JCB: ARTICLE
mBet3p is required for homotypic COPII vesicle
tethering in mammalian cells
Sidney Yu,1,2 Ayano Satoh,2 Marc Pypaert,2 Karl Mullen,3 Jesse C. Hay,3 and Susan Ferro-Novick1,2
1
Howard Hughes Medical Institute and 2Department of Cell Biology, Yale University School of Medicine, New Haven, CT 06519
Division of Biological Sciences, The University of Montana, Missoula, MT 59812
3
THE JOURNAL OF CELL BIOLOGY
T
RAPPI is a large complex that mediates the tethering
of COPII vesicles to the Golgi (heterotypic tethering)
in the yeast Saccharomyces cerevisiae. In mammalian cells, COPII vesicles derived from the transitional
endoplasmic reticulum (tER) do not tether directly to the
Golgi, instead, they appear to tether to each other (homotypic tethering) to form vesicular tubular clusters (VTCs).
We show that mammalian Bet3p (mBet3p), which is the
most highly conserved TRAPP subunit, resides on the tER
and adjacent VTCs. The inactivation of mBet3p results in
the accumulation of cargo in membranes that colocalize
with the COPII coat. Furthermore, using an assay that reconstitutes VTC biogenesis in vitro, we demonstrate that
mBet3p is required for the tethering and fusion of COPII
vesicles to each other. Consistent with the proposal that
mBet3p is required for VTC biogenesis, we find that
ERGIC-53 (VTC marker) and Golgi architecture are
disrupted in siRNA-treated mBet3p-depleted cells. These
findings imply that the TRAPPI complex is essential for
VTC biogenesis.
Introduction
Vesicle trafic from the ER to the Golgi complex is mediated by
the sequential action of the COPII and COPI coat protein complexes (Aridor et al., 1995; Presley et al., 1997; Scales et al.,
1997). COPII (coat protein II) vesicles form at the transitional
ER (tER), which is a specialized subdomain of the ER that is
distinct from the rough ER (Bannykh et al., 1996). These vesicles may tether and fuse to each other (homotypic fusion) to
form the vesicular tubular clusters (VTCs), which is a pre-Golgi
structure (also called the intermediate compartment) that lies
adjacent to the tER (Aridor et al., 1995; Presley et al., 1997;
Scales et al., 1997; Stephens et al., 2000; Xu and Hay, 2004).
COPI (coat protein I) is then recruited onto VTCs before coated
structures move cargo to the Golgi apparatus (Presley et al.,
1997; Scales et al., 1997; Ben-Tekaya et al., 2005; Sannerud
et al., 2006). Although components of the ER–Golgi traficking
machinery are highly conserved from yeast to man (FerroNovick and Jahn, 1994), no equivalent to a pre-Golgi compartment has been identiied in the yeast Saccharomyces cerevisiae.
COPII vesicle tethering/fusion has been reconstituted in vitro
with yeast membrane fractions (Barlowe, 1997; Barrowman
et al., 2000). However, in these assays, vesicles released from
Correspondence to Susan Ferro-Novick: susan.ferronovick@yale.edu
Abbreviations used in this paper: MVB, multivesicular body; tER, transitional ER;
VTC, vesicular tubular cluster.
The online version of this article contains supplemental material.
© The Rockefeller University Press $8.00
The Journal of Cell Biology, Vol. 174, No. 3, July 31, 2006 359–368
http://www.jcb.org/cgi/doi/10.1083/jcb.200603044
the ER in vitro are thought to fuse with Golgi membranes
(heterotypic fusion).
Tethering factors have been proposed to mediate the initial interaction of a transport vesicle with its acceptor compartment and are implicated in maintaining the identity of organelles
(Whyte and Munro, 2002). Tethers are either large oligomeric
complexes or long coiled-coil proteins that activate or act downstream of the GTPases (Rabs) that regulate membrane trafic
(Pfeffer, 1999; Whyte and Munro, 2002; Sztul and Lupashin,
2006). Most peripherally associate with membranes and are recruited directly onto the compartment on which they function
(Whyte and Munro, 2002). Although many tethers have been
identiied, the mechanism of vesicle tethering has remained
elusive. To address questions concerning the speciicity of vesicle
trafic and organelle identity, we have chosen to focus our studies on the tether called TRAPP. The 10 subunits of TRAPP were
initially identiied in the yeast S. cerevisiae (Sacher et al., 1998,
2000). TRAPP subunits are found in two complexes (TRAPPI
and TRAPPII) that act at different stages of membrane trafic.
TRAPPI acts in ER–Golgi trafic, whereas TRAPPII is required
for trafic through the early endosome (Sacher et al., 2001;
Cai et al., 2005).
