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INTRODUCTION |
Multiple proteins participate in the sorting and concentration of
cargo at the endoplasmic reticulum
(ER)1 exit sites,
translocation of transport intermediates on microtubules, and the
docking/fusion of transport intermediates to their target membranes
(1). Proteins that regulate the docking and fusion events include the
soluble Ras-related GTPases (Rab proteins) (2-6), NSF (the
N-ethylmaleimide-sensitive fusion factor), and soluble NSF
attachment proteins (SNAPs), and the transmembrane vesicular- and
target-soluble NSF attachment protein receptors (SNAREs)
(7-10). In mammalian cells, transport from the ER to Golgi requires
the action of the Rab1 GTPase (2) and the SNAREs, rbet1 (11), syntaxin5
(12), Sec22b/ERS-24 (13), GOS-28/GS28 (14), and membrin (15). In
addition, the peripherally associated membrane protein p115 is required
for ER to Golgi transport at the level of vesicular tubular clusters
(VTCs) downstream of the Rab1-requiring step and upstream of the
Ca2+-dependent step (16). The recruitment of
p115 to membranes is mediated by Rab1, and p115 can interact directly
with a subset (membrin, rbet 1, and syntaxin5) of the ER-Golgi SNAREs
(17).
In addition to its VTC association, p115 directly interacts with two
Golgi membrane proteins, GM130 and giantin (18, 19). GM130 is an
extended rod-like protein (~130 nm) with coiled-coil domains,
initially identified as a component of an insoluble Golgi matrix (20).
GM130 is anchored by its C-terminal domain to the cytoplasmic face of
the membrane through interaction with the myristoylated protein
GRASP-65 (the Golgi reassembly stacking protein 65) (21, 22). The p115
binding domain of GM130 lies within its N-terminal 74 amino acids (23,
24). At the ultrastructural level, GM130 localizes predominantly to the
cis-Golgi (20). This distribution partially overlaps with
p115 labeling the cis-Golgi. However, p115 appears more
concentrated on structures associated with the cis-most
aspect of the cis-cisterna (18). The other p115 binding
partner, giantin, is an integral component of the Golgi membrane that
contains a large N-terminal cytoplasmic domain (>350 kDa) and a
C-terminal membrane anchor domain (25-27). Giantin is predicted to
form a segmented coiled-coil dimer rod of ~250 nm. The p115 binding
domain has been mapped to the N-terminal 70 amino acids of giantin
(24). Giantin has been localized to tubular-cisternal Golgi elements
that might represent fenestrated connections between cisternal stacks
(26). Such regions of the Golgi have been proposed to be specialized
budding domains (28, 29). Confocal analysis indicates that giantin and
p115 do not extensively colocalize in the Golgi region (30).
The functions of GM130 and giantin have been studied predominantly in a
cell-free assay that is based on the observation that Golgi complex
disassembles during mitosis and subsequently re-assembles during late
telophase (31). In this assay, isolated Golgi membranes are treated
with mitotic cytosols to generate Golgi fragments that under
experimentally controlled conditions (in the presence of NSF,
- and
-soluble NSF attachment proteins (SNAPs), and p115) re-assemble into
cisternal elements and stacks (32). The interaction between p115 and
GM130 appears functionally relevant in this assay since the addition of
the N-terminal GM130 peptide that binds p115 inhibits cisternal
regrowth and cisternal stacking (23, 33). Likewise, the addition of the
p115 binding N-terminal giantin peptide inhibits cisternal regrowth and
cisternal stacking (34), suggesting a functional interaction between
p115 and giantin. The requirement for both GM130 and giantin function
in the assay is further supported by the ability of anti-GM130 or
anti-giantin antibodies to block cisternal regrowth and subsequent
cisternal stacking (33).
The role of GM130 and giantin in membrane transport has been largely
unexplored, but their close relationship with p115 suggests that these
proteins might also participate in ER-Golgi traffic. The involvement of
GM130 in traffic was suggested by the finding that expression of a
GM130 mutant lacking the p115 binding N terminus inhibited the surface
delivery of VSV-G protein and resulted in the disappearance of Golgi
cisternae with the concurrent accumulation of small vesicles in the
Golgi region (35). The role of giantin in trafficking has not been
previously analyzed.
