From the Departments of Cell and Molecular Biology, The Scripps Research Institute, La Jolla, California 92037
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ABSTRACT |
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The COPII coat complex found on endoplasmic
reticulum (ER)-derived vesicles plays a critical role in cargo
selection. We now address the potential role of biosynthetic cargo in
modulating COPII coat assembly and vesicle budding. The ER accumulation
of vesicular stomatitis glycoprotein (VSV-G), a transmembrane protein, or the soluble PiZ variant of Newly synthesized (biosynthetic) cargo translocated into the
endoplasmic reticulum (ER)1
is exported to downstream compartments by COPII vesicle carriers (reviewed in Refs. 1 and 2). Incorporation into vesicles is dependent
on protein folding, a process that is stringently monitored by the ER
(3). During export, biosynthetic cargo is sorted from resident ER
proteins and selected for incorporation into vesicles by interacting
with the Sar1 and Sec23/24 components of the COPII coat prior to
vesicle formation (4, 5). Subsequently, the selected cargo incorporated
into these pre-budding complexes is packaged into vesicles upon
addition of the Sec13/31 complex. Because both membrane-associated
proteins and soluble cargo proteins become physically associated with a
pre-budding complex prior to export, it becomes important to determine
whether biosynthetic cargo or cargo receptors can regulate vesicle coat
assembly (4, 5), thereby linking cargo sorting to vesicle formation
from the ER.
A role for cargo in directing vesicle formation was previously observed
in both the late secretory pathway and the endocytic pathway (reviewed
in Ref. 6). The transferrin receptor contains sorting determinants that
are capable of interacting with the AP-2 complex that mediates
clathrin-dependent endocytosis from the plasma membrane
(reviewed in Ref. 7). Overexpression of transferrin leads to increased
AP-2/clathrin coat recruitment to the plasma membrane and
clathrin-coated endocytic vesicle formation (8, 9). Cargo proteins and
cargo receptors in the trans-Golgi network (TGN), including mannose
6-phosphate receptors and the major histocompatibility class II protein
complex, contain sorting determinants that are capable of binding to
the AP-1 adaptor complex (7). The AP-1 complex mediates
clathrin-dependent export of these proteins from the TGN
(10-12). Overexpression or elimination of these cargo proteins affects
both AP-1 coat recruitment and clathrin-coated vesicle formation.
To determine whether cargo exported from the ER is capable of
modulating COPII vesicle formation, we have taken advantage of
vesicular stomatitis virus (VSV)-infected cells to control biosynthetic
cargo availability. VSV-infected cells express a single surface
glycoprotein, VSV-G. The transport of both the wild-type and a mutant
form of VSV-G, strain tsO45, has been extensively utilized to define
the basic biochemical components and principles of operation of the
secretory pathway (reviewed in Refs. 2, 13, and 14). In particular,
tsO45 VSV-G (VSV-Gts) accumulates in the ER due to a
temperature-sensitive folding defect at the restrictive temperature of
39.5 °C. Upon shift to the permissive temperature (32 °C) folding
resumes, allowing us to synchronize exit from the ER. The ability to
synchronize movement of VSV-Gts has played an important
role in defining the normal folding intermediates associated with the
molecular chaperones calnexin and BiP preceding ER export (15-18). In
addition, because host protein synthesis is blocked following VSV
infection of cells, VSV-Gts is the major glycoprotein
transiting the secretory pathway. Therefore, VSV-Gts allows
us to follow the effects of a single well defined biosynthetic cargo
molecule on COPII recruitment and vesicle budding when it has
accumulated in the ER as a folding intermediate or when it is
subsequently matured to the folded state for transport (4, 19-23).
We have now applied a variety of techniques to monitor the effect of
biosynthetic cargo on vesicle budding from secretory compartments.
These include the following: (i) quantitative stereology to follow
membrane flow at the ultra-structural level in vivo (24),
(ii) biochemical assays to monitor ER vesicle budding in
vitro (25), (iii) an assay to monitor the interactions of cargo
with COPII components in the ER prior to vesicle formation (4), and
(iv) an assay that allows us to follow the recruitment of COPI
components to downstream pre-Golgi and Golgi compartments that may be
affected by export from the ER. The effects of availability of a
membrane-bound cargo molecule such as VSV-G on ER export was also
compared with that of an endogenous soluble cargo molecule, the PiZ
variant of Materials--
Calphostin C, Roche 31-8220, staurosporine, and
phorbol 12-myristate 13-acetate were obtained from Calbiochem.
Calphostin C was made fresh every 2 weeks. Both reagents were added
from stock solutions in dimethyl sulfoxide (Me2SO).
Digitonin was obtained from Wako BioProducts (Richmond, VA).
Endoglycosidase H was obtained from Boehringer Mannheim. GS beads were
obtained from Amersham Pharmacia Biotech. Other antibodies used in this
study were generous gifts from the following laboratories: a polyclonal
antibody against Sec23p from R. Schekman, University of California,
Berkeley (Berkeley, CA); a polyclonal antibody against p58 from J. Saraste, University of Bergen (Oslo, Norway); a monoclonal antibody to
VSV-G from T. Kreis (28); a monoclonal antibody against Transmission Electron Microscopy and
Morphometry--
Measurement of the basic cell morphometric
parameters, the number of ER and Golgi-derived budding profiles, and
the number and size of pre-Golgi intermediates were determined as
described (24)
Proteins--
Recombinant Sar1 and GST-Sec23 proteins utilized
in this study were prepared as described (4).
Virus Infection--
Normal rat kidney (NRK) cells were infected
with VSV as described (22, 30). Briefly, cells were infected at a
multiplicity of 10-20 plaque-forming units with wild-type or the tsO45
strain of vesicular stomatitis virus (21) for 4 h at the
restrictive temperature. For measurement of transport in
vivo, protocols were as described under "Results" and in the
figure legends. For measurement of transport in vitro,
preparation of cell homogenates was as described (25).
ER Budding Assay--
COPII vesicle formation reactions using
NRK microsomes were performed and budding quantitated using antibodies
specific for Sec23, p58, and VSV-G using Western blotting (25). To
enhance preservation of the temperature-sensitive phenotype observed
in vivo, in some experiments (see "Results"), 1 mM dithiothreitol was added to cells during the final 15 min of incubation at 39.5 °C. Cells were harvested, washed, and
homogenized in the presence of 1 mM dithiothreitol.
Subsequently, membranes were washed twice in transport buffer lacking
dithiothreitol prior to freezing as described(25).
Preparation of Membranes and GST Complex Isolation--
Isolation of the pre-budding complex
with GST-Sec23 was performed as described (4).
The Steady-state Level of Buds on the ER Is Sensitive to
Biosynthetic Cargo--
An ER-derived bud is defined as an elevation
on the surface of the ER with a width of 60-80 nm that is extruded by
50% of its diameter and covered with a characteristic coat on the
external leaflet of the membrane (24) (Fig.
1A, asterisks) (see
"Experimental Procedures"). Such buds are found in specialized
sites of the ER referred to as "transitional regions" near the
Golgi (35) or "export complexes" found in the peripheral cytoplasm
(24).
To assess the effect of cargo on ER export in vivo, we
examined whether the number of buds found on the ER surface can be modulated by biosynthetic cargo using electron microscopy and quantitative stereology. To validate this approach, we first determined the effect of reagents that block general protein export from the ER.