Bet3p, which is the most highly conserved TRAPP
component, is 54% identical to its mammalian counterpart
and stably localizes to Golgi membranes (Sacher et al., 1998;
Barrowman et al., 2000). In yeast, the temperature-sensitive
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bet3-1 mutant blocks ER–Golgi trafic at 37°C (Rossi et al.,
1995). Although mammalian Bet3p (mBet3p) has been shown
to act in ER–Golgi trafic after COPII vesicle budding and
before Rab1, it is unknown if it participates in VTC biogenesis (Loh et al., 2005). We show that mBet3p is enriched at
the tER and adjacent VTCs. Microinjected α-mBet3p results
in the accumulation of cargo in structures that colocalize with
the COPII coat. Furthermore, the inactivation of mBet3p blocks
homotypic COPII vesicle tethering in vitro. These indings imply that mBet3p mediates the biogenesis of VTCs by linking
COPII vesicles to each other.
Results
The localization of mBet3p is resistant
to brefeldin A (BFA)
Previous studies identiied a cytosolic pool of mBet3p (Sacher
et al., 2000; Loh et al., 2005). However, the enrichment of this
protein to a particular intracellular structure was not demonstrated in these studies. To begin to address the role of mBet3p
in VTC biogenesis, we prepared polyclonal antibodies to this
TRAPP subunit to determine the intracellular compartment
where mBet3p resides.
Immunoluorescence microscopy revealed that mBet3p
localizes to punctate structures in the perinuclear region of
COS-7 cells (Fig. 1 A). This localization was not observed when
antibody was pretreated to a nitrocellulose strip containing
Figure 1. mBet3p partially colocalizes with markers of the pre-Golgi
compartment. (A) Anti-mBet3p antibody specifically recognizes mBet3p.
Affinity-purified α-mBet3p was mock treated, or pretreated with His6-mBet3p
(mBet3p depleted). The antibody was then used to label endogenous
mBet3p in COS-7 cells by indirect immunofluorescence. Nuclei were identified by DAPI staining. (B) Colocalization of endogenous mBet3p (red images) with ERGIC-53 (top, green) and COPI (bottom, green) to pre-Golgi
and Golgi membranes in BSC-1 cells. See merged image on right. The insets are magnified in the next row. Bars, 10 μm.
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Figure 2. The localization of mBet3p is largely resistant to BFA. NRK cells
were treated with 5 μg/ml BFA for 1 h, and the localization of mBet3p
(left), COPI (top right), and Man II (bottom right) were determined by indirect immunofluorescence. Bars, 10 μm.
immobilized recombinant mBet3p (Fig. 1 A). In HeLa cells, the
localization of mBet3p was predominantly cytosolic. However,
when the cytosolic pool of mBet3p was removed by digitonin
before ixation, its perinuclear localization was revealed
(unpublished data). The perinuclear localization of mBet3p
was also observed in several other mammalian cell lines, including BSC-1 (Fig. 1 B) and NRK cells (Fig. 2). mBet3p was
largely found adjacent to ERGIC-53, which is a marker for the
pre-Golgi compartment (Schweizer et al., 1988), and COPI
(Fig. 1 B). COPI is a heptameric coat complex that is recruited
to pre-Golgi and early Golgi structures (Oprins et al., 1993).
Interestingly, by immunoluorescence, mBet3p has a punctate
appearance rather than a continuous ribbonlike pattern, which
is typical of Golgi proteins.
Although the Golgi disassembles in the presence of the
drug BFA, we found that, surprisingly, the localization of
mBet3p was largely resistant to BFA (Fig. 2, top and bottom).
The observation that the localization of a component of a COPII
tethering complex is resistant to BFA prompted us to examine if
mBet3p is associated with the tER. The tER is a BFA-resistant
subdomain of the ER that specializes in the formation of COPII
vesicles (Saraste and Svensson, 1991; Bannykh et al., 1996;
Ward et al., 2001). As a marker for the tER we used Sec31p,
which is a subunit of the COPII coat (Barlowe et al., 1994).
Sec31p-labeled structures were most abundant in the vicinity
of the Golgi apparatus and largely colocalized with mBet3p
(Fig. 3 A and Fig. S1, available at http://www.jcb.org/cgi/
content/full/jcb.200603044/DC1).
mBet3p appears to be recruited
to the tER region
To address the relationship of mBet3p to the tER, we asked if
the localization of mBet3p was perturbed by conditions that
are known to disrupt or alter tER sites. When cells are treated
with the microtubule-disrupting agent nocodazole, the tER disperses and tER sites cluster near Golgi fragments (Hammond
and Glick, 2000). Although the cytosolic pool of mBet3p
appeared to increase in the presence of nocodazole, mBet3p
largely remained associated with the tER (Fig. 3 B, top) and
clustered near the Golgi marker GM130 (Fig. 3 B, bottom).
The colocalization of mBet3p with the tER marker Sec31p
is illustrated in Video 1 (available at http://www.jcb.org/cgi/
content/full/jcb.200603044/DC1), which is a 3D reconstruction
of confocal images obtained from serial sections. To determine
if the localization of mBet3p is changed by other conditions that
are known to alter the localization of the tER, we microinjected
Sar1p H79G into cells. Sar1p H79G is the constitutively active GTP-bound form of Sar1p that blocks export from the ER
(Kuge et al., 1994). In cells expressing Sar1p H79G, ER exit
sites have been shown to cluster (Ward et al., 2001). Golgi glycosylation enzymes like mannosidase II (Man II) are recycled
back to the ER (Seemann et al., 2000a), whereas the localization
of the golgin GM130 remains unchanged (Fig. 3 C; Miles et al.,
2001; Ward et al., 2001). Unlike Man II and GM130, mBet3p
always clustered with the ER exit sites (Fig. 3 C).