We have examined the role of GM130 and giantin in ER-Golgi traffic by
utilizing a VSV-G ts045 based semi-intact cell transport assay we
previously used to document p115 requirement at the VTC stage of
transport (16). Transport was inhibited by peptides corresponding to
the p115 binding N-terminal domains of either GM130 or giantin and by
anti-GM130 and anti-giantin antibodies. In all cases, the inhibition
occurred before VSV-G protein delivery to the mannosidase II-containing
medial/trans-Golgi compartment. Both anti-GM130
and anti-giantin antibodies inhibited transport subsequently to the
stage inhibited by anti-p115 antibodies. In agreement, morphological
data showed that the anti-GM130 and the anti-giantin antibodies inhibit
at the Golgi level at stages downstream from the VTC step inhibited by
anti-p115 antibodies. Analysis of the kinetics of inhibition indicates
that GM130 and giantin act at temporally different stages, with the
GM130-requiring stage preceding the giantin-requiring stage. Our
results document the novel requirement for GM130 and giantin in
ER-Golgi transport and suggest that GM130 and giantin do not function simultaneously.
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EXPERIMENTAL PROCEDURES |
Antibodies--
Rabbit polyclonal antibodies against p115 (36)
and against GM130 (18) were affinity-purified. For affinity
purification, lysates of bacteria expressing glutathione
S-transferase (GST)-p115 fusion protein or GM130 were
separated by SDS-PAGE and transferred to nitrocellulose. Nitrocellulose
strips containing GST-p115 or GM130 were incubated with immune serum in
phosphate-buffered saline, 5% fat-free dried milk, 0.1% Tween 20 for
3 h at room temperature. Bound antibodies were eluted with 0.1 M glycine, pH 3.0, neutralized with 1/10 volume 1 M phosphate buffer, pH 7.4, and dialyzed against 25 mM HEPES-KOH, pH 7.2. The antibodies were concentrated and then used in transport assays. Monoclonal anti-giantin G1/133 antibody
has been described previously (25). Polyclonal antibodies against
mannosidase II were kindly provided by Dr. Marilyn Farquhar (University
of California at San Diego). Monoclonal antibodies against mannosidase
II were from BabCO (Berkeley, CA). Monoclonal antibodies against VSV-G
protein (P5D4) were kindly provided by Dr. Kathryn Howell (University
of Colorado Health Sciences Center, Denver, CO). Goat anti-rat
and anti-mouse antibodies conjugated with fluorescein isothiocyanate or
rhodamine were purchased from Jackson ImmunoResearch (West Grove, PA).
GST Fusion Constructs--
GM130 fragment encoding amino acids 1 to 270 was generated with the polymerase chain reaction using the
primer pair 5'-CCGGATCCGAATGTCGGAAGAAACCAG with
5'-GTTTCTAGACCATGATTTTCACCTCGTC and a template encoding rat GM130
cDNA in a pBluescript-II vector (18). GST-GM130/1-270 was
engineered by inserting the GM130 fragment into the
BamHI-SalI restriction sites of the pGEX-5X-1
vector (Amersham Pharmacia Biotech). GST-giantin fragment
(GST-GTN/1-261) containing coils I and II has been described
previously (24). GST fusion protein expression and purification was
performed according to the manufacturer's (Amersham Pharmacia Biotech) protocol.
Semi-intact Cell ER-Golgi Transport Assay--
The ER to Golgi
transport assay was performed as described previously (16, 37, 38).
Briefly, NRK cells were grown on 10-cm Petri dishes (80-90%
confluent) and infected with the temperature-sensitive strain of the
vesicular stomatitis virus, ts045 VSV at 32 °C for 3-4 h (39). The
cells were pulse-labeled with Tran35S-label
(200 mCi/ml; ICN, Irvine, CA) at the restrictive temperature (42 °C)
for 10 min, chased with complete medium for 5 min, and perforated by
hypotonic swelling and scraping. Transport reactions were performed in
a final total volume of 40 µl in a buffer containing 25 mM HEPES-KOH, pH 7.2, 75 mM potassium acetate,
2.5 mM magnesium acetate, 5 mM EGTA, 1.8 mM CaCl2, 1 mM
N-acetylglucosamine, ATP regeneration system (1 mM ATP, 5 mM creatine phosphate, and 0.2 IU of
rabbit muscle creatine phosphokinase), 5 µl of rat liver cytosol, and
5 µl of semi-intact cells in 50 mM Hepes-KOH, pH 7.2, 90 mM potassium acetate. Transport was initiated by
transferring cells to 32 °C. After 90 min of incubation, cells were
pelleted, resuspended in appropriated buffer, and digested with
endoglycosidase H (endo-H) as described previously (37). The samples
were analyzed on 8% SDS/PAGE and by fluorography. Transport was
quantitated using a GS-700 imaging densitometer (Bio-Rad). In some
experiments, increasing concentrations of GST fusion peptides were
added to a complete transport mixture containing cytosol and incubated on ice for 30 min to allow the peptides to interact with cytosolic p115. The mixtures were then added to the semi-intact cells, and transport was initiated at 32 °C. In some experiments, increasing concentrations of antibodies were added to a complete transport reaction containing semi-intact cells, transport mixture, and cytosol
and incubated on ice for 30 min to allow the antibodies to interact
with cellular GM130 or giantin. Transport was then initiated at
32 °C. Kinetic staging experiments were performed as described
previously (40, 41).