We have previously established using biochemical approaches that both
calphostin C and okadaic acid (OKA) are potent inhibitors of the export
of the type 1 transmembrane protein vesicular stomatitis virus
glycoprotein (VSV-G) from the ER, whereas phorbol 12-myristate 13-acetate is stimulatory (36, 37). The effects of these reagents on
the formation of ER buds as assessed by quantitative morphometry paralleled those seen in previous biochemical assays. In control cells,
we detected on average 248 ± 25 buds per cell at steady state.
Treatment of cells with OKA reduced by >95% the number of budding
structures on the ER (12 ± 5 buds per cells). This result is
similar to the recently reported morphological effects of OKA on export
of the pre-Golgi intermediate marker protein p53/58 from the ER (38).
Calphostin C led to an 85% reduction (40 ± 10 buds per cell,
respectively), whereas phorbol 12-myristate 13-acetate treatment
resulted in a 2-fold increase in their number (480 ± 11). Changes
in ER export also led to corresponding alterations in the abundance and
size of downstream pre-Golgi intermediates. These structures are
composed morphologically of clusters of vesicles and tubules (Fig.
1A, arrowheads) (20, 24, 39, 40) (see "Experimental
Procedures"). In the absence of inhibitors, cells contained on
average 67 ± 7 pre-Golgi intermediates at steady state. This
value was reduced to 4 ± 1 and 14 ± 4 in the presence of
OKA and calphostin C, respectively.
By having established that the number of ER buds scored by quantitative
morphometry serves as a reliable measure of protein and membrane flow
from the ER through the secretory pathway, we next followed the effect
of a single biosynthetic cargo species on ER export. For this purpose,
we infected cells with the tsO45 strain of vesicular stomatitis virus
(VSVts) which encodes a temperature-sensitive mutant of the
type 1 transmembrane surface glycoprotein VSV-G (VSV-Gts).
When expressed at the restrictive temperature (39.5 °C),
VSV-Gts accumulates in the ER due to a folding defect (41).
Accumulation in the ER at 39.5 °C is not a consequence of
aggregation as VSV-Gts is freely diffusible (42). Because
virus infection inhibits host protein synthesis (43), this approach
allows us to simultaneously reduce endogenous biosynthetic cargo and
introduce a single major cargo species into the ER. Upon shift to the
permissive temperature (32 °C), VSV-Gts exits the
ER via COPII vesicular carriers (19, 21, 22, 25). If the
availability of cargo for export can influence the formation of
COPII-derived vesicular carriers in vivo, then we should
detect differences between the number of ER buds and downstream intermediates found at the restrictive and permissive temperatures.
We first examined the export of wild-type VSV-G to rule out the trivial
concern that viral pathogenesis may interfere in some unexpected way
with the appearance of budding structures. Budding activity (as defined
by the number of budding profiles per µm2 ER membrane
surface) in mock-infected or wild-type-infected cells at 39.5 °C was
generally 2-2.5-fold greater than that observed at 32 °C (Fig.
1B). The abundance of pre-Golgi intermediates in mock-infected NRK cells was also greater (~1.8-fold) at 39.5 °C than at 32 °C (Fig. 1D, compare lane a to
lane c), corresponding to the enhanced level of ER export.
Consistent with this result, the rate of processing of wild-type VSV-G
oligosaccharides by Golgi-associated enzymes was ~2.5-fold faster at
39.5 °C than at 32 °C (data not shown). Thus, the transport
activities observed in control and wild-type virus-infected cells were
comparable, and in both cases, budding activity was
temperature-dependent.
In contrast to the lack of effects of wild-type VSV-G on budding when
compared with mock-infected cells, the number of ER buds in
tsO45-infected cells retained at 39.5 °C was nearly 3-4-fold lower
(a 65-75% decrease) (Fig. 1C, lane b) than that observed in mock-infected (Fig. 1C, lane a) or wild-type VSV-infected
cells (Fig. 1B, lane b). No changes in any of the
basic cell morphometric parameters (volume and surface area) were
detected under these conditions (not shown). Although the surface area
and volume of the ER in tsO45-infected cells held at 39.5 °C
increased only slightly (~5%), we observed an ~50% reduction in
the number of pre-Golgi intermediates (Fig. 1D, lane
b), a 40% (± 5%) decrease in the size of the Golgi stack,
and a 60% (± 10%) reduction in the number of Golgi buds relative to
mock-infected cells. These results suggest that the reduction in ER
budding activity observed in tsO45-infected cells held at 39.5 °C
(Fig. 1C, lane b) results in a corresponding reduction in
size and budding activity of downstream organelles.
Because export from the ER appears to be sensitive to cargo, we would
predict that transfer of tsO45-infected cells to 32 °C should
restore budding activity and membrane flow. Indeed, a 5-min shift to
32 °C led to a wave of export activity, with a 1.5-fold increase
(Fig. 1C, lane d) in vesicle budding over that observed in
mock-infected cells held at 32 °C (Fig. 1B, lane c). This
wave of export was reflected in a corresponding ~1.4-fold increase in
pre-Golgi intermediates (Fig. 1D, lane d) and an ~1.6-fold increase in Golgi size and the number of Golgi-associated buds (not
shown). This value returned to the steady-state level seen in control
cells within 30 min (Fig. 1C, lane e). We conclude that a
major biosynthetic cargo molecule such as VSV-Gts can
affect general membrane flow from the ER by preventing or enhancing ER budding.
The Availability of Soluble Biosynthetic Cargo Affects COPII
Vesicle Formation--
The effects of VSV-Gts, a
transmembrane protein, on export led us to also characterize the
potential role of accumulation of an endogenous soluble protein, in
this case the human PiZ variant of
To mimic the more exaggerated long term accumulation of
To test first for any potential nonspecific effects of CHX on
transport, we examined whether exposure of tsO45 virus-infected cells
to CHX for up to 4 h at the restrictive temperature alters the
subsequent kinetics of ER to Golgi transport of VSV-Gts at
the permissive temperature in the presence of the drug. As shown in
Fig. 2B, VSV-G accumulated at the restrictive temperature for 4.5 h was radiolabeled at 39.5 °C and incubated in the
presence of CHX for an additional 4 h prior to transfer to
32 °C. Strikingly, no inhibitory effect was observed compared with
the transport kinetics of the control that was not incubated for an
additional 4 h in the presence of CHX. A similar lack of effect
was observed when wild-type virus-infected cells were treated up to
2.5 h with CHX, and the rate of transport was assessed immediately
following removal of the drug (data not shown). These results led us to conclude that ER to Golgi transport factors are stable following exposure to CHX for up to 4 h.
Because rapidly transported cargo such as the soluble molecule albumin
or VSV-G require ~3 h to be effectively drained from the ER in the
presence of CHX (37, 47, 48) (data not shown), whereas many other cargo
molecules exit the ER with considerably longer half-times (49, 50), we
anticipate that treatment with CHX for up to 4 h should only have
partial effects on the formation ER-derived buds. Consistent with this
prediction, only an ~30% reduction was observed after a 4-h
treatment of uninfected NRK cells (210 ± 20 buds per cell (minus
CHX); 130 ± 15 buds per cell (plus CHX)) (Fig. 2C).