Together, these indings imply that mBet3p is recruited to
membranes at or near the tER. Like Sec31p, its localization is
Figure 3. The localization of mBet3p is disrupted by reagents that are known to disrupt the tER. (A) The localization of mBet3p (left, red) largely overlaps
(Merge) with Sec31p (middle, green) in BSC-1 cells. The insets are magnified on the bottom right. (B) BSC-1 cells, treated for 1 h with 10 μg/ml nocodazole, were fixed and stained with antibodies directed against mBet3p (red) and Sec31p (top, green), and mBet3p (red) and GM130 (bottom, green).
The insets are magnified on the right. (C) The localization of mBet3p clusters in response to microinjected Sar1p H79G, which is a GTP-locked mutant. NRK
cells were microinjected with 1.5 mg/ml Sar1p H79G. mBet3p (top), Man II (middle), and GM130 (bottom) were visualized by indirect immunofluorescence.
All injected cells are marked with an asterisk. Bars, 10 μm.
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the ER and other organelles (Fig. 4 B). Although no signiicant
labeling was observed on Golgi cisternae (Fig. 4 B), mBet3p
was found on tubules with a dense lumen in proximity to multivesicular bodies (MVBs). Some of these tubules were connected to MVBs (Fig. 4 B and Fig. S2 A, available at http://www.
jcb.org/cgi/content/full/jcb.200603044/DC1). This observation
is consistent with the inding that there is more than one pool of
mBet3p (Sacher et al., 2001; Loh et al., 2005). It is also consistent with our recent inding that the yeast TRAPPII complex,
which also contains Bet3p, localizes to the early endosome
(Cai et al., 2005).
mBet3p is required for COPII vesicle
coisolation in vitro
Figure 4. mBet3p is enriched at the tER and adjacent pre-Golgi
membranes. (A) Immuno-EM in NRK cells confirmed that mBet3p (5-nm gold
particles) resides on the tER region labeled with Sec31p (10-nm gold
particles). The arrow points to a COPII vesicle containing mBet3p. Bar,
100 nm. (B) mBet3p resides on the tER, and not ER, membranes (see
Materials and methods for a description of the quantitation). NE, nuclear
envelope; PM, plasma membrane; mito, mitochondrial outer membrane;
Golgi, Golgi cisternae; endo, endosomal structures.
resistant to BFA and sensitive to reagents that block export from
the ER or disrupt tER sites. To more precisely determine the localization of mBet3p, immuno-EM was performed. For this
analysis, the tER was deined as a cluster of Sec31p-labeled
(large gold particles) membranes. Immuno-EM demonstrated
that COPII vesicles and COPII budding proiles (Fig. 4 A,
arrow) contained mBet3p (small gold particles). The observed
localization of mBet3p to the tER was signiicant, as quantitation
revealed that the labeling density of mBet3p at the tER was
7.6–22 times higher than the labeling density of mBet3p over
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In vitro transport studies have shown that mBet3p acts after
COPII vesicle budding, but before the GTPase Rab1 (Loh
et al., 2005). However, the role of mBet3p in VTC biogenesis
was not addressed in these earlier studies. Homotypic COPII
vesicle tethering and fusion have recently been reconstituted
in vitro (Xu and Hay, 2004). Using this in vitro assay, we directly addressed whether mBet3p is required for the interaction of COPII vesicles with each other. For this experiment,
COPII vesicle populations that were marked in two different
ways were generated from permeabilized cells. One population of vesicles contained Myc-tagged temperature-sensitive
vesicular stomatitis virus glycoprotein (ts045 VSV-Myc), and a
second population of vesicles contained untagged radiolabeled
VSV-G* (Fig. 5 A). After the vesicles were formed in a irststage incubation, the permeabilized donor cells were removed
and the differently marked vesicle populations were combined
and incubated together in a second-stage incubation in the presence of cytosol and an ATP-regenerating system. VSV-G*–
containing radiolabeled vesicles that interact with VSV-G–Myc
vesicles were coisolated from the supernatant with anti-Myc
antibody and detected by autoradiography. When the vesicles
fused, heterotrimeric VSV-G that contained both VSV-G–Myc
and VSV-G* was formed and detected by immunoprecipitation
in the presence of detergent (Xu and Hay, 2004). Interestingly,
α-mBet3p, but not control IgG, inhibited the coisolation of vesicles and their fusion when added to the second-stage incubation (Fig. 5 B). Furthermore, this inhibition was completely
reversed when the antibody was added in the presence of excess
recombinant His6-mBet3p (Fig. S2 B). In contrast, antibody directed against the ER–Golgi SNARE syntaxin-5 inhibited the
fusion of COPII vesicles without dramatically affecting their
coisolation (Fig. 5 B). In support of the proposal that mBet3p
mediates homotypic COPII vesicle tethering, Western blot analysis revealed the presence of mBet3p on the reaction product
(Fig. 5 C, bottom), nascent VTCs that also contained VSV-G–
Myc (Fig. 5 C, top). The localization of mBet3p to nascent
VTCs was speciic, as mBet3p was not present on the untagged
control or when vesicle budding was blocked with the GDPlocked form of Sar1 (Fig. 5 C, bottom).
