 2.5 µg of PC).
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G Proteins Are Present in a Detergent-insoluble Complex--
What
could be the basis for this exclusion of heterotrimeric G proteins from
COPI-coated vesicles? Because G proteins do not have an obvious
sorting, retention, or retrieval signal, we reasoned that other
interaction(s) would be the most likely explanation for Golgi
localization of heterotrimeric G proteins and their segregation from
COPI-coated vesicles. We therefore analyzed Golgi membranes after mild
detergent-treatment for the presence of complexes that contain G
proteins. Several different detergents were tested for their inability
to solubilize G proteins from Golgi membranes under conditions that
lead to solubilization of Golgi membranes. Solubilization of Golgi
membranes with Triton X-100 or CHAPS and subsequent centrifugation
resulted in a detergent-insoluble fraction containing ~50 and ~20%
of total G
i3, respectively (Fig.
6A, lanes 4 and
6). Other detergents such as octylglucoside, sodium deoxycholate (Fig. 6A, lanes 5 and 7)
and cholate (Fig. 6B, lane 4) almost
quantitatively solubilized the endogenous G
i3 and
G
subunits. Conditions for detergent insolubility in Triton X-100 were further investigated by solubilization of Golgi membranes under
various conditions. As shown in Fig. 6B, the presence of AlF4
did not alter the insolubility of
G
i3, indicating that its presence in the
detergent-insoluble fraction is independent of its state of activity.
The presence of G
i3 in the detergent-insoluble fraction is, however, affected by the presence of divalent cations. In the
presence of Mg2+, hardly any G
i3 could be
recovered in the detergent-insoluble fraction (Fig. 6B,
lane 8). These data indicate that the
G
i3-containing detergent-insoluble fraction is different
from another Golgi-localized detergent insoluble fraction, the
so-called Golgi matrix (31), which is isolated from Golgi membranes by
use of Triton X-100 and in the presence of Mg2+. It has
been shown that high salt concentrations disrupt the Golgi matrix (32).
In contrast, the addition of high salt did not disrupt the
detergent-insoluble complex (Fig. 6B, lane 7). 40-50% of G
i3 remains associated with the
detergent-insoluble pellet under these conditions, further indicating
that this complex is distinct from the Golgi matrix.

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Fig. 6.
Heterotrimeric G proteins are present in
detergent-insoluble Golgi fractions. CHO Golgi membranes
(A and B) or a total membrane fraction from NRK
cells (B) were solubilized in various detergent-containing
buffers, and the detergent-insoluble fraction was isolated as described
under "Experimental Procedures." The incubations were analyzed by
SDS-PAGE and subsequent Western blotting using the indicated
antibodies. A, lanes 1-3, CHO Golgi membranes
without solubilization (lane 1, 1.25 µg or 25% of input;
lane 2, 2.5 µg or 50% of input; lane 3, 5 µg
or 100% of input); lanes 4-7, detergent-insoluble fraction
from CHO Golgi membranes after solubilization in the following buffers:
lane 4, 1% Triton X-100 in PEN buffer; lane 5,
60 mM octylglucoside in PEN buffer; lane 6, 20 mM CHAPS in PEN buffer; lane 7, 9 mM
desoxycholate in 25 mM Hepes, 150 mM NaCl, pH
7.5. B, lanes 1-3, CHO Golgi membranes
(upper panel, 5 µg of total protein) or NRK total
membranes (lower panel, 20 µg of protein) without
solubilization (lane 1, 25% of input; lane 2,
50% of input; lane 3, 100% of input); Lanes
4-9, detergent-insoluble fraction from CHO Golgi membranes or NRK
total membrane fraction after solubilization in the following buffers:
lane 4, 1% cholate in 10 mM Hepes, pH 8.0, 20 mM -mercaptoethanol, 1 mM EDTA (conditions
used to solubilize and purify G proteins); lane 5, 1%
Triton X-100 in PEN buffer; lane 6, same as lane
5 but in the presence of AlF4 ;
lane 7, same as lane 5 but in the presence of 500 mM NaCl; lane 8, 1% Triton X-100 in 50 mM MOPS, pH 7.0, 0.1 mM Mg2+, 1 mM dithiothreitol, and 10% sucrose (conditions used to
isolate the Golgi matrix (31)); lane 9, same as lane
8 but in the presence of 500 mM NaCl to disrupt the
Golgi matrix (32). CHO Golgi membranes and a total membrane fraction
from NRK cells were prepared as described under "Experimental
Procedures."
