From the Departments of Cell and
§ Molecular Biology, The Scripps Research Institute,
La Jolla, California 92037 and the
Department of Anatomy,
University of California, San Francisco, California 941143
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ABSTRACT |
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Efficient export of vesicular stomatitis virus
glycoprotein (VSV-G), a type I transmembrane protein, from the
endoplasmic reticulum requires a di-acidic code (DXE)
located in the cytosolic carboxyl-terminal tail (Nishimura, N., and
Balch, W. E. (1997) Science 277, 556-558). Mutation
of the DXE code by mutation to AXA did not
prevent VSV-G recruitment to pre-budding complexes formed in the
presence of the activated form of the Sar1 and the Sec23/24 complex,
components of the COPII budding machinery. However, the signal was
required at a subsequent concentration step preceding vesicle fission.
By using green fluorescence protein-tagged VSV-G to image movement in a
single cell, we found that VSV-G lacking the DXE code fails
to be concentrated into COPII vesicles. As a result, the normal
5-10-fold increase in the steady-state concentration of VSV-G in
downstream pre-Golgi intermediates and Golgi compartments was lost.
These results demonstrate for the first time that inactivation of the
DXE signal uncouples early cargo selection steps from
concentration into COPII vesicles. We propose that two sequential steps
are required for efficient export from the endoplasmic reticulum.
Multiple pathways in the endoplasmic reticulum
(ER),1 including folding and
degradation, compete for transport of newly synthesized cargo to the
Golgi. What are the molecular mechanisms directing efficient export?
Movement of cargo between compartments of the secretory pathway
involves selective sorting and concentration into vesicle carriers (1).
Newly synthesized cargo that is translocated into the ER is
incorporated into COPII-coated vesicles (2). Cargo exiting the ER
includes both endogenous transport components that must be recycled
from post-ER compartments for reuse and biosynthetic proteins that are
delivered to downstream subcellular destinations and the cell surface.
Cargo is sorted from resident ER proteins and selected for export by
interacting with COPII coat components (3, 4). Selective recruitment of
cargo to pre-budding protein complexes occurs in response to activation
of the Sar1 GTPase to the GTP-bound form. This step is coordinated with
the recruitment of the cytosolic Sec23/24 protein complex. These
proteins, in combination with additional membrane proteins and the
cytosolic Sec13/31 complex, promote membrane invagination and vesicle
fission. Although some of the basic coat components involved in COPII
coat assembly have been identified, the mechanism by which biosynthetic
cargo is selected for and concentrated into COPII vesicles is unknown.
A role for sorting signals was first demonstrated for clathrin-mediated
vesicle formation at the plasma membrane (reviewed in Ref. 5). Here,
tyrosine-based motifs direct both the selection and concentration of
cargo molecules into clathrin-coated vesicles through the interaction
with the µ2 chain of AP2 complexes (reviewed in Ref. 6).
Similarly, the selection and concentration of recycling cargo into
COPI-coated vesicles that mediate retrograde transport between Golgi
compartments and from pre-Golgi compartments to the ER involve the
KKXX motif (7) (reviewed in Ref. 8). Current evidence
suggests that recycling cargo is concentrated by binding to the Signals involved in the selection of cargo for export from the ER are
only beginning to emerge. Peptides containing a double Phe (FF) motif
found in the cytoplasmic domain of the mammalian p24 family and p53/58
proteins that recycle between the ER and the Golgi have been shown to
bind the Sec23 COPII component in vitro (10, 11). Mutation
of these residues effects trafficking of these proteins in
vivo (10, 12). Curiously, the FF motif in the yeast p24 family
member Emp24p has been reported to attenuate the effect of a separate
export signal composed of adjacent Leu-Val (LV) residues present at the
extreme carboxyl terminus (13). We have recently found that a number of
newly synthesized type 1 transmembrane proteins destined for the cell
surface, including vesicular stomatitis glycoprotein (VSV-G), require a
di-acidic motif (DXE) in their cytoplasmic domains for
efficient export (14, 15). When the DXE code is mutated to
AXA, VSV-G exits the ER at ~10-fold slower rates. Unlike
the FF motif, the di-acidic code is sufficient to direct export from
the ER. Addition of the code to the Whereas clathrin recognizes fully folded cargo for selective delivery
to distinct destinations, export of newly synthesized cargo from the ER
is faced with competing pathways including those directing completion
of folding ("quality control" (16)) and those that target proteins
for degradation (17). Moreover, unlike clathrin-mediated carriers,
ER-derived vesicles are delivered to a common destination, the Golgi
stack. Thus, cargo sorting from the ER is a problem unique to the COPII
machinery. The functional relationships between signals that target
biosynthetic cargo to each of the ER-specific pathways are unknown.
Signal strength for each route will be expected to affect the kinetics
of export from the ER. Moreover, it is now evident that a number of
endogenous factors, some of which recycle between the ER and the Golgi,
temporally associate with biosynthetic cargo and are essential for
export (18-22). Thus, multiple signals on cargo may be required to
direct interaction with specialized accessory factors as well as the general COPII machinery to solicit efficient export.
We have recently shown that VSV-G interacts with a subset of COPII
components, the Sar1 GTPase and the Sec23/24 complex, to form
pre-budding complexes that are subsequently assembled in mature COPII
vesicles budding from the ER (3). From these and related studies in
yeast (4), it is clear that components of the COPII machinery
participate in cargo selection and concentration. Whether the
DXE signal directs the initial selection event,
concentration, or both remains a fundamental question that needs to be addressed.
To analyze the mechanism of the DXE signal in cargo
selection and concentration during ER export, we have taken advantage of cells expressing VSV-G. The transport of VSV-G has been extensively utilized to define the basic biochemical components and principles of
operation of the secretory pathway (reviewed in Refs. 1 and 15). In
particular, the tsO45 strain of VSV-G (VSV-Gts) can be
accumulated in the ER due to a temperature-sensitive folding defect
when cells are incubated at the restrictive temperature of 39.5 °C.
Upon shift to the permissive temperature of 32 °C its folding
resumes, allowing VSV-Gts to exit the ER. The ability to
synchronize movement of VSV-Gts allows us to follow a
single well defined biosynthetic cargo molecule through the secretory
pathway (3, 23-26).
In order to visualize export directly from the ER, we have tagged both
the tsO45-VSV-G wild-type (Gts-DXE) and the
AXA mutant (Gts-AXA) with green
fluorescence protein (GFP). By monitoring the real time movement of
GFP-VSV-Gts within the same cell in vivo and
in vitro using video microscopy, we find that the
DXE motif plays a key role in the concentration of VSV-G
into COPII-coated vesicles at ER export sites. Loss of concentration of
the AXA mutant is not a consequence of inability of the
mutant to be recruited to pre-budding complexes formed in the presence
of activated Sar1 and Sec23/24. Our ability to uncouple cargo selection
by the COPII machinery from a downstream concentration event(s)
preceding vesicle fission leads us to propose a minimal two-step model
for ER export of cargo.
Reagents--
Texas Red goat anti-mouse and anti-rabbit IgG
conjugates were obtained from Molecular Probes (Eugene, OR).