A single-stage transport assay was also performed in the
presence of α-mBet3p and vesicle release was monitored from
the permeabilized cells. In this experiment, α-mBet3p was
added to the cells in vitro before vesicles were formed. Studies in
Figure 5. mBet3p links COPII vesicles to each
other. (A) COPII vesicle coisolation in vitro.
Vesicles marked in two different ways were
released from permeabilized NRK cells during the stage I incubation. During the stage
II incubation, vesicles tethered to each other
and subsequently fused. (B) Anti-Bet3p antibody blocks the coisolation of COPII vesicles.
Two COPII vesicle populations were formed
in vitro in a first-stage incubation, as previously described (Xu and Hay, 2004). The two
vesicle populations were then combined and
preincubated together for 20 min on ice in the
absence or presence of antibody. After the incubation, one sample was kept on ice, while
the other samples were incubated at 32°C for
60 min in a second-stage incubation. 1, one
population of vesicles was untagged instead of
Myc-tagged; 2, incubated on ice for 60 min;
3, complete reaction incubated without antibody; 4, contained α-mBet3p (168 nM; 5μg
per reaction); 5, contained α-syntaxin-5
(168 nM); 6, contained rabbit IgG (168 nM).
(C) Bet3p is present on nascent VTCs formed
in vitro. Stage I reactions were performed in
the presence or absence of GDP-locked Sar1
T39N. Lane 1, complete reaction; lane 2,
complete reaction plus GDP-locked Sar1 T39N
(800 nM); lane 3, vesicles were formed from
NRK cells expressing untagged VSV-G. Vesicles
released during stage I were then incubated in
stage II (60 min at 32°C) and immunoisolated
with anti-Myc antibody. The final immunoisolates were subjected to Western blot analysis
to detect mBet3p (bottom) and VSV-Myc (top).
(D) Anti-Bet3p antibody stimulates the release
of COPII vesicles from permeabilized cells
when added during the stage I incubation.
COPII vesicle populations were formed in vitro
using permeabilized NRK cells as in B, only
the incubation was performed for 90 min (as
described in Fig. 1 A of Xu and Hay, 2004).
The permeabilized cells were removed by centrifugation, and the released vesicles were immunoisolated with an antibody to the integral
membrane protein p24. The VSV-G* in the precipitate was quantified by autoradiography and reported as the percentage of the total VSV-G* in the
starting cells. 1, complete reaction without antibody; 2, Sar1 T39N was included in the reaction at a concentration of 1 μM; 3, complete reaction plus
110 nM anti-mBet3 antibody. Error bars are the SEM.
both mammalian cells and yeast have previously demonstrated
that blocking the consumption of COPII vesicles increases
their release from permeabilized cells in vitro (Barlowe, 1997;
Xu and Hay, 2004). Consistent with the inding that COPII
vesicles fail to fuse when mBet3p is inactivated, we observed
that α-mBet3p stimulated the release of vesicles from the cells
(Fig. 5 D). This inding also demonstrates that mBet3p is not
required for vesicle budding and is consistent with a previous
study that showed α-mBet3p acts after COPII vesicles bud from
the ER in vitro (Sacher et al., 1998; Loh et al., 2005).
Accumulated cargo colocalizes with
Sec31p when mBet3p is inactivated in vivo
In vitro, α-mBet3p blocks the conversion of VSV-G from an
endo H–sensitive ER form to an endo H–resistant Golgi form
(Loh et al., 2005). However, the role of mBet3p in vivo has not
been addressed. To determine where cargo accumulates when
mBet3p is inactivated in vivo, we monitored the traficking of
two different marker proteins, tsO45 VSV-G and the plasma
membrane marker CD8. One marker was used to quantitate the
traficking defect when α-mBet3p was injected into cells, and
the other marker was used to address where cargo accumulates.
Quantitation of tsO45 VSV-G transport in HeLa cells revealed
that the inactivation of mBet3p resulted in a nearly 70% decrease in the traficking of YFP-tagged tsO45 VSV-G to the cell
surface (Fig. 6 A).
To address where cargo accumulates when mBet3p is
inactivated, cDNA encoding CD8 was microinjected into the
nuclei of BSC-1 cells. The CD8 mRNA was then allowed to
accumulate in the presence of cycloheximide before α-mBet3p,
or control IgG, was microinjected (Fig. 6 B). After 1 h, the cycloheximide was removed and CD8 was synthesized during a
second incubation. Newly synthesized CD8 was then chased
out of the ER during a inal incubation in the presence of cycloheximide (Pelletier et al., 2000). In control cells, CD8 was
found at the cell surface and the perinuclear region of the cell
(Fig. 6 B; IgG control). Perinuclear CD8 colocalized with
Golgin-84 in IgG injected cells (unpublished data). In the
COPII VESICLE TETHERING • YU ET AL.
