Correspondence to: Zvulun Elazar, Department of Biological Chemistry, The Weizmann Institute of Science, Rehovot, 76100 Israel., bmzevi{at}weizmann.weizmann.ac.il (E-mail), 972-8-9343682 (phone), 972-8-9344112 (fax)
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Abstract |
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Intracellular transport of newly synthesized and mature proteins via vesicles is controlled by a large group of proteins. Here we describe a ubiquitous rat proteinendoplasmic reticulum (ER) and Golgi 30-kD protein (ERG30)which shares structural characteristics with VAP-33, a 33-kD protein from Aplysia californica which was shown to interact with the synaptic protein VAMP. The transmembrane topology of the 30-kD ERG30 corresponds to a type II integral membrane protein, whose cytoplasmic NH2 terminus contains a predicted coiled-coil motif. We localized ERG30 to the ER and to pre-Golgi intermediates by biochemical and immunocytochemical methods. Consistent with a role in vesicular transport, anti-ERG30 antibodies specifically inhibit intra-Golgi transport in vitro, leading to significant accumulation of COPI-coated vesicles. It appears that ERG30 functions early in the secretory pathway, probably within the Golgi and between the Golgi and the ER.
Key Words: endoplasmic reticulum, Golgi, coated vesicles, secretion, transport intermediates
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Introduction |
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TRANSPORT of proteins between different organelles of eukaryotic cells is mediated by coated vesicles that bud from one membrane compartment and are targeted to and fuse with an appropriate acceptor organelle (
Budding of vesicles is regulated by small GTPases and mediated by cytosolic coat proteins (COP)1 that assemble on the donor membrane (Rothman and Wieland, 1996;
Docking of vesicles to the appropriate target membrane involves interaction between integral membrane proteins located on the vesicle, called v-SNAREs, and the target membrane proteins, t-SNAREs (
Following pairing, the v/t-SNARE complex binds two soluble factors: NEM-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP). These, in turn, catalyze the disassembly of the SNARE complex (
The vesicle-associated membrane protein (VAMP)associated protein of 33 kD (VAP-33) was identified in Aplysia californica (
In the present study we describe a VAP-33related protein which we denote ERG30, and demonstrate its type II transmembrane topology. We found that ERG30 is localized in the ER and in pre-Golgi intermediates. Functional in vitro assays attribute to ERG30 a role in COPI vesicle transport. We put forward the hypothesis that ERG30 is involved in intra-Golgi transport and in retrograde transport of proteins between the Golgi and the ER.
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Materials and Methods |
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Construction of Bait Plasmids
ERG30 was cloned by the two-hybrid system using the cytosolic portion of Neu differentiation factor ß4a (NDFß4a290-662) as a bait. NDFß4a290-662 was generated by PCR with EcoRI and BamHI ends using the following primers: 5'-CCGGAATTCACCAAGAAGCAGCGGCAG-3' and 5'-CGCGGATCCTTATACAGCAATAGGGTC-3'. The resulting PCR product was digested with EcoRI and BamHI and cloned into the appropriate sites in the pGBT9 vector (Clontech) downstream from the GAL4 DNA binding domain. This plasmid was transformed into the two-hybrid strain HF7c reporter strain (Clontech), and tested for expression of the fusion protein by Western analysis. The inserted fragment was sequenced to verify that no mutation had occurred because of PCR and to confirm the correct reading frame of the resultant fusion protein.
Construction of a Rat Brain cDNA Library in the pACT Vector
A cDNA library was constructed from 5 µg of oligo (dT)-selected mRNA, using a Stratagene kit. RNA was prepared from rat brain by the guanidinium thiocyanate-phenol-chloroform extraction method. The mRNA was purified and used as a template for cDNA synthesis. The resulting dscDNA was methylated by XhoI methylase and ligated to an EcoRI linker, thus generating an EcoRI site at the 5' end of the cDNA and an XhoI site at the 3'. The average size was ~2.5 kb. The purified dscDNA was ligated to ACT. The library titer was ~1.4 x 107 pfu. The titer of the library after amplification was 3 x 109 pfu/ml. In vivo excision was performed from the phagemid
ACT to pACT.
Two-Hybrid Screen
The HF7c yeast strain carrying the bait plasmid pGBT9-NDFß4a290-662 was transformed with the rat brain cDNA library generated in pACT AD vector (Clontech). Transformation efficiency was assessed by plating small aliquots onto SCD plates lacking tryptophan, leucine, and histidine and supplemented with 10 mM 3-AT. The yeast colonies were transferred to nitrocellulose filters (BA85; Schleicher and Schuell), immersed in liquid nitrogen for 5 s, and incubated at 30°C on a 3-mm Whatman paper soaked with 60 mM Na2HPO4, 40 mM NaH2P04, pH 7.0, 10 mM KCl, 1 mM MgSO4, 50 mM ß-mercaptoethanol, and 1 mg/ml X-GAL. Colonies containing the interacting pair of proteins became blue within 26 h. From an initial screen of 200,000 colonies, we isolated 5 individual clones containing different cDNAs corresponding to the same mRNA. To isolate library plasmids from positive clones, cells were grown in SC liquid media lacking leucine (to allow loss of bait, but not of the library plasmid), and plasmid DNA was prepared and transformed at low dilution into HB101 competent Escherichia coli cells. The transformants were selected on a M9 minimal medium containing 50 µg/ml Amp. Plasmids isolated were then used to retransform SFY526 yeast cells either alone or with pGBT9-NDFß4a290-662. Transformants were assayed for ß-galactosidase activity. cDNA isolated from the positive clones was subcloned to pBluescript-II, and then sequenced using an Applied Biosystems 373A automated DNA sequencer and Applied Biosystems Taq Dye DeoxyTM Terminator cycle sequencing kit. Of the five colonies isolated by this screen, clone pACT17 contained the complete coding sequence of ERG30. RT-PCR performed on rat brain mRNA, using a specific oligonucleotide derived from pACT17, confirmed that the sequence obtained from the cDNA library was the full-length cDNA.