Horseradish peroxidase (HRP)-conjugated Fab fragments of goat
anti-rabbit antibodies were purchased from BioSys (Compiègne,
France). Anti-HA antibody (12CA5) was obtained from Roche Molecular
Biochemicals. Polyclonal antibodies recognizing DNA Constructs--
The carboxyl terminus of tsO45-VSV-G
wild-type (Gts-DXE) and AXA mutant
(Gts-AXA) (14) were tagged with Myc and HA
epitope by polymerase chain reaction. The resulting clones
(Myc-Gts-DXE and
HA-Gts-AXA) were subcloned into pAdtet7 vector
for recombinant adenovirus production. The S65T variant of GFP (30) was
fused to the carboxyl terminus of Gts-DXE or
Gts-AXA with an amino acid spacer (Ala) by
standard two-step polymerase chain reaction (31). The resulting clones
(GFP-Gts-DXE and
GFP-Gts-AXA) were subcloned into pSFV3 vector
for recombinant SFV production. All constructs used in this study were
verified by DNA sequencing.
Pre-budding Complex Isolation--
Microsomes were prepared from
recombinant adenovirus (Myc-Gts-DXE or
HA-Gts-HA)-infected tTAHeLa cells as described (27, 29).
Pre-budding complex isolation was performed as described (3). Briefly, microsomes (180-200 µg) were salt-washed and incubated in a
transport buffer containing GST-Sar1 H79G (7 µg), Sec23/24 (5 µg),
and 1 mM GTP for 30 min at the indicated temperature. The
microsomes were collected by centrifugation and solubilized by 1%
digitonin. Subsequently pre-budding complex containing GST-Sar1 H79G
was isolated on GS beads. Myc-Gts-DXE and
HA-Gts-AXA recovered in pre-budding complex were
determined by Western blotting as described below.
Western Blotting--
For pre-budding complex isolation, eluates
from GS beads and 1/4 of the total lysate were separated by
7.5% SDS-polyacrylamide gel electrophoresis, transferred to
nitrocellulose membranes, and immunoblotted using T25I (for
Myc-Gts-DXE) or 12CA5 (for
HA-Gts-AXA) antibody. For
GFP-Gts-DXE and
GFP-Gts-AXA expression, BHK cell lysates were
immunoblotted using anti-GFP antibody. Blots were developed using an
ECL kit (Amersham Pharmacia Biotech) and quantitated using a
Densitometer SI (Molecular Dynamics, Sunnyvale, CA).
Indirect Immunofluorescence--
Indirect immunofluorescence was
performed as described (26). Cells were fixed in 2% formaldehyde in
PBS and blocked with 5% goat serum in PBS (PBS/goat serum). To detect
VSV-G reached to the cell surface, cells were incubated with a
monoclonal antibody against VSV-G (8G5) in the absence of saponin and
visualized with Texas Red goat anti-mouse IgG conjugate. To detect
Immunoelectron Microscopy--
For immunoperoxidase
cytochemistry, cells were fixed in 3% formaldehyde, 0.025%
glutaraldehyde in PBS for 60 min, washed with PBS containing 0.05 M glycine, and permeabilized with 0.05% saponin in PBS
containing 0.2% BSA (PBS/BSA). Then cells were incubated with a
polyclonal antibody against VSV-G overnight at 4 °C and with
HRP-conjugated secondary antibodies for 2 h. After several washes
with PBS/BSA, cells were fixed with 2.5% glutaraldehyde in 0.1 M sodium cacodylate (pH 7.4) for 30 min, washed with 0.1 M Tris/HCl (pH 7.2), and incubated with 1 mg/ml
diaminobenzidine in 0.1 M Tris/HCl (pH 7.2) containing
0.01% H2O2 for 10 min. After washing out
diaminobenzidine to terminate HRP reaction, cells were postfixed in 0.1 M sodium cacodylate (pH 7.4), cacodylate containing 1%
reduced OsO4, and 1% KFeCN. Finally cells were treated with 1% tannic acid for 1 h, dehydrated, and embedded in Epon 812 as described (38). Thin sections were cut as described (38), stained
with lead citrate, and viewed on JEOL 1200EX-II electron microscope
(JEOL, Peabody, MA) at 80 kV. In order to quantitate the effects of
DXE mutation, 25 cells expressing VSV-G in the ER were
chosen, and all of the Golgi stacks within these cells were examined
for the presence of HRP reaction products. Immunogold (6 nm) electron
microscopy and quantification were performed as described (32).
Imaging of Fluorescence--
Indirect immunofluorescence and
time lapse imaging of GFP was performed using an inverted microscope
(Carl Zeiss Axiovert 100TV, Thornwood, NY) equipped with a CCD camera
(Photometrics PXL, Tucson, AZ), a motorized objective lens (Carl Zeiss,
Thornwood, NY), a temperature controller (20/20 Technology Inc.,
Wilmington, NC), and an automated stage (Ludl Electronic Products Ltd.,
Hawthrone, NY). Isee (Inovision, Raleigh, NC) was used to control both
image acquisition and an automated stage on a UNIX computer (Silicon Graphics O2; Mountain View, CA). Pixel intensities of ER, pre-Golgi intermediates, and Golgi were determined from raw data using NIH Image
1.62 program.
Morphological Analysis of Transport in Vivo--
Morphological
analysis of VSV-G transport in vivo was performed as
described (33). Briefly, BHK cells on coverslips in 35-mm dish were
infected with recombinant vaccinia virus encoding T7 RNA polymerase
(vTF7-3) and transfected with pAR plasmid encoding either GFP-tagged
Gts-DXE (GFP-Gts-DXE) or
Gts-AXA (GFP-Gts-AXA) at
39.5 °C. After 6 h transfection, cells were subjected to either
indirect immunofluorescence, immunoelectron microscopy, or time-lapse
imaging. For indirect immunofluorescence and immunoelectron microscopy,
transport was initiated by transferring cells to 15, 20, or 32 °C
and terminated by transferring to ice. For time-lapse imaging, cells
were put on an automated stage pre-equilibrated at 4 °C and overlaid
with an ice-cold medium without phenol red. Transport was initiated by
raising its temperature to 32 °C, and time-lapse images of one cell
were acquired every 1 min up to 120 min.
Morphological Analysis of Transport in Vitro--
Morphological
analysis of VSV-G transport in vitro was performed as
described (26). Briefly, NRK cells on coverslips in 35-m dish were
infected with recombinant SFV encoding either
GFP-Gts-DXE or
GFP-Gts-AXA at 39.5 °C. After 24 h
infection, cells were permeabilized with digitonin, transferred to an
automated stage pre-equilibrated at 4 °C, and overlaid with a
transport mixture (25 mM Hepes/KOH (pH 7.2), 75 mM KOAc, 2.5 mM Mg(OAc)2, 5 mM EGTA, 1.8 mM CaCl2, 1 mM ATP, 5 mM creatine phosphate, 0.2 IU rabbit
muscle creatine phosphokinase, 2.5 mg/ml rat liver cytosol (34)).
Before initiating transport by raising the temperature to 32 °C, 10 cells were identified, and their positions were recorded by an Isee
(Inovision, Raleigh, NC) automated stage controller. Subsequently
transport was initiated, and time-lapse images of each cell were
acquired every 10 min up to 60 min.