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Figure 6. Cargo accumulates in structures that colocalize
with COPII when mBet3p is inactivated in vivo. (A) The trafficking of YFP-tagged tsO45 VSV-G to the cell surface was quantitated in HeLa cells that were microinjected with α-mBet3p or
control IgG, as described in the Materials and methods. Error
bars are the SD. (B) Intracellular trafficking was monitored
in BSC-1 cells that were injected with cDNA encoding CD8.
After CD8 mRNA accumulated, the cells were injected with
α-mBet3p or IgG. Subsequent to a 1-h incubation at 37°C, the
cells were washed and the synthesis and transport of CD8
was continued for 3 h. Finally, CD8 was chased for 45 min in
the presence of cycloheximide. The cells were stained for
CD8, Sec31p, or Golgin-84. To visualize Sec31p and Golgin-84,
antibodies were directly conjugated to Alexa Fluor 594
and viewed in the red channel. To visualize CD8, the secondary antibody was conjugated to Alexa Fluor 488 and viewed
in the green channel. The inset shows the merge of CD8,
Sec31p, and Golgin-84 in the α-mBet3p–injected cell.
The merged image is on the right. Bar, 20 μm.
presence of α-mBet3p, Golgin-84 localization was fragmented.
This inding is consistent with the observation that α-mBet3p
disrupts the Golgi (see the next section). When mBet3p was inactivated, CD8 accumulated in spots that surround the nucleus.
These spots largely colocalized with Sec31p, but were adjacent
to Golgin-84–containing membranes (Fig. 6 B), implying that
cargo accumulates in membranes that contain the COPII coat.
These results are also consistent with previous studies showing
that α-mBet3p blocks the traficking of VSV-G before it is converted to an endo H–resistant form in the Golgi (Loh et al.,
2005). Thus, both in vitro and in vivo traficking studies support
the hypothesis that mBet3p is required for the interaction of
COPII vesicles with each other.
tER sites disperse and Golgi architecture
is disrupted by perturbation of mBet3p
We also determined the consequences of depleting mammalian
cells of mBet3p by speciically reducing its expression with
siRNA. Two different siRNA duplexes were used for these studies, BetC (Fig. 7 A) and BetA (not depicted). Similar results were
obtained with both siRNA duplexes. BetC, but not the control
duplex (luciferase), reduced the expression of mBet3p (Fig. 7 A).
The loss of mBet3p did not lead to a reduction of Sec31p or
the ER protein ER60 (Fig. 7 A). However, the localization of
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Sec31p was dispersed, and its intense perinuclear localization,
which marks the tER, was lost (Fig. 7 B). In addition, pre-Golgi
and Golgi compartments were disrupted (Fig. 7 C and Fig. S2 C).
The localization of β-tubulin, however, remained unaltered
(Fig. S2 D). Similar effects on the Golgi were obtained when
cells were micro-injected with α-mBet3p (Fig. S3, available at
http://www.jcb.org/cgi/content/full/jcb.200603044/DC1).
EM analysis conirmed that the depletion of mBet3p disrupts Golgi architecture (Fig. 7 D, compare the Golgi [G] in the
top and bottom images), and leads to a dramatic accumulation
of vesicles (Fig. 7 D, arrows). Although tER was dificult
to identify by EM in the siRNA-treated mBet3p-depleted
cells, highly dilated rough ER was observed (Fig. S4, available
at http://www.jcb.org/cgi/content/full/jcb.200603044/DC1).
Quantiication revealed a threefold decrease in the amount of
membrane that represents Golgi cisternae. These indings provide in vivo evidence that mBet3p links COPII vesicles to each
other at or near the tER, an event that appears to be required for
the maintenance of Golgi structure.
Discussion
Although the late stages of ER–Golgi trafic have been visualized by live cell imaging (Presley et al., 1997; Scales et al., 1997),
Figure 7. tER sites disperse and Golgi architecture is disrupted by perturbation of mBet3p. Western blot analysis revealed that siRNA targeted to mBet3p
(BetC), but not luciferase, resulted in a decrease of mBet3p in COS-7 cells. (A) A >90% decrease of mBet3p, but not Sec31p or the ER protein ER60, was
observed. Lane 1, mock-treated cells; lane 2, cells transfected with siRNA targeted to luciferase; lane 3, cells transfected with siRNA targeted to mBet3p.
Similar results were obtained with BetA (not depicted) or BetC oligonucleotides. (B) The intense perinuclear localization of Sec31p is lost in cells depleted
of mBet3p by RNAi. siRNA targeted to mBet3p (bottom) or luciferase as a control (top) were transfected into COS-7 cells. siRNA-transfected cells were
stained with antibodies directed against mBet3p (left) and Sec31p (middle). The inset is magnified on the right. (C) The pre-Golgi compartment is disrupted
in COS-7 cells transfected with mBet3-specific siRNA. siRNA targeted to luciferase (left) and mBet3 (right) were transfected into COS-7 cells and stained
with α-mBet3p (top) and ERGIC-53 (bottom). (D) Golgi structure was disrupted in siRNA-treated HeLa cells that are depleted of mBet3p. EM analysis was
performed on HeLa cells transfected with siRNA targeted to luciferase as a control (top) or mBet3p (BetC, bottom). The empty arrow points to a Golgi stack (top).