p115 Binding Experiments--
To test binding of cytosolic p115
to cell membranes used in the semi-intact cells transport assay, NRK
cells were perforated (by hypotonic swelling and scraping as above) and
rinsed with 0.5 M KCl to remove endogenous p115. In some
experiments, increasing concentrations of GST-GM130/1-270 fusion
peptide were added to a complete transport mixture containing cytosol
and incubated on ice for 30 min to allow the peptide to interact with
cytosolic p115. The mixtures were then added to semi-intact cells, and
the cells were incubated for 60 min at 32 °C. Cells were collected by pelleting and rinsed (two times) with transport buffer, and the cell
pellet was processed for SDS-PAGE. The amount of membrane-associated p115 was detected by Western blotting and quantified using GS-700 imaging densitometer (Bio-Rad). In some experiments antibodies were
added to the semi-intact cells and incubated on ice for 30 min to allow
the antibodies to interact with cellular GM130 or giantin. The cells
were then supplemented with complete transport mixture and cytosol and
incubated for 60 min at 32 °C. Cells were then processed as above to
measure membrane-associated p115.
Morphological Analysis of VSV-G Protein Transport--
NRK cells
plated on coverslips were infected with ts045 VSV at 32 °C for 30 min followed by an incubation at 42 °C for 3 h and then shifted
to ice and permeabilized with digitonin (20 mg/ml), as described
previously (16, 42). Coverslips were incubated at 32 °C for 90 min
in transport mixtures supplemented with various IgGs, as indicated
under "Results." Transport was terminated by transferring
coverslips to ice and fixing the cells in 3% paraformaldehyde, phosphate-buffered saline for 10 min. The coverslips were then processed for double-label immunofluorescence as described previously (16). Cell area and the mean fluorescence were measured with the
IP Lab Spectrum software (Signal Analytics). The total cell area, the Golgi region area (defined by the presence of a select Golgi
marker; see Fig. 6 legend), and the area of colocalization between the VSV-G protein and the Golgi marker were defined manually. These data are presented as a mean of analyses from five representative cells.
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RESULTS |
VSV-G Protein Transport Is Inhibited by the N-terminal Peptide of
GM130 and the N-terminal Peptide of Giantin--
To test whether the
p115-interacting proteins GM130 and giantin participate in membrane
traffic, we added recombinant GM130 or giantin peptide constructs to
the semi-intact cell transport assay used previously to document p115
requirement during VSV-G protein transport (16). The peptide constructs
were generated as GST fusion proteins and contained either the
N-terminal 270 amino acids of GM130 (GST-GM130/1-270) or the first 261 amino acids of giantin (GST-GTN/1-261) fused to the C terminus of GST (Fig. 1A). We previously
showed that each construct contains a domain sufficient and necessary
for p115 binding (24). The fusion constructs and a control GST protein
were expressed in bacteria and purified nearly to homogeneity as judged
by Coomassie Blue staining (Fig. 1B). The SDS-PAGE
mobilities of the fusion constructs are appropriate for the specific
composition of each peptide (GM130/1-270 is predicted to be 29,565 daltons, whereas GTN/1-261 is predicted to be 30,144 daltons).

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Fig. 1.
The N-terminal peptides of GM130 and giantin
inhibit VSV-G transport to the Golgi. A, structure of
full-length GM130 (GM130/1-987) and giantin (GTN/1-3259) compared
with the respective GST-GM130 and GST-giantin N-terminal fusion
peptides. B, GST and GST fusion peptides were expressed in
bacteria and purified. Coomassie Blue-stained SDS-PAGE gel shows the
size of the fusion peptides and their purity. C, ER to Golgi
transport assay was performed in semi-intact NRK cells. Transport is
measured as the percentage of VSV-G protein processed from the
endo-H-sensitive to the endo-H-resistant form. Transport reaction with
complete transport mixture (lane 2) is set as 100%, and
transport with mixture without ATP (lane 1) is set as 0%.
Reactions were preincubated in presence of GST (lane 3),
GST-GM130/1-270 (lanes 4-6), or GST-giantin/1-260
(lanes 7-9). Transport of VSV-G protein was inhibited in
the presence of GST-N130/1-270 or GST-giantin/1-260 peptides. Results
are presented as the mean of three separate experiments ± S.E.