The Role of the Di-acidic Exit Code in Recruitment of VSV-G into
Pre-budding Complexes--
VSV-G is a type I transmembrane
glycoprotein containing two N-linked oligosaccharides in the
lumenal domain and a 29-amino acid cytoplasmic tail. We have recently
shown that the cytoplasmic tail of VSV-G contains a di-acidic export
signal (DXE) that is sufficient to direct the export of
cargo from the ER (14). Substitution of this DXE code with
Ala (AXA) results in an ~5-10-fold reduction in the
rate of ER export based on the kinetics of processing of its
N-linked oligosaccharides from endoglycosidase H (endo
H)-sensitive species found in the ER to endo H-resistant forms found in
the cis/medial Golgi compartments. Consistent with the reduced rate of
processing in Golgi, we have shown that at an early time point (10 min)
following temperature shift, only trace levels of the AXA
mutant can be detected in the Golgi stack using indirect
immunofluorescence (14). However, this mutation does not affect the
total extent of the transport. Pulse-labeled AXA mutant
present in the ER can be recovered in fully processed Golgi forms at
longer incubation times (60-90 min) (14).
To begin to explore the mechanism by which the DXE signal
directs efficient ER export, we first examined whether the
DXE code was necessary for the recovery of VSV-G in
detergent-soluble pre-budding complexes. These are intermediates in the
pathway required for the selection and concentration of VSV-G in COPII
vesicles (3). Recovery in pre-budding complexes is specific as ER
resident proteins are efficiently excluded from these intermediates
(3). They can be readily detected when ER membranes are incubated in
the presence of a subset of COPII components, the Sec23/24 complex and
a glutathione S-transferase (GST)-tagged Sar1[H79G], a
mutant that has a markedly reduced rate of GTP hydrolysis (Sar1-GTP) (27). Sar1-GTP stabilizes the assembled pre-budding complex for
isolation on glutathione-Sepharose (GS) beads in the presence of
detergent (3). We have recently demonstrated that interaction of cargo
with these COPII components precedes completion of folding and
oligomerization, suggesting that recruitment to pre-budding complexes
represents an early step in the export pathway (32).
To generate ER microsomes containing wild-type or AXA mutant
VSV-G, tTA-HeLa cells were infected with recombinant adenovirus expressing either Myc-tagged VSV-Gts-DXE
(Myc-Gts -DXE) or HA-tagged
VSV-Gts-AXA (HA-Gts
-AXA). VSV-Gts (Gts) is a
temperature-sensitive variant of wild-type G protein produced by tsO45
virus. VSV-Gts fails to exit the ER when cells expressing
the protein are incubated at the restrictive temperature (39.5 °C).
Transfer of cells to the permissive temperature (32°) leads to
synchronous export (24, 26, 33). The ability to synchronize export
allows us to control the movement of VSV-G from the ER into early
compartments of the secretory pathway including pre-Golgi intermediates
and Golgi cisternae (23, 24, 26, 34). We have used VSV-infected cells
expressing Gts extensively to demonstrate the central role
of the Sar1 GTPase and other COPII components in the formation of VSV-G
containing COPII-coated vesicles (3, 14, 27). Neither the Myc nor the
HA tags were found to have any effect on the kinetics of transport of
the untagged Gts-DXE or
Gts-AXA mutant to the Golgi based on processing
to endo H-resistant forms (data not shown).
Following infection at the restrictive temperature to restrict either
the Myc-Gts DXE or HA-Gts
AXA to the ER, microsomes were prepared and incubated
in vitro 30 min in the presence of GST-Sar1-GTP and Sec23/24
as described (3) to generate pre-budding complexes. As expected,
Myc-Gts DXE was not recovered in pre-budding
complexes when microsomes were incubated on ice, a condition that also
does not support COPII recruitment or vesicle budding from the ER (27)
(Fig. 1). In contrast,
Myc-Gts-DXE was recovered at both the permissive
and restrictive temperatures (~18% of total based on quantitative
immunoblotting) (Fig. 1). This result is consistent with our recent
observations that untagged VSV-Gts can be efficiently
captured in pre-budding complexes in both the folded and unfolded
states (32).
Interestingly, the HA-Gts-AXA was also
efficiently recovered in the pre-budding complexes formed in the
presence of Sar1-GTP at both the permissive and restrictive
temperatures (~20% of total) (Fig. 1). Under these conditions less
than 10% of the HA-Gts-AXA has exited the ER at
the permissive temperature compared with >90% for
Myc-Gts-DXE (14). Although we were surprised at
our ability to recover VSV-G in pre-budding complexes given the
possibility that it was a COPII recognition motif (14), this suggests
that the DXE code is also required for a new, previously
undetected, step after formation of pre-budding complexes to promote
efficient ER export.
Inactivation of the Di-acidic Exit Code Markedly Reduces the
Steady-state Concentration of VSV-G in Golgi Compartments--
To
identity novel step(s) requiring DXE function for
accelerated ER export, we tagged the tsO45 variant of VSV-G with green fluorescent protein (GFP) (GFP-Gts) in order to follow
transport in real time in single cells using fluorescence microscopy
(35). Baby hamster kidney (BHK) cells were transiently transfected with
GFP-Gts containing either the wild-type DXE exit
code (GFP-Gts-DXE) or the mutant
(GFP-Gts-AXA). Both the
GFP-Gts-DXE and
GFP-Gts-AXA were restricted to the ER when cells
were incubated at 39.5 °C based on sensitivity to endo H and ER
localization using indirect immunofluorescence (data not shown; see
below). Transfer to the permissive temperature resulted in the
processing of GFP-Gts-DXE and
GFP-Gts-AXA to endo H-resistant forms. The
kinetics of processing of GFP-tagged Gts paralleled that of
their respective untagged wild-type and mutant forms (14) (data not
shown). These results demonstrate that the tag has no effect on the
overall properties or kinetics of transport of tsO45 VSV-G as observed
previously (35). When transiently transfected cells were analyzed using
immunoblotting, we found that the GFP-Gts-AXA
construct was expressed on average at an ~2-fold greater efficiency
than that of the GFP-Gts-DXE (Fig.
2A, inset in panel a).