The number of buds in wild-type VSV-infected NRK cells was reduced
similarly (375 ± 20 buds per cell (minus CHX) to 190 ± 15 buds per cell (plus CHX)), emphasizing that viral-infected cells behave
identically to uninfected cells. Identical results were observed in rat
basophilic leukemia cells treated with CHX (data not shown). When
AATwt-expressing cells were treated with CHX, we also
observed a modest ~25-30% reduction in budding profiles (Fig.
2A, black bars, compare lane a to lane
d). In all of these controls, the reduction in the number of
detectable ER buds was not a consequence of slower transport leading to
flattening of the budding profile, as the mean height distribution of
residual buds did not change (62 ± 22 nm (minus CHX)), 63 ± 30 nm (plus CHX)), and inhibition was fully reversible.
In contrast to the above results, the effects of CHX on
AATPiZ-expressing cells were more dramatic, leading
to an ~45-50% decrease in budding profiles (Fig. 2A, gray
bars, compare lane e to lane f) (44, 51). When compared with the
AATwt-expressing cells, this level of accumulation resulted
in a 60% decrease in budding (Fig. 2A, compare lane
a to lane h). Thus, while the partial (~30%)
reduction of budding in response to general cargo reduction by
treatment of cells with CHX is consistent with its effects on vesicle
formation in vivo in yeast (46), it cannot account for the
nearly 2.5-or 4-fold reduction observed during following accumulation
of the PiZ variant in the presence of CHX (Fig. 2A) or
VSV-Gts at the restrictive temperature (Fig. 1), respectively.
Release of COPII-coated Vesicles in Vitro Is Linked to VSV-G
Export--
Given the compelling effects of VSV-Gts on ER
export in vivo, we examined whether these events can be
reconstituted in vitro to study its potential mechanism
biochemically. For this purpose, we utilized a budding assay that
reconstitutes the release of VSV-Gts into COPII-coated
vesicles from mammalian microsomes (25). In brief, to follow vesicle
budding in vitro a post-nuclear supernatant fraction is
prepared from homogenates of cells infected at 39.5 °C with tsO45
virus to accumulate VSV-Gts exclusively in the ER.
Following washing to remove cytosolic components, membranes are
incubated in a transport mixture containing cytosol and an energy
source in the form of ATP. The fraction of VSV-Gts exported
from the ER is measured using differential centrifugation to separate
more rapidly sedimenting ER and Golgi membranes that are recovered in a
medium speed pellet (MSP) from slowly sedimenting ER-derived COPII
vesicles that remain in the medium speed supernatant (MSS). The amount
of VSV-Gts in each fraction can be quantitated using
SDS-polyacrylamide gel electrophoresis and Western blotting (25).
Fig. 3 illustrates that the in
vitro budding reaction fully reconstitutes the
temperature-sensitive export of VSV-Gts observed in
vivo. At early time points at 32 °C, a transient peak of
VSV-Gts (~20% of total VSV-G) appears in COPII vesicles
in the MSS (Fig. 3, open squares). At later time points this
pool declines and is recovered in the MSP. We have previously
demonstrated that the appearance of VSV-G in the MSP is concomitant
with processing of VSV-Gts to Golgi-modified forms (25). In
contrast, incubation of microsomes at 39.5 °C strongly (>80%)
inhibited export of VSV-Gts from the ER (Fig. 3,
closed squares), consistent with the fact that under these
conditions VSV-Gts was not processed to Golgi-modified
forms (data not shown).
Because the release of VSV-Gts from ER microsomes was
inhibited at the restrictive temperature, we examined whether other
proteins that exit the ER were similarly affected. We have previously
shown that p58, a transmembrane protein that actively recycles between the ER and pre/cis-Golgi elements (19, 25, 40), is a component of COPII
vesicles (25). p58 does not bind VSV-G at either the permissive or
restrictive temperatures (data not shown), is not required for VSV-G
export from the ER (52), and therefore serves as an independent marker
for COPII vesicle formation.
We examined the rate and extent of release of p58 into the MSS at the
permissive and restrictive temperatures in mock- and tsO45 VSV-infected
cells. In mock-infected cells, incubation at 39.5 °C led to a
stimulation of p58 recovery in COPII vesicles over that observed at
32 °C (Fig. 4A, dotted lines;
closed squares (39.5°), open squares (32 °C)).
Similar results were observed using microsomes prepared from
wild-type-infected cells (not shown). In contrast, export of p58 in
microsomes prepared from tsO45 VSV-infected cells was markedly reduced
at 39.5 °C compared with 32 °C (Fig. 4A, solid lines;
closed circles (39.5°), open circles (32 °C)). Up
to 70-80% inhibition was observed at the peak of p58 release. These
in vitro results are consistent with our stereological
analysis of budding in vivo (Fig. 1) and provide independent
biochemical evidence that the export of at least one endogenous
component of the transport machinery is responsive to cargo regulation
of COPII vesicle formation.
If the formation of ER-derived COPII vesicles is linked to cargo
availability as suggested by the above experiments, one would anticipate that the general release of COPII vesicles from tsO45 VSV-infected cells would be inhibited at 39.5 °C. Therefore, we analyzed the effect of cargo availability on COPII budding from the ER
by following the recovery of the membrane-bound COPII component Sec23
in the MSS of our budding assay. In controls, when microsomes prepared
from mock-infected cells were incubated at 39.5 °C, we observed a
significant stimulation (~1.5-fold) of the release of
Sec23-containing COPII-coated carriers over that observed at 32 °C
(Fig. 4B, compare lane a to lane b). A
similar result was obtained using wild-type virus-infected cells (not
shown), values reflecting the temperature sensitivity of ER budding
in vitro (25). In contrast, microsomes prepared from
tsO45-infected cells, when incubated at 39.5 °C, exhibited an
~2.5-fold decrease in Sec23 abundance in the MSS compared with
32 °C (Fig. 4B, compare lane c and lane
d), and an ~4-fold decrease compared with microsomes prepared
from mock-infected cells (Fig. 4B, compare lane d
to lane b). These results are consistent with our
stereological analysis of ER budding in vivo (Fig. 1)
suggesting that the level of transportable biosynthetic cargo is
coupled to the general formation of COPII vesicular carriers.
The Activated Form of Sar1 Promotes Stable Coat Assembly but Will
Not Promote COPII Vesicle Formation in the Absence of Available
Biosynthetic Cargo--
Under typical incubation conditions, the
assembly and disassembly of COPII coats on membranes is regulated by
the GTPase cycle of the Sar1 GTPase (19, 25, 53). In our
microsome-based assay, the release of Sec23-containing COPII vesicles
into the MSS occurs with a transient peak at ~10 min (Fig.
5A, open circles). In this
case, the abundance of Sec23 in the MSS rapidly declines to background
levels, reflecting the fact that COPII coats are rapidly lost from
vesicles (19, 25, 53). To determine whether the formation of
Sec23-containing vesicles was indeed dependent on COPII coat assembly,
we supplemented the assay with the GDP-restricted mutant of Sar1
(Sar1[T31N]) (to be referred to as Sar1-GDP) which inhibits the
activation of endogenous Sar1 in the reaction and prevents the release
of both VSV-Gts and p58 into the MSS (25). As shown in Fig.
5A (open squares), it also blocks the general
release of Sec23-coated carriers. Thus, Sar1-dependent
budding recapitulates budding events observed in vivo.