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Increasing concentrations of GST-GM130/1-270 or GST-GTN/1-261 were
added to cytosol and incubated on ice for 20 min to allow the peptides
to bind to cytosolic p115. The mixtures were then used in the
semi-intact cell transport assay. Transport was measured by following
the processing of VSV-G protein from a core-glycosylated (endo-H-sensitive) ER form to a more mature (endo-H-resistant) form
upon its arrival in a Golgi compartment containing mannosidase II. In
agreement with previously published results (6, 14), when transport was
measured with untreated cytosol, ~60% of VSV-G protein was processed
to endo-H-resistant form, and this was set as 100% relative transport
(Fig. 1C, lane 2). When transport was analyzed in the absence of ATP (with an ATP-depleting system), more
than 90% of VSV-G protein remained endo-H-sensitive, and this was set
as 0% processing (lane 1). The addition of 10 µM GST had negligible effect on transport (lane
3). In contrast, addition of increasing amounts of the
GST-GM130/1-270 construct led to a dose-dependent
inhibition of VSV-G protein transport (lanes 4-6). Adding
the GST-GTN/1-261 construct to the transport assay was also
inhibitory. At the lowest concentration tested (2 µM,
lane 7), the fusion peptide had no effect on
transport, but increasing the concentration to 6 or 8 µM
led to a significant (>80%) inhibition of VSV-G protein-processing
(lanes 8 and 9, respectively).
These data suggest that interactions between p115 and GM130 and between
p115 and giantin are required for transport and that both GM130 and
giantin function in ER-Golgi traffic. However, we have recently shown
that the N-terminal domain of GM130 and the N-terminal domain of
giantin interact with the same C-terminal acidic domain of p115 and
compete for binding (24). Therefore, the addition of either the
GST-GM130/1-270 or the GST-GTN/1-261 fusion peptide will prevent the
interaction of p115 with both GM130 and giantin. Consequently,
inhibition of transport by either peptide may not distinguish between
GM130 alone, giantin alone, or both proteins as required for transport.
VSV-G Protein Transport Is Inhibited by Anti-GM130 Antibodies and
Anti-giantin Antibodies--
To further characterize the role of GM130
and giantin in transport, anti-GM130, and anti-giantin antibodies were
added to the semi-intact cell assay. Increasing amounts of
affinity-purified polyclonal anti-GM130 antibodies were added to
transport mixtures containing cytosol. The mixtures were added to
permeabilized cells, and the cells were incubated on ice for 30 min to
allow the antibodies to bind cellular GM130. Cells were then shifted to
32 °C for 90 min. As shown in Fig.
2A, lanes 3-6, the
addition of anti-GM130 antibodies inhibited transport of VSV-G protein
in a dose-dependent manner. Transport was blocked 70% when
0.48 µg was present in the assay (lane 6). Preincubation
of the antibodies with recombinant full-length GM130 immobilized on
nitrocellulose strips efficiently neutralized the inhibitory effect
(compare lanes 2 and 7). In contrast, the
antibodies remained inhibitory when pre-incubated with recombinant
full-length p115 immobilized on nitrocellulose strips (data not
shown).

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Fig. 2.
GM130 and giantin antibodies inhibit VSV-G
transport to the Golgi. ER to Golgi transport assay was performed
in semi-intact NRK cells as described under "Experimental
Procedures." Transport is measured as the percentage of VSV-G protein
processed from the endo-H-sensitive to the endo-H-resistant form.
Transport reaction with complete transport mixture (ATP+) is
set as 100% (lane 2, panels A and B),
transport with mixture without ATP (ATP ) is set as 0%
(lane 1, panels A and B). Reactions
were supplemented with increasing amounts of affinity-purified
antibodies. Transport of VSV-G protein was inhibited in the presence of
anti-GM130 antibody (panel A, lanes 3-6) or
anti-giantin antibodies (panel B, lanes 3-6).
Inhibitory effect is neutralized by preincubating GM130 antibody with
recombinant full-length GST-GM130 (panel A, lane
7) or by boiling giantin antibody (panel B, lane
7). Results are presented as the mean of three separate
experiments ± S.E.
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The addition of monoclonal antibodies directed against giantin also
inhibited VSV-G protein transport. As shown in Fig. 2B, lanes 3-6, the addition of 0.1- 0.8 µg of anti-giantin
antibodies inhibited relative transport of VSV-G protein 10-90%.
Heat-inactivating the antibodies neutralized their inhibitory effect
(compare lanes 2 and 7). Analogous results were
obtained with affinity-purified polyclonal anti-giantin antibodies
(data not shown). These data indicate that anti-GM130 and anti-giantin
antibodies inhibit VSV-G protein transport and suggest that both GM130
and giantin participate in VSV-G protein movement from the ER to a
mannosidase II-containing Golgi compartment.