To determine the steady-state distribution of wild-type and mutant
VSV-G, following transfection at 39.5 °C to accumulate protein in
the ER, cells were incubated at the permissive temperature of 32 °C
for 120 min. This time frame is sufficient to mobilize both mutant and
wild-type forms of VSV-G to the cell surface (14). Under these
conditions we found that GFP-Gts-DXE showed the
typical increased fluorescence in Golgi compartments over that observed
in the ER (Fig. 2A, panel a). Golgi localization of
concentrated GFP-Gts-DXE was confirmed by
co-localization with the cis/medial Golgi marker protein
In contrast to GFP-Gts-DXE, delivery of
GFP-Gts-AXA to the Golgi was difficult to detect
using indirect immunofluorescence despite its ~2-fold expression
relative to GFP-Gts-DXE (Fig. 2A, panel
c). The normal distribution observed for
These results are quantitated in Fig. 2B where
GFP-Gts-DXE was found to be concentrated
7.2-fold relative to the ER (Fig. 2B, lane a), whereas the
GFP-Gts-AXA mutant was detected at levels
1.2-fold that found in the ER (Fig. 2B, lane b). No
difference in the relative concentration of the GFP-Gts-AXA Is Not Concentrated in the TGN
at 20 °C--
GFP-Gts-AXA failed to be
concentrated in Golgi compartments in cells incubated at 32 °C
relative to levels observed in the ER. It remained possible that we
could not detect this event morphologically due to a more rapid
transport of GFP-Gts-AXA from the Golgi to the
cell surface compared with its rate of export from the ER. To address
this concern, we took advantage of the well established effects of
incubation at 20 °C on exit from the Golgi stack. At 20 °C, the
transport of VSV-G and other cargo are kinetically inhibited in the
trans Golgi network (TGN) with little transport to the cell surface
(39). This results in the accumulation of cargo in the TGN and
elaboration of the trans-most tubular elements of the Golgi.
When cells transfected with GFP-Gts-DXE were
transferred from 39.5 to 20 °C for 120 min,
GFP-Gts-DXE accumulated in the TGN as expected
(Fig. 3A, panel a).
In contrast, GFP-Gts-AXA showed only a slight
increase in fluorescence over the background ER levels (Fig. 3A,
panel b). The apparent slight increase is likely to be due to the
overlapping elements of the Golgi stack that are frequently found in
compacted forms in peri-nuclear loci. In this experiment,
GFP-Gts-DXE was concentrated 5.5-fold relative
to the ER level (Fig. 3C, lane a), whereas the
AXA was found at 1.4-fold that observed in the ER (Fig.
3C, lane b). Thus, incubation at 20 °C failed to show any
significant concentration of GFP-Gts-AXA in
trans-most Golgi compartments.
GFP-Gts AXA Is Not Concentrated in Pre-Golgi
Intermediates at 15 °C--
Although the inability to detect
concentrated VSV-G in Golgi compartments is consistent with a role for
the DXE code in directing concentration during ER export, it
is necessary to directly assess its effect on the movement of VSV-G
from the ER. For this purpose, we took advantage of previous studies
where we and others (26, 38, 40, 41) have demonstrated that cargo
exiting the ER in COPII vesicles accumulates in numerous punctate
pre-Golgi intermediates. These consist of compact clusters of
vesicular-tubular elements that form when cells are incubated at the
reduced temperature of 15 °C. Current evidence suggests that the
tubular elements are likely to arise from the fusion of COPII vesicles
(34) that can be readily visualized using indirect immunofluorescence
(23, 26, 40, 42). Visualization of the dynamic movement of pre-Golgi intermediates containing GFP-VSV-Gts on microtubules in
living cells (35) has shown that peripheral vesicular-tubular clusters
function to deliver cargo to the central Golgi region (reviewed in Ref.
15).
When BHK cells transfected with GFP-Gts-DXE at
39.5 °C were incubated at 15 °C for 120 min, they showed the
typical accumulation of VSV-G in numerous punctate structures
throughout the cell (Fig. 3B, panel a). These co-localized
with the pre-Golgi intermediate marker protein syntaxin 5 (Syn5) (Fig.
3B, panel b) (34). In contrast, in
GFP-Gts-AXA-transfected cells no structures
containing GFP (Fig. 3B, panel c) could be observed, despite
the abundance of Syn5-containing elements (Fig. 3B, panel
d). On average, the number of Syn5-containing elements that
accumulate at 15 °C in
GFP-Gts-AXA-transfected cells was the same as
that observed in GFP-Gts-DXE-transfected cells
(~60-100 punctate elements per cell). This indicates that
transfection with GFP-Gts-AXA did not affect the
ability of the cell to generate Syn5-containing pre-Golgi intermediates
at 15 °C. When quantitated, GFP-Gts-DXE was
concentrated 5.8-fold over that observed in the ER (Fig. 3C, lane
a, whereas an increase in GFP-Gts-AXA
concentration was not detectable (Fig. 3C, lane b)). The concentration of Syn5 in pre-Golgi intermediates that accumulate at
15 °C were identical in GFP-Gts-DXE- and
GFP-Gts-AXA-expressing cells (Fig. 3C,
lanes c and d). The inability to detect concentrated
GFP-Gts-AXA in pre-Golgi intermediates under
conditions where these structures form normally provides direct
evidence that DXE participates in the efficient delivery to
these structures.
Real Time Imaging Confirms the Requirement for the DXE
Code in Concentration--
The above results utilized fixed time
points to visualize directly the distribution of VSV-G morphologically
in vivo. It remained possible that the movement of
GFP-Gts-AXA through pre-Golgi intermediates to
the Golgi was a transient event. Moreover, incubation at reduced
temperature may have in some unexpected way exacerbated the reduced
kinetics of transport of the AXA mutant. To circumvent these
potential concerns, we examined the continuous real time movement of
GFP-Gts-DXE and
GFP-Gts-AXA in living cells over a complete time
course of 120 min.
BHK cells were transfected with either
GFP-Gts-DXE or
GFP-Gts-AXA at 39.5 °C to restrict the
protein to the ER. Subsequently, cells were shifted to the permissive
temperature, and the movement of VSV-G was followed in real time using
fluorescence video microscopy. Representative time frames are shown in
Fig. 4.
GFP-Gts-DXE showed rapid concentration in
pre-Golgi intermediates (Fig. 4, a-e) as reported
previously (35). This was particularly evident by the 10-min (Fig.
4d, arrowheads) and 15-min (Fig. 4e,
arrowheads) time points. In contrast, no detectable concentration
of GFP-Gts-AXA was observed in any structure
during a 45-min incubation at 32 °C (Fig. 4, f-j) or up
to 120 min of incubation (data not shown). This is a time period
sufficient for complete processing of pulsed
GFP-Gts-AXA in the ER to Golgi-modified endo
H-resistant forms (14) and for efficient delivery to the cell surface
(Fig. 4j, arrow; Fig. 2C) (14). A real
time visualization of export of GFP-Gts-DXE and
GFP-Gts-AXA to pre-Golgi intermediates can be
found on the Internet.2
The appearance of VSV-G in pre-Golgi intermediates was quantitated over
a time course of 75 min (Fig. 5,
closed circles). Following a brief lag period, the number of
VSV-G containing punctate structures increased with time to a peak
level of ~50 elements at the 30-min time point which declined
slightly to a steady-state level of ~45 in this typical series. This
number is consistent with the average number of pre-Golgi intermediates
detectable in living cells using quantitative stereology (38). The
slight decline in intermediates may reflect consolidation of VSV-G in the more perinuclear Golgi regions following microtubule-mediated transit to this region of the cell (35). In contrast, no detectable intermediates containing concentrated VSV-G were discernible at any
time point (Fig. 5, open circles). These in vivo
data are consistent with an essential role for the DXE motif
in concentration of VSV-G in COPII vesicles.
The Reduced Concentration of GFP-Gts-AXA in
the Golgi Can Be Detected by Electron Microscopy--
To analyze the
effects of the di-acidic code on the concentration of VSV-G in post-ER
compartments using electron microscopy, we employed two approaches as
follows: semi-quantitative immunoperoxidase cytochemistry and
quantitative immunoelectron microscopy.