Given the importance of Sar1 activation in cargo export, we examined
whether a pre-activated form of Sar1 could bypass the block in
ER-derived vesicle formation imposed by incubation of ER prepared from
VSVts-infected cells at 39.5 °C. For this purpose, we
supplemented the assay with Sar1[H79G] (19), a mutant that is
restricted to the GTP-bound state (to be referred to as Sar1-GTP). We
have previously shown that the Sar1-GTP-restricted mutant does not further stimulate the rate or extent of formation of vesicles containing VSV-Gts or p58 at 32 °C (25). However, we did
find that in the presence of the Sar1-GTP mutant, VSV-G/p58 released
into COPII vesicles at 32 °C accumulates in the MSS as the delivery
of vesicles to the Golgi is largely blocked in response to the
inability of coats to be disassembled (19, 25).
When we examined the effect of Sar1-GTP on general vesicle recovery in
the MSS at 32 °C using Sec23 as a marker, we observed an ~3-fold
increase over incubations performed in the absence of the activated
mutant (Fig. 5A, compare closed squares to
open circles), reflecting the stabilization of the COPII
coat on vesicle membranes by the activated GTPase. The addition
of Sar1-GTP at 39.5 °C led to a similar 3-fold increment in the
recovery of Sec23 in the MSS (Fig. 5B, compare lane
c to lane d) when compared with the effects of Sar1-GTP
at 32 °C (Fig. 5B, compare lane a to
lane b). Again, this reflects the stabilization of the COPII
coat to vesicles formed at the higher temperature. Strikingly, the
addition of Sar1-GTP to the assay did not lead to either
VSV-Gts (Fig. 5C, compare lane b to lane
c) or p58 release into the MSS at 39.5 °C (Fig. 5C,
compare lane e to lane f)
and therefore has no effect on the extent of general vesicle release.
Thus, addition of an activated form of Sar1 was not sufficient to
by-pass the absence of a transportable form of biosynthetic cargo in
the ER, emphasizing the importance of VSV-Gts in regulating
these events.
VSV-G Can Interact with the COPII Machinery in the Unfolded
State--
Accumulation of VSV-Gts in the ER appears to
play a dominant inhibitory role in the formation of COPII vesicles.
Because in viral-infected cells VSV-Gts is the major form
of cargo for export, it may have a previously unsuspected interaction
with the COPII budding machinery even at the restrictive temperature.
To examine this possibility, we took advantage of recent studies where
we demonstrated that under conditions that do not support vesicle
budding from the ER, VSV-Gts is selected for ER export by
interacting with Sec23 or the Sec23/24 complex in a
Sar1-dependent manner (4). In these studies, we demonstrated that incubation of VSV-Gts containing ER
membranes at the permissive temperature in the presence of glutathione
S-transferase-tagged Sec23 (GST-Sec23) can lead to the
efficient recovery of VSV-Gts in a detergent-soluble
pre-budding protein complex on glutathione-Sepharose beads (GS beads).
Recovery on GS beads is Sar1A-dependent and does not occur
when membranes are retained on ice (4). Moreover, under these
conditions, the VSV-Gts/Sec23-containing complex excludes
ER resident proteins such as BIP, calnexin, and ribophorin II,
indicative of efficient sorting from resident ER proteins (4). On the
average, 10-20% of the total soluble VSV-G can be recovered on GS
beads at 32 °C (4).
To determine whether a pre-budding complex containing
VSV-Gts and COPII components can be detected at the
restrictive temperature, membranes were washed to remove any residual
bound Sar1, Sec23/24, and Sec13/31 complex (4). Washed membranes remain
export-competent and retained their temperature sensitivity as
incubation with cytosol and Sar1-GTP led to the accumulation of
VSV-G-containing vesicles at 32 °C but not at 39.5 °C (Fig.
6A). To determine when cargo
becomes associated with the Sec23 partial coat, microsomes incubated at
32 or 39.5 °C with recombinant GST-Sec23 and/or Sar1A mutants were
pelleted, washed, and solubilized in a detergent containing buffer.
Following centrifugation to remove insoluble material, the supernatant
was incubated with glutathione-Sepharose (GS) beads, and bound
GST-Sec23 was quantitated using Western blotting (4). As shown in Fig.
6B, recovery of VSV-Gts or Sec23 was not
observed in samples held on ice (Fig. 6B, lane a).
Strikingly, the level of recovery of VSV-Gts on GS beads at
the restrictive temperature (39.5 °C) was similar to the level of
recovery at the permissive temperature (32 °C) (Fig. 6B, upper
panel, compare lane b to lane d).
Recovery was specific for the COPII as it was blocked by incubation
with the inactivated, Sar1-GDP mutant (Fig. 6B, lanes c and
e). Moreover, in both cases, the recruitment of
VSV-Gts was directly correlated with the recruitment of
GST-Sec23 (Fig. 6B, lower panel, compare lanes b
to c and lanes d to e).
Thus, although budding may be largely blocked at 39.5 °C in
vitro (Fig. 3), recruitment of the COPII component Sec23 and
VSV-Gts to a pre-budding complex is not. These results
support the interpretation that misfolded VSV-G can sequester the COPII
budding machinery. By establishing conditions to control the export of
a major biosynthetic cargo species from the ER, we have identified a
novel intermediate preceding completion of protein folding.
Biosynthetic Cargo Reduction Leads to Changes in Vesicle Budding
from Downstream Compartments--
The substantive effects of
VSV-Gts on ER budding in vivo and in
vitro (Fig. 1 and Fig. 4) and its ability to sequester COPII components at the restrictive temperature (Fig. 6) led us to assess whether biosynthetic cargo availability could be correlated with the
function of downstream secretory compartments directing recycling of
transport components. Consistent with this possibility, we had already
noted a decrease in vivo in the abundance of pre-Golgi intermediates in tsO45-infected cells held at the restrictive temperature (Fig. 1). In addition, using indirect immunofluorescence, we have found that the pre-Golgi intermediate marker protein p58 as
well as the COPI subunit
Previous studies have shown that incubation of Golgi membranes in the
presence of cytosol, ATP, and GTP
We first examined the recruitment of COPI to control (uninfected)
microsomes. We found that COPI present on microsomes held on ice can be
removed by a high salt wash (250 mM KOAc) indicative of a
low affinity, inactive form (Fig.
7A, open circles).
In contrast, when microsomes were incubated in the presence of cytosol,
ATP, and GTP
We next examined the response of downstream compartments to regulated
release of VSV-Gts from the ER. We first tested whether a
reduction in transported cargo in microsomes prepared from cells
infected with the tsO45 virus at 39.5 °C would lead to a decrease in
COPI binding. Microsomes prepared from mock-infected cells incubated
for 5 min at 39.5 °C efficiently recruited the high salt-resistant
form of COPI (Fig. 7C, open squares). In
contrast, recruitment of the high salt-resistant form of COPI was
3-4-fold lower in microsomes prepared from tsO45-infected cells
incubated for 5 min at 39.5 °C (Fig. 7C, closed circles)
and did not increase upon further incubation (not shown). When these
microsomes were incubated at 32 °C for 5 min, no recruitment was
observed (Fig. 7D, closed circles), whereas after incubation
for 15-20 min, high salt-resistant COPI binding was comparable to that
observed in microsomes prepared from mock-infected control cells (Fig.