Anti-GM130 Antibodies and Anti-giantin Antibodies Do Not Inhibit
p115 Membrane Binding--
We have shown previously that p115 binds to
Golgi membranes by interacting with the N termini of GM130 and giantin
(18, 24) and that p115 is required for ER to Golgi traffic of VSV-G protein (16). Therefore, the anti-GM130 and anti-giantin antibodies could inhibit VSV-G protein transport by preventing p115 membrane binding. To address this issue, transport mixture (without cytosol) was
supplemented with increasing amounts of anti-GM130 or anti-giantin antibodies, then added to perforated NRK cells that have been washed
with 0.5 M KCl to remove membrane-associated p115. The cells were preincubated on ice for 30 min to allow the antibodies to
bind cellular GM130 or giantin. Cytosol was added as the source of
p115, and the cells were incubated at 32 °C for 60 min to allow p115
binding. The amount of p115 recovered with the membranes was determined
by Western blotting and was quantitated relative to the amount of
calnexin (used to standardize the amount of cells) present in each
sample. As shown in Fig. 3A,
lanes 3-6, increasing amounts of affinity-purified
anti-GM130 antibodies did not influence the binding of p115 to
membranes. Equivalent amounts of bound p115 were recovered in those
samples as in control samples (lane 1) or containing
membranes treated with an irrelevant antibody against mannosidase II
(lane 7). Cells that did not receive cytosol showed no
detectable p115 (lane 2), indicating that the KCl wash removed all endogenous p115 and that cytosol was the only source of
p115 in the assay. Similarly, p115 binding was observed when increasing
concentrations of anti-giantin antibodies were added to the assay (Fig.
3B). No signal was observed when cells were omitted from the
assay (lane 7).

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Fig. 3.
p115 binding to membranes is not altered by
anti-GM130 or anti-giantin antibodies. Semi-intact NRK cells were
washed with 0.5 M KCl to remove membrane-associated p115.
Cells were incubated with anti-GM130 or anti-giantin antibodies for 30 min at 4 °C. Cytosol was then added, and the cells were incubated
for 60 min at 32 °C. Cells were recovered by centrifugation and
rinsed, and the amount of p115 and calnexin was quantitated by
densitometry after separation by SDS-PAGE and Western blot. Ratio of
p115 to calnexin in control samples (lanes 1 in panel
A and B) or without peptide is set as 100%. p115 was
not detected in samples incubated in absence of cytosol (lanes
2, panels A, B, and C).
A, p115 binding to membranes is not modified in the presence
of increasing concentrations of anti-GM130 antibodies (lanes
3-6). Control antibody against mannosidase II (lane 7)
does not influence p115 binding to membranes. B, increasing
concentrations of anti-giantin antibodies (lanes 3-6) do
not affect the amount of p115 bound to membranes. p115 was not detected
in samples incubated without cells (lane 7). C,
p115 binding to membranes decreased in the presence of increasing
concentrations of the GST-GM130/1-270 peptide (lanes 3-6).
GST does not inhibit p115 binding (lane 7). Results are
presented as the mean of two separate experiments ± S.E.
Representative immunoblots are shown.
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The effect of the GST-GM130/1-270 peptide on p115 binding was tested
as a control. We predicted that GST-GM130/1-270 will bind to cytosolic
p115 and compete for its interaction with GM130 and giantin on Golgi
membranes, thus reducing p115 binding to membranes. Increasing
concentrations of the GST-GM130/1-270 peptide were added to cytosol
and incubated on ice for 30 min to allow the peptide to interact with
p115. The mixtures were then added to the perforated and washed cells,
and the amount of p115 recovered with membranes was determined by
Western blotting. As shown in Fig. 3C, lane 1,
control membranes bound p115. Samples processed in an analogous manner
but without cytosol do not contain detectable p115 (lane 2).
As shown in lanes 3-6, the addition of the GST-GM130/1-270 peptide inhibited p115 binding to membranes. The relative binding was
reduced to ~85% in the presence of 0.10 µM peptide,
~75% in presence of 0.17 µM peptide, ~50% in
presence of 0.25 µM peptide, and 30% in presence of 0.33 µM peptide. Inhibition was specific since GST (0.50 µM) alone did not inhibit p115 binding to membranes (lane 7). These results indicate that the anti-GM130 and
anti-giantin antibodies do not prevent p115 membrane interactions and
directly inhibit the transport functions of GM130 and giantin.
Anti-p115, Anti-GM130, and Anti-giantin Antibodies Inhibit VSV-G
Protein Transport at Temporally Distinct Steps--
The requirement
for various transport factors during transport through the secretory
pathway can be sequentially ordered by adding specific inhibitory
reagents at different times during traffic and measuring the subsequent
transport of VSV-G protein in the presence of the inhibitor. For
factors that function early, the addition of inhibitors will block at
early time points but not after the VSV-G protein has moved past the
stage requiring the factor. For factors that act late, the addition of
inhibitors will block at early and late time points, since VSV-G
protein has not yet moved pass the stage requiring the factor.