BHK cells were transfected with either
GFP-Gts-DXE or
GFP-Gts-AXA at 39.5 °C and shifted to
32 °C for 120 min to reach a steady-state distribution in all
cellular compartments. Subsequently, these cells were prepared for
electron microscopy under identical conditions. By using
immunoperoxidase labeling with an antibody directed to the lumenal
domain of VSV-G, both GFP-Gts-DXE and
GFP-Gts-AXA could be detected in a weak, diffuse
distribution in the ER (Fig. 6,
A-C, arrows). In cells transfected with
GFP-Gts-DXE, horseradish peroxidase (HRP)
reaction products were heavily concentrated in Golgi compartments
compared with the ER (Fig. 6A). In 25 transfected cells
examined, 95% (96/101) of the Golgi stacks detected contained
concentrated VSV-G throughout all cisternae. In contrast, cells
transfected with the GFP-Gts-AXA construct
showed a uniformly weak deposition of HRP reaction product in the Golgi
(Fig. 6, B and C). In 25 transfected cells containing VSV-G in the ER, 49% (48/98) of Golgi stacks contained minimally detectable levels of VSV-G in at least one of the Golgi compartments. Given the fact that the average expression level of
GFP-Gts-AXA is approximately 2-fold that of
GFP-Gts-DXE (Fig. 2A, inset in
panel a), these results are completely consistent with the low
levels of fluorescence in GFP-Gts-AXA-expressing
cells using light microscopy.
To quantitate the fold difference in concentration between
GFP-Gts-DXE and
GFP-Gts-AXA in the Golgi of transfected cells,
we prepared cells as described above, and we determined the
distribution of VSV-G using 6-nm gold particles in conjunction with
cryoimmunoelectron microscopy and stereology (38). Typical images (Fig.
6D (GFP-Gts-DXE) and E
(GFP-Gts-AXA)) revealed a striking difference in
their relative concentrations. Whereas
GFP-Gts-DXE was present at an average
concentration of 82 gold particles per µm2,
GFP-Gts-AXA was present at a concentration of
7.7 gold particles per µm2, corresponding to an
~10-fold difference. Given our previous results demonstrating that
the level of concentration of VSV-G detected in Golgi compartments
directly reflects the concentration of VSV-G in COPII vesicles exiting
the ER (3, 24, 38), these data provide high resolution evidence for the
importance of the DXE code in promoting concentration during
ER export.
Export of GFP-Gts from the ER Can Be Visualized in Real
Time in Vitro Using Permeabilized Cells--
In order to begin to gain
biochemical insight into the concentration process, we developed a new
approach to follow the real time movement of VSV-G into pre-Golgi
intermediates in vitro using permeabilized cells. We have
previously demonstrated the utility of permeabilized cells to follow
morphologically the concentration of VSV-G into punctate structures
that correspond to pre-Golgi intermediates observed in vivo
(23, 24, 26, 38). By using this approach, we have shown that cargo
selection involves the COPII budding machinery whose assembly is
controlled by the activation of the Sar1 GTPase (3, 27, 38, 43).
To follow movement of VSV-G in vitro, normal rat kidney
(NRK) cells expressing either GFP-Gts-DXE or
GFP-Gts-AXA were incubated at 39.5 °C to
retain VSV-G in the ER. Subsequently, the coverslip containing cells
was transferred to ice, permeabilized, and incubated in
vitro in the presence of cytosol and ATP. The movement of
GFP-Gts from the ER to post-ER compartments within the same
cell was visualized using video microscopy following shift from 4 to
32 °C. Under these conditions, the transport of
GFP-Gts-DXE in vitro from the ER to
pre-Golgi intermediates could be readily detected. At the first time
point, corresponding to approximately 5 min after temperature shift
(the time period required for acquisition of the first image),
GFP-Gts-DXE could be weakly detected in small
fluorescence puncta indicative of initiation of ER export (Fig.
7A, arrowheads). By 20 min,
numerous brightly fluorescent puncta were detected in
GFP-Gts-DXE-containing cells (Fig. 7A,
arrowheads). In contrast, GFP-Gts-AXA could
not be detected in fluorescence puncta by the 60- (Fig. 7B)
or 120-min time points (data not shown).
When quantitated (Fig. 7C),
GFP-Gts-DXE-containing puncta reach a maximum
level of ~30-40 intermediates per cell by the 20-min time point.
These results are consistent with the ~5-fold reduced kinetics of
processing of GFP-Gts-AXA to endo H-resistant
forms in vitro compared with
GFP-Gts-DXE (data not shown). Therefore, cytosol
and ATP-dependent export in vitro faithfully
reconstitute the requirement for the DXE motif in
concentration of VSV-G in pre-Golgi intermediates observed in
vivo.
The Concentration Defect in GFP-Gts-AXA Cannot Be
Suppressed by the Addition of Activated
Sar1-GTP--
Gts-AXA can be recruited to a
pre-budding complex in the presence of the Sar1-GTP mutant at levels
equivalent to Gts-DXE yet fails to be
concentrated before completion of COPII vesicle fission from the ER
membrane. If the DXE-dependent concentration step is completed by the interaction between cargo and COPII components Sar1-GTP and Sec23/24, then the efficiency of recruitment of
Gts-AXA to pre-Golgi intermediates in
permeabilized cells should be indistinguishable from
Gts-DXE when incubated in the presence of the
Sar1-GTP mutant. Alternatively, if the concentration step utilizes
additional factors recruited by the DXE signal,
Gts-AXA should not be concentrated in pre-Golgi
intermediates as efficiently as Gts-DXE. To
address this issue, we incubated permeabilized cells with the Sar1-GTP mutant.
Incubation of permeabilized cells expressing
GFP-Gts-DXE in the presence of ATP, cytosol, and
the Sar1-GTP mutant resulted in the appearance of concentrated VSV-G in
numerous fluorescent puncta by 30 min of incubation (Fig.
8A, arrowheads). We have
previously shown that these are composed of clusters of coated COPII
vesicles due to the ability of the Sar1-GTP to prevent vesicle
uncoating (3, 27, 38). In contrast, we were unable to detect
GFP-Gts-AXA in a concentrated form in pre-Golgi
intermediates (Fig. 8A) at this early time point (30 min)
when the AXA mutant can be fully recovered in pre-budding
complexes containing the Sar1-GTP. This suggests that Sar1-GTP could
not bypass inactivation of the DXE signal in the
concentration step. However, at later time points (60 min)
GFP-Gts-AXA was detectable in punctate pre-Golgi
intermediates (Fig. 8B, arrowheads). This lag is consistent
with the 5-10-fold reduction in the kinetics of transport of
Gts-AXA from the ER to Golgi in vivo.
Thus, whereas activation of Sar1 to the GTP-bound form is necessary for
cargo selection (3), it is not sufficient to rapidly concentrate VSV-G
into COPII vesicles. This suggests that DXE signal plays an
important role in recruiting an additional factor(s) that mediates
cargo concentration for efficient ER export.