7D, open circles and squares). The lag period
observed in the recruitment of high salt-resistant forms of COPI at
32 °C by microsomes prepared from infected cells is consistent with
the time period (~15-20 min) we have previously shown to be required
to efficiently mobilize VSV-Gts to pre-Golgi intermediates
in semi-intact cells (19, 20, 22). Thus, the observed changes in the
recruitment of COPI is consistent with the ability of cargo to modulate
COPII vesicle formation through its recruitment to pre-budding intermediates.
Biosynthetic cargo exiting the ER includes a wide collection of
newly synthesized molecules, protein and lipids, that are mobilized
from the ER to distinct cellular and extracellular destinations. Other
forms of cargo incorporated into COPII vesicles also includes proteins
that are continuously recycled between ER and Golgi compartments. Recycling proteins include transport machinery proteins involved in
cargo selection, in vesicle formation, and in targeting and fusion
(reviewed in Refs. 55 and 56). These proteins, like biosynthetic cargo,
are packaged into the budding vesicle by interacting either directly or
indirectly with COPII components (4, 5, 63). Given this complexity, we
introduced one cargo molecule, VSV-G, into the ER. Due to the ability
of VSV to inhibit host-protein synthesis, VSV-G becomes the major cargo
mobilized through the secretory pathway. Moreover, the transport of
VSV-G has been extensively characterized and used to define the basic
components and principles of the secretory pathway (2, 13). In
non-infected cells, biosynthetic cargo accumulation and its potential
effects on vesicle budding will be tightly controlled by activation of
the ER degradation machinery (64) and, possibly, the ER overload
response (65). In contrast, in VSV-infected cells these checkpoints are
largely bypassed through the ability of the virus to prevent host
protein synthesis, allowing us to accumulate VSV-Gts in the
ER in a fully functional form. This experimental system, as with many
biochemical approaches, represents an exaggeration of the normal course
of cargo-related events in the ER. However, it provides us with a
unique window to examine the relationship between a defined and
controllable form of cargo and the existence of novel intermediates
involved in COPII vesicle budding.
We have presented in vivo evidence using quantitative
morphometry that the availability of biosynthetic cargo in the ER can both prevent and enhance the formation of COPII vesicles that mediate
ER export. This effect was reconstituted in vitro by
following the movement of two separate markers for ER export: the COPII component Sec23, a marker for general vesicle formation, and the endogenous recycling protein p58, which is transported independently of
VSV-G. Although speculated to be a general mannose-binding lectin,
(66), it does not form a complex with
VSV-G.2 Moreover, recent
studies now suggest that p58, a major component of COPII vesicles (25,
52), may have a selective role in transport (67, 68). A strong
correlation was observed between the export of p58, the formation of
COPII vesicles in vitro, and COPII vesicle formation
in vivo as measured using quantitative stereology in response to VSV-Gts. These observations support our
conclusion that a single biosynthetic cargo molecule such as VSV-G can
modulate general COPII vesicle budding. Importantly, the inhibition of
budding observed during accumulation of cargo in the ER was readily
reversible. The folding of VSV-Gts following transfer to
the permissive temperature led to rapid and efficient stimulation of
COPII vesicle production. This important result indicates that the
increased availability of VSV-G led to efficient coupling to the COPII
machinery. Moreover, these results negate the possibility that the
accumulation of cargo in a non-transportable form had an indirect
effect by irreversibly "poisoning" the budding machinery in
vivo or in vitro. Because biosynthetic cargo can have a
dominant effect over the movement of an endogenous component such as
p58, it is apparent that general vesicular traffic is tightly modulated
in virus-infected cells. Whether VSV-G can exert a similar dominant
effect on other biosynthetic cargo molecules remains to be tested.
Given that virus infection inhibits host protein synthesis, as controls
we examined the effects of CHX on the level of ER budding profiles
in vivo. In these experiments, although we found that
treatment with CHX for up to 4 h had no general effect on the
kinetics of ER to Golgi transport, treatment did lead to a reduction in
ER budding profiles of ~30%. It is apparent that the decrease
observed by cargo reduction alone cannot account for the much larger
2.5-4-fold effects of the accumulation of cargo in the ER observed
with VSV-Gts or the PiZ variant of The limited decrease in budding in response to the treatment with CHX
only may only reflect the loss of export activity of rapidly
transported endogenous cargo such as albumin. Many other proteins,
including cell-surface receptors and soluble proteins, are exported at
significantly slower rates from the ER (49, 50). Alternatively,
in vivo, a pool of recycling components involved in
maintaining the function of downstream pre-Golgi and Golgi compartments
may be in continuous flux through the ER. Short term treatments with
CHX, such as used in the present experiments, would be expected to have
only a minor effect on the transport of these proteins. Our results
with CHX showing a partial reduction in COPII budding in mammalian
cells are agreement with those obtained in in vivo studies
in yeast in response to CHX (46). However, in vitro studies
in yeast using an assay that reconstitutes ER budding could not
reproduce this effect, leading to the suggestion that cargo recruitment
is uncoupled from vesicle formation (69). This in vitro
result is now consistent with the more recent observations by Schekman
and colleagues (70) that purified yeast COPII components can
self-assemble on liposomes and yeast ER membranes on ice to generate
empty coated vesicles. This degree of uncoupling of assembly of the
COPII coat from other endogenous components therefore may markedly
limit the sensitivity of in vitro assays to detect the effects of modest cargo reduction observed in CHX-treated membranes.
Recently, in vitro binding experiments using purified COPII
components has led to the suggestion that coat assembly is promoted by
snap- receptor (SNARE) components and
that this may be key in vesicle formation (71). In contrast, we find
that COPII recruitment and vesicle formation under physiological
conditions is largely sensitive to the availability of biosynthetic
cargo. Moreover, genetic studies to date suggest that SNARE components
largely function in vivo following vesicle formation (72,
73). Whereas an explanation for these apparently contradictory findings
between in vivo and in vitro studies on SNARE
function remains to be resolved, if we view SNARE components as
essential cargo whose recruitment needs to be physiologically
coordinated with biosynthetic cargo, then these results and our
in vivo and in vitro results are strongly consistent with a potential general role for many forms of cargo in
modulating COPII vesicle formation.
To provide additional evidence that membrane flow from the ER can be
controlled by biosynthetic cargo, we found that both the reduction and
stimulation of ER export was proportionally conserved in all of the
immediate downstream intermediates. These include changes in the
general abundance of pre-Golgi intermediates, budding profiles on the
CGN, and the size and volume of the Golgi stack in vivo in
virus-infected cells held at the restrictive temperature to retain
VSV-Gts in the ER. We have observed similar modest effects
following a 4-h treatment with
CHX,3 results in agreement
with studies by Howell and colleagues (74). Moreover, we also observed
a corresponding decrease in the ability of residual pre-Golgi and Golgi
compartments to recruit COPI coats in vivo and in
vitro. This may reflect the expected loss in the need for
retrograde recycling of ER-derived components or, potentially, for
bi-directional transport as COPI has been suggested to promote anterograde transport through the Golgi stack (75). In support of the
possibility that ER export affects the function of downstream compartments, a recent study has demonstrated that a recycling soluble
cargo molecule that contains the retrograde sorting determinant KDEL is
required for activation of the Golgi-localized receptor ERD2 to
initiate retrieval to the ER (76, 77). In this case, activation has
been proposed to modulate ARF1 GTPase activity, which, in turn,
controls COPI recruitment and the function of ERD2 in retrieval.