To determine whether p115, GM130, and giantin act together or in a
sequential fashion, we compared kinetics of traffic in the presence of
inhibitory antibodies. Perforated NRK cells were supplemented with
complete transport mixtures and incubated at 32 °C for different
times (
t) to allow transport. At each time point, one sample of
cells was transferred to ice (control), another sample received
anti-p115 antibodies (anti-p115), another sample received anti-GM130
antibodies (anti-GM130), and another sample received anti-giantin
(anti-giantin) antibodies. The control samples were incubated on ice
for a total of 90 min, whereas the other samples were incubated at
32 °C for a total of 90 min. As shown in Fig.
4, VSV-G transport from the ER to the
medial/trans-Golgi compartments containing mannosidase II
required a 20-30-min lag period at 32 °C, as previously published
for control cells (40). Only after this time, processing of the VSV-G
protein oligosaccharide chains can be detected. After 55 min of
incubation at 32 °C, ~50% relative transport is observed, and
maximal transport is obtained by 70 min. The amount of transport after
a 90-min incubation at 32 °C (60% of VSV-G protein is processed at
that point) is the maximal amount of processing that could be observed
in all control samples and is set as 100% relative transport.

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Fig. 4.
Anti-p115, anti-GM130, and anti-giantin
antibodies inhibit VSV-G protein transport at temporally distinct
steps. Semi-intact NRK cells prepared as described under
"Experimental Procedures" were incubated at 32 °C to initiate
transport. At the indicated times ( t), cells were transferred to ice
(control), or supplemented with 0.4 µg of anti-p115
antibodies, 0.48 µg of anti-GM130 antibodies, or 0.80 µg of
anti-giantin antibodies and incubated for a total of 90 min at
32 °C. Transport was measured as the relative percentage of VSV-G
protein processed from the endo-H-sensitive to the endo-H-resistant
form. Total transport after 90 min ( t = 90) is set as 100%,
and transport at 0 min ( t = 0) is set as 0%. Results are
presented as the mean of three separate experiments ± S.E.
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VSV-G protein transport was completely inhibited when anti-p115
antibodies were added at the beginning of incubation at 32 °C or
after 5 or 10 min of incubation at 32 °C (Fig. 4). When the
antibodies were added 15 min after the start of the 32 °C incubation, ~30% relative transport was observed. Almost 50%
relative transport was seen when anti-p115 antibodies were added 30 min after the start of the 32 °C incubation, and >70% relative
transport was detected when antibodies were added 45 min after the
start of the 32 °C incubation. These results indicate that ~50%
of the VSV-G protein was transported beyond the step inhibited by the anti-p115 antibodies during the first 30 min of incubation, and more
than 70% of VSV-G protein passed it by 45 min.
A slightly shifted response curve was obtained with anti-GM130
antibodies (Fig. 4). Like anti-p115 antibodies, anti-GM130 antibodies
completely inhibited transport when added at the beginning of
incubation at 32 °C or after 5 or 10 min of incubation at 32 °C.
However, when anti-GM130 antibodies were added 15 min after the start
of the 32 °C incubation, only ~17% relative transport was
observed, whereas ~30% relative transport was seen when anti-p115 antibodies were added at that time point. This result suggests that
p115 is required before GM130, since the VSV-G protein passes the
p115-inhibited stage faster than it passes the GM130-inhibited stage.
The inhibition by anti-GM130 antibodies at later time points paralleled
the inhibition by anti-p115 antibodies. Together, the data suggest that
p115 acts independently of GM130 at an early stage (at ~15 min of
transport) but that the two proteins might interact at a later event in
transport (at ~30 min of transport).
A significantly different response curve was obtained with anti-giantin
antibodies (Fig. 4). Unlike the anti-p115 and anti-GM130 antibodies,
which showed only partial inhibition when added after 15 min of
32 °C incubation, anti-giantin antibodies remained completely inhibitory when added at that time point. Even when added 30 or 45 min
after the start of the 32 °C incubation, at a time when the addition
of anti-p115 or anti-GM130 antibodies had limited inhibitory effect,
anti-giantin antibodies remained strongly inhibitory. For example,
~37% relative transport was obtained when anti-giantin antibodies
were added after 45 min of incubation as compared with >75% relative
transport when anti-p115 or anti-GM130 antibodies were added at the
same time point. These data suggest that p115, GM130, and giantin
function at sequential stages of transport, with the p115-requiring and
the GM130-requiring stages preceding the giantin-requiring stage.