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Our isolated Golgi membranes are contaminated with approximately
5-10% plasma membrane.2 We
could not exclude the possibility that the insolubility of G
i3 is due to the presence of this protein in caveolae,
which are present in the plasma membrane and which exhibit similar
insolubility properties (33, 34). We therefore analyzed the
characteristics of detergent insolubility of G
i3 present
in NRK cells as well. In this cell-line, G
i3 is
predominantly localized to Golgi membranes, and no plasma membrane
localization could be observed (9). As shown in Fig. 6B, the
characteristics of insolubility in Triton X-100 are very similar when
comparing CHO Golgi membranes with a membrane fraction from NRK
cells.
 |
DISCUSSION |
Reconstitution of intra-Golgi protein transport revealed the
presence of both a GTP
S- and an
AlF4
-sensitive factor that inhibits the
transport of the VSV G protein and results in an accumulation of
COPI-coated vesicles (8). The GTP
S-sensitive factor was previously
identified as a member of the ARF-family (2, 27), which in its purified
form cannot be activated by AlF4
(7).
Recently, it was shown that AlF4
can act on
small molecular weight GTP-binding proteins such as Ras when it
associates with its corresponding GTPase-activating protein (35, 36).
By analogy, ARF complexed to its GTPase-activating protein might be a
potential target for AlF4
in our in
vitro assay system. In this case, GTP
S and
AlF4
would act on the same protein (complex).
The experiments described here, however, make it unlikely that
AlF4
mimics the GTP
S effect for several
reasons. (i) In contrast to ARF, the
AlF4
-sensitive factor resides on Golgi
membranes (Fig. 2), (ii) whereas ARF is involved in the recruitment of
coatomer to Golgi membranes (3, 4, 37) and uncoating of COPI-coated
vesicles (5), the AlF4
-sensitive factor might
be involved in the actual fusion step (Fig. 2). With the transport
assay, one can differentiate between vesicle-mediated transport and
uncoupled fusion. The latter likely reflects the actual fusion step as
soluble NSF attachment protein proteins and NSF are involved in this
process (38). The uncoupled fusion reaction dominates in the transport
assay under conditions of limited amounts of cytosol, which results in
limited amounts of the coat proteins ARF and coatomer. As shown in Fig.
2, with reduced amounts of cytosol, GTP
S does not inhibit the
transport assay, and this signal reflects uncoupled fusion. Thus, under transport (or rather fusion) conditions that are independent of ARF,
AlF4
does inhibit the transport assay as
effectively as under conditions of vesicle-mediated transport. These
data show that the AlF4
effect is distinct
from ARF. We also take these data as preliminary evidence for the
involvement of an AlF4
-sensitive factor in
the fusion reaction. In agreement with this, we find that
AlF4
also inhibits the vesicle-mediated
reaction in the presence of saturating amounts of cytosol (Fig. 2), and
(iii) determination of transport resistant-intermediates also shows a
difference between GTP
S and
AlF4
, with the
AlF4
inhibition occurring after the GTP
S
inhibition (Fig. 4). Surprisingly, the
AlF4
-resistant intermediate occurs before the
NEM-resistant intermediate, suggesting that
AlF4
inhibits before the action of NSF, which
itself is part of the fusion reaction. NSF is part of a 20 S particle
containing the cognate v- and t-SNARE proteins, and hydrolysis of its
bound ATP results in disassembly of the SNARE complex (39). Therefore it is tempting to speculate that the
AlF4
-sensitive factor is involved in the
assembly of the SNARE complex. This can be either during the actual
recognition of a v-SNARE and its corresponding t-SNARE (docking
reaction) or during the recruitment of v-SNAREs into transport
vesicles. It cannot be excluded that the actual target of
AlF4
is not a heterotrimeric G protein but
rather a phosphate-binding protein like phosphatases (40). Phosphatases
are generally inhibited by NaF itself in the millimolar range, without
the addition of aluminum (41). Inhibition of the transport assay,
however, is absolutely dependent on the presence of both
F
and Al3+-ions (8).
Recently, heterotrimeric G proteins have been implicated in the
regulation of the Golgi structure (12, 13). In contrast to our data
indicating that G proteins act downstream of ARF, it was speculated
that these G proteins act upstream in the process of COPI vesicle
formation (13). Thus, it is possible that trimeric G proteins act at
multiple stages in the secretory pathway. Alternatively, the G
protein-mediated signaling pathways involved in the maintenance of the
Golgi structure operate independently of G proteins involved in protein
secretion.
Segregation of Golgi-localized G Proteins from COPI-coated
Vesicles--
To determine the presence of G proteins in COPI-coated
vesicles, we compared equal amounts of Golgi membranes with COPI-coated vesicles based on their PC content. We find that the segregation of
trimeric G proteins from COPI-coated vesicles seems more efficient than
has been described for Golgi-resident proteins (42).
The experimental system does not allow for discrimination between G
proteins in their inactive (GDP) and active (GTP) conformation. GTP
S
is needed to generate sufficient amounts of COPI-coated vesicles for a
solid quantification of the G protein subunits. The portion of G
proteins activated under our experimental conditions is unknown.