Collectively, the combined results demonstrate a functional
relationship between the availability of ER biosynthetic cargo and the
level of membrane traffic between subsequent downstream secretory compartments.
To account for the observed changes in COPII vesicle formation in
response to the availability of biosynthetic cargo, we analyzed the
ability of VSV-G that was accumulated in the ER to interact with the
COPII components found in pre-budding complexes (4). To our surprise,
we observed comparable VSV-G recovery in Sar1/Sec23 containing
pre-budding complexes at both the transport permissive (32 °C) and
transport-impaired (39.5°) temperatures. This unexpected association
of misfolded cargo with the ER export machinery is compatible with the
previously reported low, yet detectable, transport of misfolded cargo
proteins such as VSV-Gts at the restrictive temperature.
Such transport is observed in vivo only when excessive
infection conditions are used to saturate the folding pathway, leading
to export of monomeric VSV-G and its subsequent retrieval from
pre-Golgi intermediates through its interaction with the ER chaperone
BiP (18, 78). Moreover, our results are consistent with the observation
that even the misfolded PiZ variant of AAT can escape the ER, albeit at
much reduced levels (~15% of wild type) (27). Although a dominant effect of the PiZ variant on the export of wild-type AAT in
heterozygous PiMZ individuals is not observed (27), this may simply be
due to the efficient packaging of the wild-type protein through
interaction with the COPII machinery even in the presence of a reduced
level of vesicle carriers budding from the ER (reviewed in Ref. 79). This interpretation is consistent with the ability of the biosynthetic cargo selection machinery to efficiently sort transport-competent from
transport-incompetent forms of VSV-G (80). Indeed, the defect leading
to liver disease may be a consequence of loss of normal function of the
AAT-specific selection machinery (81) in the absence of cargo,
resulting in direct or indirect effects on the generation of COPII vesicles.
The observed effect of VSV-Gts on vesicle formation
in vivo and in vitro demonstrates a more direct
link between protein folding events and those involved in biosynthetic
cargo selection than previously anticipated. Our results, combined with
the observation that degradation of ER proteins is coupled to the
translocation pore-component Sec61 (82), now suggest that protein
import, folding, and export may be an integrated process. We anticipate that the effect of VSV-Gts on general budding at the
restrictive temperature is at least partially exerted by non-productive
interactions with Sar1 and Sec23/24. However, our ability to both
prevent and enhance export by temperature shift demonstrates that such
interactions are likely to be typical of the normal pathway involved in
cargo selection (4). We propose that biosynthetic cargo such as VSV-G
can therefore modulate COPII vesicle formation.
Is the observed ability of biosynthetic cargo accumulated in the ER to
modulate vesicle formation a property that is shared at other export
sites in the exocytic and endocytic pathways? In the case of clathrin,
a similar regulation has been observed. Studies of trans-Golgi network
(TGN) export sites have demonstrated that cargo molecules such as
mannose 6-phosphate receptors and major histocompatibility complex II
regulate the formation of AP1-driven clathrin-coated vesicles from the
TGN in vivo (10-12). Although sorting is the primary
function for vesicle budding from the TGN, the ER must coordinate cargo
sorting and vesicle formation with quality control events associated
with protein folding, oligomerization, and the degradation of misfolded
cargo or cargo subunits that fail to be oligomerized into the mature
protein (18, 64). We suggest that the detection and disposal of
incompetent cargo by the degradative pathway may play an important role
in eliminating potential dominant negative effects of such cargo on
general ER export as we have observed for VSV-Gts and the
AATPiZ variant. Indeed, evidence for the adverse effects of
ER accumulation of cargo are found in the pathophysiology of disease. A
subset of patients who express the PiZ variant of AAT develop liver
injury (reviewed in Ref. 27). The partial effects of accumulated PiZ variant leading to a general decrease in budding raises the possibility that its accumulation may lead to liver injury by both decreasing the
ability of the ER to efficiently export other critical biosynthetic cargo, in addition to possible negative effects related to induction of
stress-related signaling responses (65). Related effects are evident in
a number of neuronal and non-neuronal degenerative diseases that are
also marked by accumulation of mutant cargo in the ER with severe
consequences on cell growth (83, 84). Additional studies will be
required to determine the general role of cargo in the development of
these diseases.
It is now apparent that a variety of mechanisms may operate in the ER
to sense, couple, and synchronize cargo processing, degradation, and
export. We have raised the possibility that coupling between
biosynthetic cargo and the COPII machinery may represent an important
step in control of ER budding which will now need to be explored more fully.
1-antitrypsin, reduced levels of general COPII vesicle formation in vivo. Consistent with
this result, conditions that prevent the export of VSV-G from the ER led to a significant inhibition of general COPII vesicle budding from
ER microsomes and the export of an endogenous recycling protein p58
in vitro. In contrast, synchronized export of VSV-G
stimulated COPII vesicle budding both in vivo and in
vitro. Under conditions where VSV-G is retained in the ER, we
find that it can to be recovered in pre-budding complexes containing
COPII components. These results suggest that the export of biosynthetic
cargo is integrated with ER functions involved in protein folding and
oligomerization. The ability of biosynthetic cargo to prevent or
enhance ER export suggests that interactions of cargo with the COPII
machinery contribute to the formation of vesicles budding from the ER.
INTRODUCTION
Top
Abstract
Introduction
References
1-antitrysin (PiZ) (26, 27) which accumulates in the ER
under pathophysiological conditions. We now demonstrate that cargo can
prevent and enhance COPII vesicle budding in vivo and
in vitro. Furthermore, we show that VSV-G held in the ER
interacts with the Sar1 and Sec23/24 components of the COPII machinery. We propose that the observed coupling between cargo and COPII components can modulate the formation of pre-budding intermediates that
are essential for vesicle formation from the ER.
EXPERIMENTAL PROCEDURES
-COP (M3A5)
from T. Kreis, University of Geneva (Geneva, Switzerland); and an
anti-peptide antibody against
-COP (EAGE) from M. Farquhar
(University of California, San Diego, CA). A polyclonal antibody
specific for VSV-G was described previously (22). A polyclonal antibody
to Sec23 was generated against a peptide (DTEHGGSQAR) (residues
707-716) or against recombinant GST-Sec23 as described (4). The stably transfected mouse hepatoma cells (line Hep 1a) expressing recombinant PiZ variant (line H1A/RSVATZ-8) (29) or normal PiM1 human AAT (line
H1A/M-15) (26) were generously provided by R. Sifers, Baylor College of
Medicine (Houston).