In the present study we have examined the biochemical role of the
di-acidic code found in the cytoplasmic tail of VSV-G (14) in mediating
the efficiency of export of cargo from the ER. We have now shown that
the code is involved in a step involving concentration in COPII-coated
vesicles. To demonstrate this point, we used combined biochemical and
morphological approaches, in particular utilizing GFP-tagged VSV-G to
visualize the ER export in real time. This approach allowed us to
follow the fate of VSV-G in a single cell during trafficking from the
ER to the cell surface in vivo. Moreover, we have now
extended this technology to visualize the movement of
GFP-Gts in vitro. This facile approach gives us
precise biochemical control over incubation conditions that are
important to physiologically mobilize VSV-G from the ER (3, 27, 38).
Our results provide new insight into the pathway directing export of
cargo from the ER.
Role of DXE in Recovery of VSV-G in Pre-budding
Complexes--
We found that mutation of the DXE code to
AXA does not affect the ability of VSV-G to be recovered in
detergent-soluble pre-budding complexes formed in vitro in
the presence of activated Sar1-GTP and purified Sec23/24 when the
AXA mutant is largely present in the ER. This selection
event is the first detectable step in the capture of VSV-G into COPII
vesicles in mammalian cells (3) and for COPII vesicle assembly by
endogenous recycling transport factors in yeast (44). Although we were
at first surprised that the DXE code was not required for
cargo selection by Sar1-GTP and Sec23/24, this result is entirely
consistent with the fact that loss of the code affects the kinetics of
transport but not the extent (14). We conclude that at least one
principal activity that is responsible for the accelerated rate of
VSV-G export from the ER is downstream from selection into pre-budding
complexes. These results raise the possibility that the multiple
signals may be required in VSV-G and other cargo for efficient export. Experiments are currently in progress to address this possibility.
DXE Is Involved in Concentration in COPII
Vesicles--
By using both indirect immunofluorescence and electron
microscopy we have demonstrated an important role for the
DXE motif in concentration of cargo in COPII vesicles.
Evidence for this conclusion stems from the observation that the
AXA mutant could not be detected in a concentrated form in
pre-Golgi and Golgi compartments under a variety of incubation
conditions designed to block and accumulate cargo in post-ER
compartments at reduced temperature. Moreover, continuous monitoring of
movement in vivo and in vitro failed to detect
even a transient appearance of concentrated AXA mutant in
the early secretory pathway over a time frame in which the
AXA mutant is efficiently processed to endo H-resistant forms and delivered to the cell surface. Two independent approaches were used to characterize the concentration of VSV-G in post-Golgi compartments using electron microscopy. Given the fact that the concentration of VSV-G in Golgi compartments is a measure of
concentration during ER export (3, 24, 38), the reduced level of HRP reaction product or gold particles in Golgi compartments expressing the
AXA mutant when compared with wild-type demonstrates a
marked deficiency in concentration during COPII vesicle budding. It
is apparent that inactivation of the code (mutation of DXE
to AXA) uncouples VSV-G from the concentration machinery
in vivo.
Requirement for Sar1-GTP in Cargo Concentration--
Because we
could not detect a difference in the recovery of wild-type VSV-G or the
AXA mutant in pre-budding complexes formed in the presence
of Sar1-GTP, we examined whether Sar1-GTP could bypass the
concentration defect following inactivation of the code. We found that
this was not the case. Incubation in the presence of Sar1-GTP did not
result in normal concentration of the AXA mutant. At early
time points (30 min), when wild-type VSV-G could be readily detected in
a concentrated form in pre-Golgi intermediates in the presence of
Sar1-GTP, the AXA mutant could not be detected. This result
is consistent with the reduced kinetics of export of the AXA
mutant (14). Because recruitment to pre-budding complexes by Sar1-GTP
appears unaffected in the AXA mutant under the current experimental conditions, it is apparent that concentration is a
distinct step in the export pathway. These results emphasize that the
DXE code is essential for rapid concentration even in the
presence of the activated Sar1-GTP mutant.
In contrast to early time points, we found that at later time points
(60 min), the AXA mutant could start to be detected in a
concentrated form in pre-Golgi intermediates in the presence of
Sar1-GTP. This result contrasts to normal incubation conditions (in the
presence of wild-type Sar1) where the AXA mutant could not
be detected in pre-Golgi intermediates or Golgi compartments even after
120 min of incubation. Suppression of the AXA mutant phenotype at later time points is not due to a general loss of fidelity
in cargo selection, as we have previously documented that Sar1-GTP has
no effect on the ability of resident ER proteins to be efficiently
excluded from COPII vesicles formed in vitro (3, 27). One
possibility to explain concentration at late time points is that
incubation with Sar1-GTP favors stable recruitment of COPII components
to ER membranes (1, 3, 45). This increase in the steady-state level of
the COPII components on the ER surface could result in efficient
"capture" of lower affinity AXA mutant without
additional DXE signal-dependent factors. An
alternative, but very speculative possibility, is that the
DXE code normally facilitates coat assembly by stabilizing
Sar1 in the GTP-bound form upon assembly of a "recruitment"
complex, thereby facilitating efficient packing of cargo by COPII complexes.
The ability of excess Sar1-GTP to alter the efficiency of recruitment
of wild-type and mutant biosynthetic cargo is consistent with recent
observations from biochemical analysis of yeast COPII components in
vesicle budding. First, COPII vesicle budding from artificial liposomes
requires ~5-fold higher concentration of activated Sar1 than that
required to generate vesicles using microsomes (45). This suggests that
the liposomes form a low affinity template for COPII coat assembly.
In vivo, biosynthetic and/or recycling cargo would be
expected to initiate these events (3, 32, 44). Second, the
incorporation of biosynthetic cargo and recycling cargo into COPII
vesicles is differentially affected by the amount of Sar1 (46). Thus,
exported proteins require different levels of COPII components for
efficient export, suggesting recruitment is affected by the relative
affinity of the protein for the COPII machinery.
Sequential Steps Direct Pre-budding Complex Formation and
Concentration--
We have shown that recruitment of VSV-G to COPII
vesicles involves selection into pre-budding complexes containing
Sar1-GTP and Sec23/24 (3) that is DXE-independent (this
study). This is followed by a DXE-dependent
concentration step, suggesting that mutation of the DXE
signal can uncouple selection from concentration. We propose that
DXE may serve as a high affinity recruitment motif for a
novel "linker" component(s), possibly SNARE proteins that have
recently been shown to bind to Sar1 in vitro (44), or it may
enhance recruitment of membrane-associated components such as Sec16
that interact with Sec23/24 and whose role in COPII vesicle assembly
remain to be defined (47-49). Alternatively, it may recruit presently
unknown factors that stabilize the formation of pre-budding complexes
or mediate stable coat assembly from pre-budding complexes. Our
combined results suggest for the first time that export from the ER may
involve at least a two-step mechanism. In this model, cargo present in
pre-budding complexes (Fig. 9, step
1) undergoes a distinct concentration event for efficient
recruitment to COPII vesicles (Fig. 9, step 2).