Vesicles are marked with an arrow (bottom). The arrowhead marks a tubule (bottom). Mini Golgi stacks are labeled G (bottom). Bars: (B and C) 10 μm;
(D) 200 nm.
little is known about the homotypic fusion of COPII vesicles
and how the pre-Golgi structure is formed from these vesicles.
We provide evidence that mBet3p, the most highly conserved
component of the TRAPP complex, plays a pivotal role in
these events.
Although it was previously shown that the SNARE
syntaxin-5 is required for homotypic vesicle fusion (Xu and Hay,
2004), the indings we report here are the irst to link the tether
TRAPP to VTC biogenesis. Our results imply that mBet3p is
recruited to the tER region to mediate homotypic COPII vesicle
tethering. Several lines of evidence support this proposal. First,
we show that the localization of mBet3p is perturbed by conditions that are known to alter or disrupt the tER, suggesting that
mBet3p may be recruited to membranes by one or more components of the tER. Second, mBet3p is required for homotypic
COPII vesicle tethering in vitro and the inactivation of mBet3p
in vivo leads to the accumulation of cargo in membranes that
colocalize with the COPII subunit Sec31p. Finally, a role for
mBet3p in VTC biogenesis is supported by data that shows the
loss of mBet3p disrupts Golgi structure and the VTC marker
ERGIC-53.
Immuno-EM revealed that mBet3p is enriched on more
than one membrane, the tER region, and endosomal structures.
Surprisingly, although yeast Bet3p was reported to reside on the
Golgi (Sacher et al., 1998; Barrowman et al., 2000), we did not
observe signiicant labeling of mBet3p on Golgi cisternae.
In the yeast S. cerevisiae, tER, Golgi, and endosomal marker
proteins are all found on punctate structures (Rossanese et al.,
1999). Yeast Bet3p was originally reported to reside on the
Golgi because a signiicant fraction of the Bet3p containing
puncta enlarged in a sec7 mutant (Sacher et al., 1998), a putative Golgi proliferating mutant (Novick et al., 1980). However,
our more recent studies suggest that Sec7p and yeast TRAPPII
(which contains Bet3p) may reside on endosomal membranes
(Cai et al., 2005). The localization of mBet3p to both the tER
region and endosomal structures is consistent with studies in
COPII VESICLE TETHERING • YU ET AL.
365
yeast showing that the two TRAPP complexes (TRAPPI and
TRAPPII) act at different stages of membrane trafic.
Yeast TRAPPI, which contains Bet3p and several other
subunits, is required for COPII vesicle tethering (Sacher et al.,
1998; Barrowman et al., 2000). Because it is dificult to morphologically distinguish membranes in the ER–Golgi branch of
the yeast secretory pathway, no equivalent to a pre-Golgi compartment has been identiied, and COPII vesicles are thought to
tether directly to the Golgi. In mammalian cells, COPII vesicles
do not fuse directly with the Golgi, instead it is thought they
fuse with each other to form VTCs (Xu and Hay, 2004). The recent inding that the ER–Golgi intermediate compartment is
stable (Ben-Takaya et al., 2005; Sannerud et al., 2006), however, has raised the possibility that homotypic fusion is not exclusive and COPII vesicles may also heterotypically fuse with
stable pre-Golgi membranes. Nonetheless, our in vitro and in
vivo indings clearly show that mBet3p is required for the biogenesis of VTCs and Golgi membranes. The high degree of homology between yeast and mammalian Bet3p also raises the
possibility that yeast COPII vesicles may undergo homotypic
tethering and fusion before they fuse with the Golgi. Elucidation
of the mechanism of COPII vesicle tethering in yeast and mammalian cells will be needed to resolve this issue. In the future it
will also be important to address how mammalian TRAPP mediates subsequent steps in membrane trafic. Experiments are
currently in progress to address these questions.
Materials and methods
Antibodies
Monoclonal antibody directed against GM130 was purchased from BD
Biosciences. CD8 antibody, monoclonal antibodies directed against the
COPI coat (CM1A10), the lumenal domain of VSV-G protein, rodent Man
II (53FC3), and Golgin-84 were obtained from G. Warren (Yale University,
New Haven, CT). Monoclonal antibody directed against ERGIC-53 was
obtained from H.-P. Hauri (University of Basel, Basel, Switzerland). Anti–
β-tubulin antibody (sc-5274) was purchased from Santa Cruz Biotechnology,
Inc. Anti-ER60 antibody was obtained from P. Kim (University of Cincinnati,
Cincinnati, OH). Monoclonal antibody directed against GalT (GTL-2)
was obtained from D. Shima (Eyetech Research Center, Woburn, MA).