-COP Binding
Reaction--
150-mm dishes of NRK cells were washed three times with
ice-cold phosphate-buffered saline and scraped with rubber policeman in
a buffer containing 10 mM Hepes (pH 7.2) and 250 mM mannitol (buffer A). The cells were pelleted,
resuspended in buffer A, and homogenized by passing the cell suspension
6 times through a ball bearing homogenizer (31). A post-nuclear
supernatant was prepared by centrifuging the homogenate at 1000 × g for 10 min at 4 °C. 15 µl (20-40 µg protein) of
post-nuclear supernatant was added to a transport reaction mixture
containing 27.5 mM Hepes-KOH (pH 7.2), 2.5 mM
MgOAc, 65 mM KOAc, 5 mM EGTA, 1.8 mM CaCl2, 1 mM ATP, 5 mM creatine phosphate, 0.2 units of rabbit muscle creatine kinase (final concentrations), and 1.5-2 mg/ml rat liver cytosol in a
final volume of 200 µl on ice. The tubes were then transferred to
32 °C for 5-20 min as indicated under "Results." The reaction was terminated by transfer to ice. 1 ml of a buffer containing 25 mM Hepes (pH 7.2), 2.5 mM MgOAc, and KOAc to
give the final salt concentration as indicated under "Results" was
added, and the tubes were vortexed and centrifuged at 16,000 × g for 10 min at 4 °C. The supernatant was aspirated, and
the tubes were centrifuged for an additional 3 min at 16,000 × g at 4 °C. The residual supernatant was removed and 25 µl of a gel sample buffer (32) added. Samples were heated for 5 min
at 95 °C and resolved using SDS-polyacrylamide gel electrophoresis
(32) on 7.5 or 10% polyacrylamide gels.
-COP was detected by
immunoblotting using the M3A5 monoclonal antibody specific for
-COP
and developed with alkaline phosphatase (33, 34).
-COP was
quantitated by densitometry using a Molecular Dynamics
Densitometer (Sunnyvale, CA).
RESULTS
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Fig. 1.
Release of VSV-G from retention in the ER
stimulates vesicle budding in vivo. A,
an electron micrograph illustrating ER budding profiles
(asterisks) and vesicular-tubular (pre-Golgi) intermediates
(arrowheads) involved in ER to Golgi transport (24).
Bar, 0.1 µM. B, cells were either
mock-infected (open bars, lanes a and
c) or infected with wild-type VSV (closed bars,
lanes b and d) for 4.5 h at 39.5 °C as
described (30). Cells were rapidly transferred to ice and fixed (0 min
(a and b)) or incubated for 5 min at 32 °C
(c and d) prior to fixation. C and
D, cells were either mock-infected (open bars, lanes
a and c) or infected with tsO45-VSV for 4.5 h at
39.5 °C (closed bars, lanes b, d, and e).
Cells were transferred to ice and fixed (0 min (a and
b)), or incubated for 5 (c and d) or
30 (e) min at the permissive temperature (32 °C) prior to
fixation. The number of ER buds (B and C) or
pre-Golgi intermediates (D) was determined as described
under "Experimental Procedures." Error bars were
determined as described (24).
1-antitrysin (AATPiZ)
(27). Unlike wild-type monomeric
1-antitrypsin (AATwt),
which is secreted rapidly from the ER, greater than 85% of the
transport-impaired PiZ variant is retained within the ER and is
subjected to rapid intracellular degradation (29). In a subset of PiZ
variant carrier patients, the degradation of the protein is markedly
reduced leading to its accumulation in the ER (27). These patients
develop severe liver injury, a problem that may potentially reflect a
more global deficiency in ER and/or cell function. Interestingly, when
we examined isogenic cell lines expressing either the wild-type
1-antitrypsin (ATTwt) or those accumulating the PiZ
variant (ATTPiZ), we found an ~30% decrease in the
number of ER buds in the PiZ variant (Fig.
2A, compare lane a
to lane e). Thus, steady-state accumulation of
ATTPiZ in the ER led to reduced levels of budding
profiles.
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Fig. 2.
ER buds are significantly reduced in response
to accumulated PiZ variant in vivo. A,
the abundance of ER budding profiles in a stably transfected hepatoma
cell line (Hepa 1A) expressing the wild-type (black bars) or
PiZ variant (gray bars) in the absence (0 h) or after
increasing time of treatment with CHX. Quantitation was performed as
described under "Experimental Procedures." B (open
squares), NRK cells infected with tsO45 VSV at 39.5 °C for
4 h were radiolabeled at 39.5 °C and chased at 32 °C in the
presence of unlabeled methionine as described (30). Closed
circles, radiolabeled cells as described above were incubated for
an additional 4 h in the presence of CHX (50 µg/ml) prior to
transfer to 32 °C. The fraction of VSV-G processed to Golgi-modified
forms was determined as described (30). C, the abundance of
ER-budding profiles (black bars) were measured in
vivo in non-infected NRK cells in the absence (0 h) or after
increasing time (up to 4 h) in the presence of CHX (50 µg/ml).
Bars indicate standard error of the mean for three
independent experiments.
1-antitrypsin found in PiZ variant patients, we took advantage of previous studies which have demonstrated that ER degradation of the PiZ
variant requires protein synthesis (44). Addition of CHX not only
prevents degradation of the PiZ variant but also reduces the content of
other cargo proteins in the ER. As such, this condition leads to a
temporary relative increase in the abundance of polymerized
AATPiZ variant in the ER (44, 45). In addition, incubation
of cells in the presence of CHX allows us to examine independently the role of general cargo reduction on ER-budding structures. This is an
important control in the case of VSV-infected cells where the virus
inhibits host protein synthesis. Previous studies in yeast have shown
that CHX treatment does lead to a partial reduction of ER-derived
vesicles (46).
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Fig. 3.
Release of VSV-G containing vesicles in
vitro is blocked at the restrictive temperature.
Microsomes prepared from tsO45 VSV-infected cells were incubated at
32 °C (open squares) or 39.5 °C (closed
squares) for the indicated times, and the release of
VSV-G-containing COPII vesicles from the ER found in the medium speed
pellet (MSP) (P in upper panel) to the medium
speed supernatant (S in upper panel) was
determined by Western blotting as described (25). The Western blot
(upper panel) was quantitated (lower panel) as
described under "Experimental Procedures."
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Fig. 4.
General ER budding in vitro
is blocked at the restrictive temperature. A,
microsomes prepared from mock-infected (MI) or tsO45
VSV-infected cells (I) were incubated at the permissive
(32 °C) or restrictive (39.5 °C) temperatures for the indicated
times, and the release of p58 from the ER found in the medium speed
pellet (MSP) (P in upper panels) to the medium
speed supernatant (MSS) (S in upper panels) was
determined by Western blotting (upper panels) and
quantitated (lower panel) as described under "Experimental
Procedures" (25). The export of p58 in mock-infected cells is
indicated by broken lines; export in virus-infected cells is
indicated by solid lines. B, microsomes prepared
from tsO45-VSV-infected cells were incubated at 32 or 39.5 °C for 10 min, and the appearance of vesicles coated with Sec23 in the MSS was
determined by Western blotting using an antibody specific for Sec23 as
described under "Experimental Procedures."
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Fig. 5.
Activated Sar1-GTP does not promote exit of
VSV-Gts from the ER. Microsomes prepared from
mock-infected cells were incubated for the indicated times with no
additions (open circles), in the presence of the
Sar1-GDP-restricted mutant (Sar1[T39N]) (open squares), or
the Sar1-GTP-restricted mutant (Sar1[H79G]) (closed
squares), and the abundance of COPII vesicles released into the
MSS was determined by Western blotting for the COPII coat subunit Sec23
as described under "Experimental Procedures." Recombinant mutant
proteins (1 µg) were prepared as described (85). B,
microsomes prepared from infected cells were incubated for 10 min at 32 (a and b) or 39.5 °C (c and
d) in the absence (a and c) or
presence (b and d) of 1 µg Sar1-GTP, and the
amount of Sec23-bound vesicles was recovered in the MSS determined by
Western blotting. C, microsomes prepared from infected cells
were incubated for 10 min at 32 (a and d) or
39.5 °C (b, c, e and f) in the absence
(a, b, d, and e) or presence (c and
f) of 1 µg of Sar1-GTP, and amount of VSV-G or p58
released into the MSS was determined by Western blotting. Typical
results of three independent experiments are shown.