The two-step model is supported by three lines of evidence. First, the
inactivation of the DXE can distinguish pre-budding complex
formation from concentration processes. Second, these two steps have
different sensitivities for the activated Sar1-GTP mutant. Whereas
Gts-AXA can be recovered in pre-budding
complexes containing the Sar1-GTP mutant as efficiently as
Gts-DXE, the inefficient kinetics of
concentration in pre-Golgi intermediates observed in the
Gts-AXA mutant cannot be restored to normal
efficiency by incubation in the presence of the Sar1-GTP. This argues
that following an initial interaction between VSV-G and a subset of
COPII components, further step(s) are required to promote a
concentration event that requires DXE-dependent
factors. Third, when Gts-DXE containing
microsomes are retained at the restrictive temperature (where
Gts is unfolded and remains in a diffuse ER reticular
distribution), Gts-DXE can be efficiently
recovered in pre-budding complexes (32). This provides an independent
line of evidence that cargo selection can be uncoupled from
concentration. Furthermore, a number of endogenous accessory factors,
some of which recycle between the ER and Golgi, are now recognized to
be essential for the export of specific biosynthetic cargo proteins,
for example NinaA, receptor-associated protein, Shr3, and p53/58 that
are required for ER export of rhodopsin, low density lipoprotein
receptor-related proteins, general amino acid permease-1, and
procathepsin C, respectively (18, 22, 50, 51). Although their mechanism
of action is currently unknown, it is possible that these factors
regulate either selection (step 1), concentration
(step 2), or novel, unidentified steps prior to COPII
vesicle formation.
Export from the ER requires discrimination between immature and mature
cargo by the COPII machinery. The use of a multi-step pathway allows
for branch points in which dysfunctional cargo can be targeted for
degradation. Moreover, it provides additional flexibility of the rate
in which different forms of cargo are exported from the ER, perhaps
independent of their folding pathways. Such a multi-step pathway may be
applicable to clathrin-mediated endocytosis of cell-surface receptors.
For example, stimulation of the
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunit of the COPI coat complex (9).
subunit of the T-cell
receptor-
, a resident ER protein, leads to recruitment into COPII
vesicles (14). Although the code is necessary for efficient export,
VSV-G transport is not completely blocked as the AXA mutant
exits the ER with ~10-fold reduced efficiency (14). This raises the
possibility that the DXE signal participates in only a
subset of interactions required for the efficient coupling of
biosynthetic cargo to the ER export machinery.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-1,2-mannosidase II,
green fluorescence protein (GFP), and a lumenal domain of VSV-G were
generous gifts from M. Farquhar (University of California, San Diego,
CA), C. Zukar (University of California, San Diego, CA), and K. Simons (EMBL, Heidelberg, Germany), respectively. A monoclonal antibody recognizing a lumenal domain of VSV-G (8G5) was kindly provided by B. Wattenberg (Upjohn, Kalamazoo, MI). Polyclonal antibodies against a
cytoplasmic tail of VSV-G (T25I) and syntaxin 5 (Syn5) was described
previously (27, 28). Purified proteins of GST-Sar1 H79G and Sec23/24
complex were prepared as described (3). Recombinant adenovirus and
Semliki forest virus (SFV) were generated using the method described in
Ref. 29 and SFV gene expression system kit (Life Technologies, Inc.),
respectively. All other reagents were purchased from Sigma.
-1,2-mannosidase II and Syn5, cells were permeabilized with 0.1%
saponin in goat serum/PBS, incubated with an appropriate antibody in
the presence of 0.1% saponin, and visualized with Texas Red goat
anti-rabbit IgG conjugate. Images were recorded digitally and
quantified as described below.
RESULTS
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ABSTRACT
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EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
The AXA mutant can be recovered
in pre-budding complexes. Microsomes prepared from tTA-HeLa cells
infected with recombinant adenovirus expressing either
Myc-Gts-DXE or HA-Gts-AXA
were incubated in the presence GST-Sar1-GTP and purified Sec23/24
complex on ice at 32 or 40 °C. Subsequently, detergent-soluble
pre-budding complexes were recovered on GS beads. VSV-G in the
pre-budding complex was detected by immunoblotting as described under
"Experimental Procedures." Total VSV-G represents 1/4 of the
total lysate used for isolation of pre-budding complexes. Quantitation
reported in the text was determined by densitometry.
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Fig. 2.
The AXA mutant is not
concentrated in the Golgi at steady state. A, BHK cells
were transfected with either GFP-Gts-DXE or
GFP-Gts-AXA at 39.5 °C. Following
transfection, cells were shifted to 32 °C for 120 min to reach
steady state where both mutant and wild-type protein are distributed
throughout the entire exocytic pathways. The relative concentration of
VSV-G in the exocytic compartments was determined by following the
distribution of GFP as described under "Experimental Procedures."
-1,2-Mannosidase II (ManII) was visualized using specific
antibody as described under "Experimental Procedures." Results are
representative of over 100 cells examined. Inset, BHK cells
were transfected with either GFP-Gts-DXE or
GFP-Gts-AXA at 39.5 °C. Following
transfection, the amount of VSV-G was determined using immunoblotting
as described under "Experimental Procedures." B,
quantitation of concentration in response to mutation of the
DXE signal. Average pixel intensity of Golgi and ER was
determined as described under "Experimental Procedures." The fold
increase relative to ER is indicated. The error bars
indicate the standard deviation of the mean for over 25 individual
determinations. C, GFP-Gts-AXA can be
readily detected at the cell surface. BHK cells were transfected with
either GFP-Gts-DXE (panel a) or
GFP-Gts-AXA (panel b) at 39.5 °C.
Following transfection, cells were shifted to 32 °C for 120 min to
reach the cell surface. VSV-G at the cell surface was visualized by
indirect immunofluorescence as described under "Experimental
Procedures." Results are representative of over 100 cells
examined.
-1,2-mannosidase II (36, 37) (Fig. 2A, panel b). Thus,
the addition of the GFP tag to the cytoplasmic tail does not interfere
with efficient delivery of GFP-Gts-DXE into
COPII vesicles during export from the ER.
-1,2-mannosidase II in
GFP-Gts-AXA transfected cells (Fig. 2A,
panel d) excludes the possibility that expression of
GFP-Gts-AXA per se disturbs the
organization of Golgi compartments.
-1,2-mannosidase II
marker compared with background was detected in
GFP-Gts-DXE- or
GFP-Gts-AXA-expressing cells (Fig. 2B,
lanes c and d). Importantly, the lack of concentration
of GFP-Gts-AXA in Golgi compartments did not
reflect a defect in the ability of the mutant to be transported to and
through the Golgi stack. GFP-Gts-AXA could
readily be detected on the cell surface at levels comparable to that of
wild type (Fig. 2C). Because concentration of cargo does not
occur during transit of cargo between compartments of the Golgi stack
(24, 38), these results raise the possibility that the DXE
motif may be required for concentration during ER export.
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Fig. 3.
GFP-Gts-AXA is not
concentrated in post-ER compartments at reduced temperature.
A, GFP-Gts-AXA is not concentrated in
the TGN at 20 °C. BHK cells were transfected with either
GFP-Gts-DXE (panel a) or
GFP-Gts-AXA (panel b) at 39.5 °C.