Anti-p115, Anti-GM130, and Anti-giantin Antibodies Inhibit VSV-G
Protein Transport at Spatially Distinct Steps--
The addition of
anti-p115 antibodies to the semi-intact cell assay inhibits VSV-G
protein transport after the protein exits the ER and enters peripheral
VTCs but before its delivery to the Golgi complex (16). To investigate
the intracellular site at which anti-GM130 and anti-giantin antibodies
block transport of VSV-G protein, morphological transport assay was
performed. We first determined the cellular loci to which the
anti-p115, anti-GM130, and anti-giantin antibodies bound in the
semi-intact cell transport assay and then defined the localization of
the VSV-G protein arrested by the presence of these antibodies. As
shown in Fig. 5A and in agreement with the in situ distribution of p115 in intact
cells (18), affinity-purified anti-p115 antibodies labeled the Golgi region and colocalized with mannosidase II and in association with
peripheral VTCs (arrowheads) lacking mannosidase II. This dual distribution suggests that the antibodies might inhibit p115 function at either or both sites. In contrast, anti-GM130 antibodies (Fig. 5B) and anti-giantin antibodies (Fig. 5C)
were found exclusively in the Golgi region where they colocalized with
mannosidase II. The Golgi localization suggests that the antibodies are
likely to block GM130-mediated and giantin-mediated events at the level of the Golgi complex. Significantly, the mannosidase II patterns appear
normal, indicating that the antibodies did not drastically perturb the
structure of the Golgi complex.

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Fig. 5.
Localization of anti-p115, anti-GM130, and
anti-giantin antibodies in semi-intact cells. Semi-intact NRK
cells were supplemented with anti-p115 (panel A), anti-GM130
(panel B), or anti-giantin (panel C) antibodies.
After transport at 32 °C for 90 min, cells were processed for
double-label immunofluorescence to visualize the added antibodies and
costained for mannosidase II. p115 antibody was detected in the Golgi
region and in peripheral punctate structures (arrowheads),
whereas anti-GM130 and anti-giantin antibodies were detected
exclusively in the Golgi region. The semi-intact cells show normal
Golgi pattern of mannosidase II staining.
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VSV-G protein is arrested in peripheral VTCs in cells supplemented with
anti-p115 antibodies (16). Fig. 6 shows a
direct comparison between the effects of the anti-p115, anti-GM130, and anti-giantin antibodies. Quantitation of the VSV-G protein signal shows
the majority in peripheral structures, with only ~8% colocalizing with the anti-p115 antibodies at the Golgi stack. In contrast, the
addition of the anti-GM130 antibodies (Fig. 6B) or the
anti-giantin antibodies (Fig. 6C) or nonimmune serum (Fig.
6D) did not inhibit VSV-G protein transport to the Golgi.
Quantitation of the VSV-G protein signal in cells supplemented with
anti-GM130 or anti-giantin antibodies indicates that ~70% of VSV-G
protein colocalizes with each antibody in the Golgi stack. Analogous
distribution was seen in cells supplemented with nonimmune antibodies.
The data show that anti-p115 antibodies block transport before Golgi
delivery, whereas anti-GM130 and anti-giantin antibodies inhibit
traffic after the VSV-G protein is transported to the Golgi stack.

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Fig. 6.
Antibodies against p115, GM130, and giantin
block ER to Golgi transport of VSV-G protein at spatially distinct
steps. ER to Golgi transport assay was performed in semi-intact
NRK cells supplemented with anti-p115 (panel A), anti-GM130
(panel B), anti-giantin (panel C), or nonimmune
serum (panel D). Localization of the added antibodies and of
VSV-G protein was monitored by double-label immunofluorescence. In
panel D, localization of mannosidase II and of VSV-G protein
was analyzed. Boxed regions of the Golgi area are shown at
higher magnification. The addition of anti-p115 antibodies prevented
VSV-G protein transport to the Golgi and resulted in VSV-G protein
accumulation in peripheral VTCs. In contrast, the addition of
anti-GM130 or anti-giantin antibodies had no effect on the transport of
VSV-G protein to the Golgi region. VSV-G protein was also transported
to the Golgi in the presence of nonimmune serum.
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DISCUSSION |
In this study, a semi-intact cell assay that measures
biochemically or morphologically the movement of the VSV-G protein was used to assess GM130 and giantin function in ER-Golgi transport. Peptides derived from the p115 binding N-terminal domain of GM130 or
the p115 binding N-terminal domain of giantin were potent transport inhibitors. In both cases, transport was arrested before the VSV-G protein acquiring endo-H resistance, indicating that the reporter protein was not processed by mannosidase II, a medial/trans
Golgi enzyme (43). These results suggest that each peptide inhibited the corresponding p115 interaction and that p115-GM130 and p115-giantin interactions are required for VSV-G transport to the Golgi. We have
recently mapped the GM130 binding domain and the giantin binding domain
to the same C-terminal acidic domain of p115 and showed that GM130 and
giantin compete for p115 binding (24). Our findings contradict those of
Dirac-Svejstrup et al. (44) who show p115 binding
simultaneously to both GM130 and giantin. The discrepancy is currently
unexplained, but it should be noted that a trimeric GM130-p115-giantin
complex has not been identified in vivo.