Therefore, we used AlF4
to activate the
complete pool of trimeric G proteins. This condition did not affect the
segregation of trimeric G proteins from COPI-coated vesicles (Fig. 5),
indicating that it is the activated form of trimeric G proteins that
are segregated from COPI-coated vesicles. It cannot be excluded,
however, that the inactive, GDP-bound trimeric G proteins enter the
vesicles and are not segregated.
The efficiency of segregation might be overestimated for some G
proteins. For example, G
s is predominantly localized to
the trans-Golgi and trans-Golgi network in
exocrine pancreas (29). Because COPI-coated vesicles are predominantly
generated from the cis-, medial-, and
trans-face of the Golgi stack (43) but not from the
trans-Golgi network, G
s might simply not be
present in the donor membranes at the site of vesicle formation.
G
i3 and G
q/11 have been localized to the
cis/medial Golgi cisternae (9, 29), and therefore these
subunits must have been segregated during the generation of COPI-coated
vesicles. The exact intracellular localization of the other G
proteins tested for their segregation remains to be determined.
Are G Proteins Segregated from COPI-coated Vesicles by Specific
Interactions with Other Proteins?--
We used G
i3 as a
marker for cis-Golgi-localized trimeric G proteins and found
that it is present in a detergent-insoluble complex. This indicates
that G
i3 interacts with other proteins and that this
interaction is not disrupted by some detergents. G
i3 is
present in the detergent-insoluble pellet, irrespective of pretreatment
of the Golgi membranes with GDP, AlF4
(Fig.
6), or with GTP
S (data not shown). This correlates well with the
finding that trimeric G proteins are segregated from the vesicles in
the absence and presence of AlF4
(Fig. 5).
How is G
i3 retained in the detergent-insoluble complex?
A critical observation is that stimulation of G proteins with
AlF4
does not change its participation in
detergent-insoluble fractions (Fig. 6). This excludes the possibility
that the G protein subunit 
is responsible for the detergent
insolubility of G
i3, because this subunit only interacts
with G
in its inactive (GDP) state (44). Another possibility is that
Golgi-localized G proteins are present in a detergent-insoluble complex
due to their acylation with myristate and palmitate. Amino-terminal
dual acylation of G
i3 with myristate and palmitate has
been reported to be a determinant for caveolar localization (45, 46).
Acylation is unlikely to be the sole determinant, since palmitoylation
is a dynamic posttranslational event, and stimulation of G
subunits
results in depalmitoylation (47, 48). Activation of G
with
AlF4
also results in de-palmitoylation of the
G
-subunit (48), yet its detergent insolubility is not affected (Fig.
6). Finally, these general mechanisms such as interaction with caveolae
or G
or dual acylation with myristate and palmitate cannot
explain the presence of G
subunits in the Golgi complex but rather
would target these proteins to the plasma membrane. Given these
considerations, it is likely that specific protein-protein interactions
cause the presence of Golgi-localized G proteins in a
detergent-insoluble complex. We are currently purifying this complex to
homogeneity. Identification of proteins that interact with the
heterotrimeric G proteins in the Golgi-derived detergent-insoluble
complex might give an indication on the G protein-mediated signal
transduction involved in protein secretion.
 |
ACKNOWLEDGEMENTS |
We acknowledge Dr. J. E. Rothman for
providing support during the early stages of this research, as it was
initiated by J. B. H. as a postdoctoral fellow in his laboratory. We
thank members of the Wieland lab for helpful discussions during the
course of this work.
 |
FOOTNOTES |
*
This work was supported by a grant from the Deutsche
Forschungsgemeinschaft and of the Human Frontier Science Program
Organization.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.
§
Recipient of a fellowship from the Alexander von Humboldt
Foundation. To whom correspondence should be addressed. Tel.:
49-6221-544688; Fax: 49-6221-544366; E-mail:
Helms{at}urz.uni-heidelberg.de.
1
The abbreviations used are: G protein,
heterotrimeric GTP-binding protein; CHO, Chinese hamster ovary; NRK,
normal rat kidney; PC, phosphatidylcholine; VSV, vesicular
stomatitis virus; NEM, N-ethylmaleimide; ARF,
ADP-ribosylation factor; GTP
S, guanosine 5'-O-(thiotriphosphate); hCgB-GFP, green fluorescent
protein-tagged human chromogranin B; PAGE, polyacrylamide gel
electrophoresis; Pipes, 1,4-piperazinediethanesulfonic acid; CHAPS,
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; MOPS,
4-morpholinepropanesulfonic acid; NSF, NEM-sensitive factor.
2
B. Brügger, unpublished data.
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