Anti-Sec31p antibody (Shugrue et al., 1999) was obtained from F. Gorelick
(Yale University, New Haven, CT), and polyclonal anti–Man II antibody
was obtained from M. Farquhar (University of California, San Diego,
San Diego, CA). Polyclonal rabbit anti-mBet3p antibody was previously described (Sacher et al., 2000). Rabbit IgG specific to mBet3p was purified
on an Affigel column (Bio-Rad Laboratories) preloaded with His6-tagged
Bet3p. Bound IgG was eluted with 0.1 M glycine, pH 2.5. For immunofluorescence, fluorescent-labeled secondary antibodies were purchased
from Invitrogen.
Fluorescence microscopy
Cells grown on 10-mm coverslips were fixed in cold methanol for 5 min (for
GTL-2 and CM1A10 antibodies only), or fixed in PBS with 3.7% formaldehyde for 15 min (for all other antibodies). Samples were quenched with
serum-free DME, rinsed with buffer A (PBS with 5 mM EDTA, 0.25% BSA,
and 0.05% NP-40) and permeabilized with buffer B (PBS with 5 mM EDTA,
0.25% BSA, 0.05% NP-40, and 0.1% Triton X-100) for 5 min. The samples
were then treated with buffer C (PBS with 0.5% BSA) for 15 min. Antibodies
were applied, and the samples were incubated at room temperature
for 1 h. After four washes in buffer C, the samples were incubated with
secondary antibody for 30 min. Four additional washes were performed
in buffer C, and the samples were mounted on glass slides in Fluoromount G
(Southern Biotech). Images were taken with an Axiophot fluorescence
microscope using a 63× oil-immersion objective and a LSM 510 confocal
microscope (both from Carl Zeiss MicroImaging, Inc.). Images were captured with a digital camera Orca ER; Hamamatsu Photonics). The images
366
JCB • VOLUME 174 • NUMBER 3 • 2006
were processed with the OpenLab 3.08 imaging software (Improvision)
and Photoshop 7.0 (Adobe).
To deplete α-mBet3p of determinants directed against mBet3p,
affinity-purified antibody (1 μg/ml in buffer C) was incubated (with
agitation) for 2 h with a nitrocellulose strip containing 60 μg of purified
His6-tagged protein. Antibody was mock treated with a blank nitrocellulose
strip as a control.
mBet3p was visualized with goat anti–rabbit secondary antibody conjugated to Cy3. GM130, COPI, β-tubulin, and ERGIC-53 were visualized
with donkey anti–mouse secondary antibody conjugated to FITC. For doublelabeling experiments with mBet3p and Sec31p, the Zenon labeling kit was
used (Invitrogen). Samples were first stained with α-mBet3p and then stained
for Sec31p, using this kit according to the manufacturer’s instructions.
Microinjection experiments
Affinity-purified α-mBet3p, control IgG, or Sar1pDN was injected into COS-7
or NRK cells as previously described (Seemann et al., 2000a). The antibodyinjected samples were fixed and stained after 2 h, whereas the Sar1pDNinjected samples were fixed and stained after 6 h.
Homotypic COPII vesicle tethering/fusion assay
Experiments were performed as previously described (Xu and Hay, 2004).
The VSV-G–Myc–expressing cells and the pulse-radiolabeled VSV-G*–
containing cells were permeabilized by scraping the cells. For the first-stage
incubation, each reaction was 1,595 μl and contained 352 μl of water,
55 μl of 0.1 M magnesium acetate, 110 μl of an ATP-regenerating system,
33 μl of 1 M Hepes, pH 7.2, 220 μl of 20/18/50 buffer (20 mM Hepes,
pH 7.2, 18 mM CaCl2, and 50 mM EGTA), 550 μl of rat liver cytosol dialyzed into 25/125 buffer (25 mM Hepes, pH 7.2, and 125 mM potassium
acetate), and 275 μl of either semi-intact NRK cell population. Reactions
were incubated at 32°C for 30 min (stage I incubation), centrifuged at
4,000 g for 1 min, and then centrifuged again at 15,000 g for 1 min. The
supernatant, which contained the vesicles, was saved. In the stage II incubation, each reaction was 200 μl and contained 72.5 μl of VSV-G–Myc
vesicles, 72.5 μl of VSV-G* vesicles, and 55 μl of 25/125 buffer or purified proteins (antibodies, GST, or GST-Sec23p) dissolved in 25/125 buffer.
After a 20-min preincubation on ice, the reactions were incubated at 32°C
for 60 min (stage II incubation). For coisolation assays, the final intact vesicle suspensions were processed for immunoisolation using biotinylated
anti-Myc antibodies and streptavidin–Sepharose. Bound vesicles were analyzed by SDS-PAGE (8%) and autoradiography, and the coisolated
VSV-G* was quantified. For heterotrimer assays, the final vesicle suspensions were incubated with 2% Triton X-100 for 20 min and then centrifuged
at 100,000 g for 30 min. The 100,000 g supernatants containing solubilized VSV-G trimers were then processed for immunoprecipitation using
biotinylated anti-Myc antibody and streptavidin–Sepharose, and the
coprecipitating VSV-G* was quantified.