Direct involvement of GM130 and giantin in VSV-G protein transport was
shown by using anti-GM130 and anti-giantin antibodies. Both antibodies
were monospecific and recognized only their corresponding antigens (18,
25). Both reagents inhibited transport before VSV-G protein acquired
endo-H resistance. Results from the Warren laboratory showing that
anti-GM130 and anti-giantin prevent p115 binding to the corresponding
antigen (19) led us to test whether our antibodies could also act in
this manner. The inhibitory effects of our antibodies were not due to
p115 displacement, since p115 membrane binding was unchanged in the
presence of the antibodies. In our studies, inhibition of VSV-G protein
transport by the antibodies appears direct and is most likely due to
functional inactivation of GM130 or giantin. The anti-GM130 and the
anti-giantin antibodies appear to inhibit events downstream of the
p115-GM130 and the p115-giantin interactions. It is possible that the
antibodies sterically hinder interactions of GM130 or giantin with
other proteins required for traffic progression. Alternatively, the antibodies might prevent p115 dissociation from GM130 and giantin by
preventing conformational changes and/or signaling events required for
p115 dissociation. This would lead to the formation of stable complexes
and could prevent subsequent events requiring unoccupied GM130 and/or giantin.
How are p115, GM130, and giantin involved in ER-Golgi traffic? A model
that fits all the available data have the three proteins functioning at
temporally and spatially different stages of transport. We propose that
p115 first acts at the level of peripheral VTCs. This is supported by
p115 localization to VTCs (18) and the arrest of cargo in peripheral
VTCs in the absence of p115 (16). The VTC localization of p115 is the
result of p115 recruitment by an activated Rab1 to COPII vesicles (17)
that subsequently form VTCs (45). The exact function of p115 at this
stage is currently unknown. The yeast homologue of p115, Uso1p, tethers COP II vesicles to Golgi membranes (46-48), suggesting that p115 might
also tether COP II vesicles. In COP II vesicles p115 interacts with
specific ER-Golgi SNAREs, membrin, syntaxin5, and rbet1 (17), but the
functional relevance of these interactions remains to be defined.
Significantly, the requirement for p115 activity at the VTC stage of
transport is independent of GM130 or giantin since VTCs lack GM130 and
giantin (16, 18).
The second site of p115 action in our model is after VTC-derived
transport intermediates move to the Golgi region. VTC-derived transport
intermediates contain p115 (16), and we propose that p115 provides
targeting specificity by interacting with GM130 localized on the
cis side of the cis-Golgi. This tethering
represents the first stage in a series of events that results in
membrane fusion. Our anti-GM130 antibodies do not prevent the
p115-GM130 interaction but inhibit transport, suggesting that GM130
participates in downstream events. The model also fits data showing
that expression of a mutant GM130 lacking the p115 binding N-terminal
domain results in the accumulation of tubulo-vesicular structures
(possibly transport intermediates) in the Golgi region (35). A block in
fusion of transport intermediates would result in such a phenotype.
The role of giantin in transport is more puzzling. Based on the
ultrastructural localization of giantin to the Golgi rims (likely to be
involved in COP I vesicle budding (28, 29)) and its presence in
isolated COP I vesicles (19), a model has been proposed in which p115
would function to cross-bridge giantin in COP I vesicles to GM130 on
Golgi membranes in a single tethering event (19). However, this model
is not supported by our kinetic results. Although both anti-GM130 and
anti-giantin antibodies block VSV-G protein transport before
mannosidase II processing, staging experiments showed that the temporal
site of inhibition by anti-giantin antibodies differs from the stage
inhibited by anti-GM130 antibodies. Anti-giantin antibodies remained
inhibitory at times when anti-GM130 antibodies no longer inhibited
transport, suggesting that GM130 and giantin function at distinct steps
of transport. We therefore propose a distinct model for p115, GM130, and giantin function in the Golgi, one based on dimeric interactions between p115 and GM130 and between p115 and giantin. We suggest that
the p115-GM130 interactions are required for the anterograde delivery
of the cargo into an early Golgi compartment and the p115-giantin
interactions for the retrograde recycling of glycosyl transferases to
that compartment from a later Golgi compartment. COP I vesicles have
been shown to contain mannosidase II (49). Such a mechanism would
functionally couple the delivery of cargo with the delivery of
processing enzymes by utilizing a common molecule, p115, required for
both events. This model would allow the coordination of the forward and
the recycling pathways by utilizing p115 as a pivotal molecule
regulating both stages. Additional analysis will be required to explore
the exact molecular roles of p115, GM130, and giantin in the secretory pathway.