Following transfection, cells were shifted to 20 °C for 120 min to
prevent the export of VSV-G from the TGN. The distribution of VSV-G was
determined by detecting the fluorescence of GFP as described under
"Experimental Procedures." Results are representative of over 100 cells examined. B, GFP-Gts-AXA is not
concentrated in the pre-Golgi intermediates at 15 °C. BHK cells were
transfected with either GFP-Gts-DXE
(panels a and b) or
GFP-Gts-AXA (panels c and
d) at 39.5 °C. Following transfection, cells were shifted
to 15 °C for 120 min to block the transport from the pre-Golgi
intermediates to the cis Golgi compartment. The distribution of VSV-G
(panels a and c) was determined by detecting the
fluorescence of GFP. Syn5 was visualized using specific antibody as
described under "Experimental Procedures" (panels b and
d). The arrowheads indicate pre-Golgi intermediates. Results
are representative of over 100 cells examined. C,
quantitation of concentration in response to mutation of the
DXE signal. Average pixel intensity of pre-Golgi
intermediates and ER were determined as described under "Experimental
Procedures." The fold increase relative to ER is indicated. The
error bars indicate the standard deviation of the mean for
over 25 individual determinations.
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Fig. 4.
GFP-Gts-AXA is not
concentrated during export from the ER at 32 °C. BHK cells
transfected with either GFP-Gts-DXE
(a-e) or GFP-Gts-AXA
(f-j) at 39.5 °C were transferred directly to ice.
Transport was initiated by shifting cells from ice to 32 °C.
Time-lapse images of VSV-G within a living cell were acquired as
described under "Experimental Procedures." The arrow in
j indicates the cell surface. The images can be visualized
in real time.2
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Fig. 5.
Quantitation of appearance of VSV-G in
punctate intermediates. The number of punctate intermediates
containing GFP-Gts-DXE (closed
circles) or GFP-Gts-AXA (open
circles) with a greater than 2-fold increase in pixel intensity
relative to the ER at 1-min intervals (closed circles) or
5-min intervals (open circles) is plotted. The 45-75-min
time point is the averaged value of punctate intermediates observed in
this time frame with the standard deviation of the mean
indicated.
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Fig. 6.
Electron microscopy of steady-state
concentration of GFP-Gts-DXE and
GFP-Gts-AXA in the Golgi compartments. BHK
cells were transfected with either GFP-Gts-DXE
(A and D) or GFP-Gts-AXA
(B, C, and E) at 39.5 °C. Following
transfection, cells were shifted to 32 °C for 120 min to reach
steady state. The distribution of VSV-G was visualized by
immunoperoxidase staining as described under "Experimental
Procedures" (A-C) or immunoelectron microscopy using 6-nm
gold particles (D and E). D and
E, visualization of the distribution of 6-nm gold particles
has been enhanced by placement of a 12-nm dot over each gold particle.
Arrows indicate the ER; G indicates Golgi
complex.
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Fig. 7.
Transport and concentration of
GFP-Gts-DXE in the pre-Golgi intermediates can
be reconstituted in vitro. A, NRK
cells expressing GFP-Gts-DXE were permeabilized
on ice and placed in ice-cold transport mixture. Transport was
initiated by changing the temperature from 4 to 32 °C as described
under "Experimental Procedures." The first time point collected,
reflecting technical limitations, corresponds to 5 min after
temperature shift. Time-lapse images of VSV-G within a permeabilized
cell were acquired for up to 120 min as described under "Experimental
Procedures." Transport in a typical cell (>50 cells examined) is
shown. The arrowheads indicate pre-Golgi intermediates.
B, GFP-Gts-AXA is not concentrated in
pre-Golgi intermediates in vitro. NRK cells expressing
GFP-Gts-AXA were treated as in A.
Transport in a typical cell (>50 cells examined) at the 5- and 60-min
time points are shown. C, quantitation of the number of
punctate intermediates containing GFP-Gts-DXE or
GFP-Gts-AXA with a greater than 2-fold increase
in pixel intensity relative to the ER at the indicated time points. The
average value and standard deviation of the mean for 5 representative
cells followed throughout an entire time course is shown.
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Fig. 8.
The concentration defect in
GFP-Gts-AXA cannot be completely suppressed in
the presence of the Sar1-GTP mutant in vitro.
A, NRK cells expressing either
GFP-Gts-DXE (DXE) or
GFP-Gts-AXA (AXA-1 and
AXA-2 (two representative cells shown)) were permeabilized
on ice and placed in ice-cold transport mixture supplemented with 1 µM activated Sar1-GTP (Sar1[H79G]). Transport was
initiated by changing the temperature from 4 to 32 °C. Time-lapse
images of VSV-G within a cell were acquired at 5 min and every 10 min
thereafter for up to 120 min as described under "Experimental
Procedures." Representative cells following at 5, 30, and 60 min of
incubation are shown. The arrowheads indicate pre-Golgi
vesicle clusters containing concentrated VSV-G. B,
quantitation of the number of punctate intermediates containing
GFP-Gts-DXE or
GFP-Gts-AXA with a greater than 2-fold increase
in pixel intensity relative to the ER at the indicated time points. The
average value and standard deviation for 5 representative cells
followed throughout an entire time course is shown.
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 9.
Model for the mechanism of DXE
function in export from the ER. In a two-step mechanism of cargo
export from the ER, an initial event directing assembly of partial
(pre-budding) coat complexes is augmented by a separate
DXE-dependent second step that directs
concentration of cargo through the activity of additional
factors.
2-adrenogenic receptor
triggers recruitment of
-arrestin from cytosol and regulates the
concentration of receptor in clathrin-coated vesicles (52). We have
recently demonstrated that cargo selection by Sar1-GTP and Sec23/24 is
an early event, as even the misfolded form of Gts can be
recovered in pre-budding complexes at the restrictive temperature (32).
These results demonstrate that cargo can modulate general COPII vesicle
formation from the ER through competition for general transport factors
that may participate in later steps of COPII vesicle assembly (32).
Studies currently in progress directed at identifying the
component(s) that mediate DXE-dependent cargo concentration in the ER should provide important insight into
such mechanisms.
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ACKNOWLEDGEMENT |
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We thank K. Mostov for generous support of adenovirus vector construction.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant GM 42336 (to W. E. B.) and by Core C of the National Cancer Institute Grant CA 58689.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.
¶ Senior Postdoctoral Fellow of the American Cancer Society.
** To whom correspondence should be addressed: Depts. of Cell and Molecular Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037. Tel: 619-784-2310; Fax: 619-784-9126; E-mail: webalch{at}scripps.edu.
2 The on-line address is as follows: www.scripps.edu/cb/balch/dxepaper. (User, balch) (password, ER).
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ABBREVIATIONS |
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The abbreviations used are: ER, endoplasmic reticulum; VSV-G, vesicular stomatitis virus glycoprotein; endo H, endoglycosidase H; BHK, baby hamster kidney; NRK, normal rat kidney; GFP, green fluorescent protein; GST, glutathione S-transferase; HRP, horseradish peroxidase; HA, hemagglutinin; PBS, phosphate-buffered saline; GS beads, glutathione-Sepharose beads; BSA, bovine serum albumin; Syn5, syntaxin 5; SFV, Semliki forest virus; TGN, trans Golgi network.
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