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Fig. 6.
VSV-G can be detected in a complex with Sec23
at 39.5 °C. A, salt-washed microsomes prepared as
described under "Experimental Procedures" were incubated at
32 °C (a) or 39.5 °C (b) for 30 min in the
presence of rat liver cytosol and 1 µg of the activated Sar1A
(GTP-restricted) mutant. The amount of VSV-Gts (percent of
total) released from the ER in COPII vesicles was determined as
described (25). B, salt-washed microsomes were incubated on
ice (a), at 32 (b and c), or
39.5 °C (d and e) for 30 min with GST-Sec23 in
the absence (a) or presence of either the GTP-restricted
(b and d) or GDP-restricted (c and
e) Sar1 mutant. Membranes were pelleted, solubilized, and
incubated with GS beads, and the amount of GST-Sec23 or
VSV-Gts recovered on beads was determined using Western
blotting as described under "Experimental Procedures." Typical
results of three independent experiments are shown.
-COP, a coat complex involved in recycling
from both pre-Golgi and Golgi compartments (2, 54-56), undergo a
significant change in their morphological distributions in response to
infection at the restrictive temperature or treatment with CHX. Whereas
p58 migrates from its typical pre-Golgi and Golgi punctate distribution
(19, 22, 39, 40) to the ER, COPI shifts from its typical peri-nuclear
membrane-bound Golgi localization (19, 54) to a diffuse cytosolic
localization (data not shown). These qualitative observations raised
the possibility that reduced membrane flow from the ER results in a
decreased need for COPI function in recycling. We therefore examined
the ability of microsomes containing pre-Golgi and Golgi membranes to
recruit COPI quantitatively as a more direct measure for these events.
S, a poorly hydrolyzable analog of
GTP which prolongs the activation of the small GTPase ARF1, enhances
and stabilizes the recruitment of COPI to pre- and cis-Golgi elements
(19, 57-60). Unlike COPI present on membranes prior to activation of
ARF1, the COPI pool actively engaged in transport is resistant to
release by treatment with high salt (61, 62). We utilized this
difference to quantitate the steady-state recruitment of activated COPI
in vitro to membranes.
S for 5 min at 32 °C to maximize the ARF1 activation
(19),
-COP was now efficiently retained by membranes following the high salt wash (Fig. 7A, closed circles). Further
incubation did not lead to an increase in COPI binding. As an
additional control, we examined the ability of CHX-treated cells to
recruit the high salt-resistant form of COPI in the absence GTP
S to
assess recruitment in response to a partial cargo reduction. At low
salt concentrations (75 mM KOAc) CHX-treated and control
cells retained identical levels of
-COP (Fig. 7B).
Incubation of control membranes for 5 min in vitro in the
absence of GTP
S showed recruitment of a pool of COPI of which
40-60% of the total membrane-associated pool of COPI was in the high
salt-resistant COPI form (Fig. 7B, open circles). In
contrast, microsomes prepared from CHX-treated cells had a 3-4-fold
reduced level of membrane-associated COPI following the high salt wash
in the absence of GTP
S (Fig. 7B, closed circles). The
observed deficiency in the recruitment of the high salt-resistant form
of COPI was not a consequence of the inability to activate the ARF1
pathway in CHX-treated cells. When these microsomes were incubated for
5 min in the presence of GTP
S, COPI coats resistant to high salt
extraction were recruited to statistically comparable levels to those
observed in control membranes (Fig. 7B, lanes a and
b). These results suggest that COPI binding is a sensitive
measure of Golgi function in response to reduced cargo in the
pathway.
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Fig. 7.
Depletion of cargo interferes with the
recruitment of -COP to membranes.
A, microsomes prepared from cell homogenates of NRK cells
were maintained on ice (open circles) or were incubated for
5 min at 32 °C in the presence of an ATP-regenerating system,
cytosol, and GTP
S (100 µM) (closed circles)
as described under "Experimental Procedures." Membranes were washed
with the indicated concentration of KOAc, and the fraction of
-COP
remaining associated with membranes was determined by Western blotting
as described under "Experimental Procedures." Values are reported
as percent of
-COP binding relative to the level of
-COP attached
to membranes following the 75 mM KOAc salt wash in cells
treated with GTP
S (maximal binding). B, microsomes
prepared from control (open circles) or CHX-treated (4 h)
(closed circles) cells were incubated for 5 min in the
presence of an ATP-regenerating system and cytosol in the absence of
GTP
S. Values are reported as percent of
-COP binding relative to
the level of
-COP present on membranes following the 75 mM KOAc salt wash. Bars a and b
illustrate the effect of the presence of 100 µM GTP
S
on
-COP recruitment in control (a) and CHX-treated
(b) membranes. C, microsomes prepared from cell
homogenates of mock-infected (squares) or tsO45 VSV-infected
(closed circles) were incubated in vitro for 5 min at the restrictive temperature (39.5 °C) as described under
"Experimental Procedures." Membranes were washed with the indicated
concentration of KOAc and the fraction (percent) of
-COP remaining
associated determined as described above. D, microsomes
prepared from mock-infected (open squares) or tsO45
VSV-infected (circles) cells were incubated for 5 (closed circles) or 20 min (open circles) in the
presence of ATP-regenerating system and cytosol. A-D, the
data presented are the average of two or more experiments with the
error bar indicating the standard error of the mean.
DISCUSSION
1-antitrypsin.
However, we cannot exclude the possibility that the reduced budding
observed in the presence of CHX (and therefore a reduced load of
readily available cargo) supports our observations with VSV-G that
normal biosynthetic cargo plays role in ER export.
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ACKNOWLEDGEMENT |
---|
The electron microscopy made extensive use of Core B supported by National Institutes of Health Grant CA 58689.
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FOOTNOTES |
---|
* This work was supported in part by Grants GM 42336 and CA 58689 from the National Institutes of Health (to W. E. B).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.
This paper is dedicated to the memory of Thomas Kreis.
Recipient of a fellowship grant from the European Molecular
Biology Organization and the Human Frontier Sciences Program.
§ Recipient of a Cystic Fibrosis Foundation Post-doctoral fellowship.
¶ Recipient of a fellowship from the Human Frontier Sciences Program and the Muscular Dystrophy Association.
To whom correspondence should be addressed: Dept. of Cell and
Molecular Biology, The Scripps Research Institute, 10666 N. Torrey
Pines Rd., La Jolla, CA 92037.
The abbreviations used are:
ER, endoplasmic
reticulum; VSV-G, vesicular stomatitis glycoprotein; TGN, trans-Golgi
network; VSV, vesicular stomatitis virus; GST, glutathione
S-transferase; NRK, normal rat kidney; OKA, okadaic acid; CHX, cycloheximide; MSP, medium speed pellet; MSS, medium speed
supernatant; GTPS, guanosine
5'-3-O-(thio)triphosphate.
2 M. Aridor and W. E. Balch, unpublished observations.
3 S. Bannykh and W. E. Balch, unpublished observations.
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