Trafficking assays
VSV-G transport was assayed as previously described (Diao et al.,
2003) with several minor exceptions. HeLa cells were first injected with
1.5 mg/ml α-mBet3p or 1.5 mg/ml control IgG; 2 h later, plasmid encoding 0.2 mg/ml YFP-tagged tsO45 VSV-G was injected. Protein transported
to the cell surface was detected with a monoclonal antibody to the lumenal
domain of VSV-G that was conjugated to Alexa Fluor 647. Total VSV-G
was detected by YFP. The extent of VSV-G transport to the cell surface was
determined by the ratio of surface to total measured fluorescence, as previously described (Seemann et al., 2000b). A total of nine cells injected with
either α-mBet3p antibody or IgG were quantitated. The trafficking of the
plasma membrane marker CD8 was monitored, as previously described
(Pelletier et al., 2000). Cells were injected with 1.5 mg/ml α-mBet3p or
1.5 mg/ml IgG after the CD8 plasmid was injected in the presence of
100 μg/ml cycloheximide. The incubation was then continued for 1 h
at 37°C.
RNAi
siRNA oligonucleotides specific for human Bet3p were designed according
to the method developed by Dharmacon Research, Inc. Two oligonucleotides,
BetA (5′-UCACUCCAAGCAUUACUAAUU-3′) and BetC (5′-GGAGACGGUGUGACAGAAAUU-3′) were chosen because they are specific and
target to regions that are conserved between human and monkey Bet3
sequences. Duplex RNA was introduced into COS-7 or HeLa cells, using
OligofectAMINE (Invitrogen) according to the manufacturer’s instructions.
3 d after transfection, the cells were analyzed. In three independent experiments, the reduction of mBet3p (determined by Western blot analysis)
ranged from 74 to 99% using BetA, and from 87 to 99% for BetC.
Samples were quantitated using Image 1.61/68 K software (National
Institutes of Health, Bethesda, MD).
EM analysis
Immuno-EM was performed in NRK cells as previously described (Folsch
et al., 2003). Estimation of labeling densities was determined on digital
images captured using a charge-coupled device camera (Morada; Soft Imaging System). At a magnification of 43,000×, sections were scanned
and 118 fields were captured at random. Transitional elements were defined as tubular vesicular clusters found in the proximity of ER membranes
labeled for Sec31p (at least two gold particles). The labeling density for
mBet3p was calculated by counting the number of 5-nm gold particles falling on or within 15 nm of membranes, and dividing these numbers by the
length of the membranes expressed in micrometers. Length was estimated
by counting the number of intersections of membranes with the vertical and
horizontal lines of a grid at an estimated distance of 110 nm between lines
(Weibel and Bolender, 1979).
HeLa cells were processed for EM analysis as previously described
(Lopez et al., 2002), with the following two exceptions: cells were scraped
after fixation, and the section thickness was 60 nm. Quantitation of Golgi
membrane was done directly at the EM. Sections were scanned horizontally in a random fashion at a magnification of 9,900×, and the number
of intersections of membrane with a fixed point were counted. For each
sample, >900 intersections with the plasma membrane were counted. The
number of intersections with stacked Golgi cisternae was divided by the
number of intersections with the plasma membrane (or nuclear envelope).
A threefold decrease in the ratio of Golgi to plasma membrane (or Golgi
to nuclear envelope) was observed in the mBet3p-depleted sample. HeLa
cells were used for this analysis because it was easier to obtain better cryosections with these cells than with COS-7 cells.
Online supplemental material
Figure S1 shows a still from Video 1. Fig. S2 shows that the loss of mBet3p
blocks COPII vesicle tethering and disrupts Golgi architecture. Fig. S3
shows that microinjected α-mBet3p, but not control IgG, disrupts the preGolgi compartment marked by ERGIC-53 in COS-7 cells. Fig. S4 shows
the rough ER is highly dilated in siRNA-treated mBet3p-depleted HeLa cells.
Video 1 shows the colocalization of mBet3p and Sec31p in nocodazoletreated cells. Online supplemental material is available at http://www.jcb.
org/cgi/content/full/jcb.200603044/DC1.
We thank Lee Walker for help in the preparation of the manuscript and Yueyi
Zhang for technical assistance. We also thank Graham Warren, Fred Gorelick, Marilyn Farquhar, David Shima, Hans-Peter Hauri, Paul Kim, Jaakko
Saraste, Derek Toomre, David Stephens, and Jennifer Lippincott-Schwartz for
antibodies and plasmids. We also thank Hong Chen for advice on the use of
siRNA and Laurence Pelletier for his primary observations, as well as Pete
Takizawa, Pietro DeCamilli, Ira Mellman, and Graham Warren, for comments
during the preparation of the manuscript. Special thanks to Graham Warren
and Pete Takizawa for their advice and stimulating discussions.
This work was supported by the Howard Hughes Medical Institute.
Salary support for S. Ferro-Novack and S. Yu were provided by the Howard
Hughes Medical Institute. A. Satoh was supported by an American Heart Association scientist development grant. Work at the University of Montana was
supported by National Institutes of Health (NIH) grant GM59378 to J.C. Hay
and the NIH–Centers of Biomedical Research Excellence Center for Structural
and Functional Neuroscience.
Submitted: 9 March 2006
Accepted: 23 June 2006
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