Construction and Expression of MBP-ERG30 Fusion Proteins
ERG30 and its truncated forms were tagged at their NH2 terminus with a maltose binding protein (MBP) domain. For that purpose, full-length ERG30, ERG30 (coiled-coil) (amino acids 1140), or ERG30 (
NH2 terminus) (amino acids 141243) was ligated into the EcoRI BamHI site of pMAL-p2 vector (New England Biolabs). The different fusion proteins were overexpressed in the JM109 strain of bacteria by growing log phase bacteria in the presence of 1 mM IPTG for 4 h. Cell extracts were prepared by sonicating the bacteria in a buffer containing 50 mM Tris (pH 8.0), 50 mM KCl, 0.1 mM EDTA, and 1% Triton X-100. Bacterial extracts were centrifuged in a Ti60 rotor (Beckman) at 45,000 rpm for 60 min, and the supernatant was passed over a 1-ml amylose column at 4°C. The column was washed with 10 column volumes of 20 mM Tris-HCl (pH 7.4), 0.4 M NaCl, and 10 mM ß-mercaptoethanol. The protein of interest was eluted with the above buffer plus 10 mM maltose.
Production of Anti-ERG30 Antibodies
An antiserum was raised in rabbits against a recombinant MBP-ERG30 protein purified from E. coli. The antiserum was first run through CNBr activated Sepharose column with covalently bound MBP, to remove anti-MBP antibodies. Anti-ERG30 antibodies were purified from the flow-through material of the first column by affinity chromatography on a CNBr-activated Sepharose column with covalently bound MBP-ERG30 fusion protein.
Subcellular Fractionation
Rat liver homogenates were fractionated over sucrose gradients as described previously (
Drug Treatment and Immunofluorescence Microscopy
NRK or CHO cells were seeded onto microscope slides 24 h before staining. For drug treatment before immunofluorescence microscopy, cells were incubated for 1 h in RPMI (NRK cells) or in -MEM (CHO cells) medium containing 10 µg/ml brefeldin A (BFA). To fix cells for microscopy, the growth medium was removed and cells were incubated for 10 min in methanol at -20°C. Cell staining involved a 1-min incubation in acetone at -20°C for permeabilization followed by incubation in a blocking solution containing 10% FCS in PBS. Cells were incubated for 2 h with the primary antibodies in 2% FCS in PBS, then washed three times with PBS and incubated in an incubation solution containing affinity-purified fluorescein- or rhodamine-labeled antibodies against mouse or rabbit IgG. Slides were finally washed three times with PBS, and mounted beneath coverslips. The stained cells were analyzed with an MRC1024 confocal microscope (BioRad).
EM/Immunocytochemistry of CHO Cells
CHO cells were grown on glass coverslips that had been coated with carbon and gelatin (
Cis- to Medial-Golgi Transport Assay
The standard assay mixture (25 µl) contained 0.4 µCi UDP-[3H] N-acetylglucosamine (America Radiolabeled Chemical), 5 µl of a 1:1 mixture of donor and acceptor CHO Golgi membranes, and crude bovine brain cytosol, as described (
Isolation of COPI Vesicles
Golgi-derived COPI vesicles were isolated as described previously (
Formation of 20S Particles
For the isolation of 20S SNARE particles we used the procedure described by SNAP and NSF-Myc in a buffer containing 25 mM Hepes-HCl, pH 7.0, 75 mM KCl, 1 mM DTT, 2 mM EDTA, 0.75% Triton X-100 and 0.5 mM ATP
S, 1% (wt/vol) polyethyleneglycol (PEG 400), and 0.5 mM PMSF for 30 min at 4°C. Mouse anti-Myc monoclonal antibodies coupled to protein GSepharose were added and the incubation continued for an additional 2 h with constant agitation. The beads were then washed with 10 vol of buffer A (25 mM Hepes-HCl, pH 7.0, 100 mM KCl, 1 mM DTT, 2 mM EDTA, 0.5% Triton X-100, and 0.5 mM ATP
S), followed by elution with buffer A containing 8 mM MgCl2 (nonspecific elution), or with buffer A containing 8 mM MgCl2 and 0.5 mM ATP to allow ATP hydrolysis (specific elution). The eluted fractions were precipitated with trichloroacetic acid, boiled, and analyzed by Western blotting using the appropriate antibodies.
Western Blot Analysis
To prepare rat tissue extracts, frozen organs were washed in cold PBS and lysed with a homogenizer (PCU Kinematica) in ice-cold protein extraction buffer containing 0.5 M ß-glycerophosphate, 15 mM EGTA, 10 mM EDTA, 1 mM ortho-vanadate, 1 mM benzamidine, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 2 µg/ml pepstatin A, and 1 mM DTT, at pH 7.4. Lysates were cleared by 15 min spin at 20,000 rpm at 4°C. Supernatants were then mixed with sample buffer and heated to 95°C for 2 min. Equal amounts of tissue extracts were subjected to SDS-PAGE (15% acrylamide), transferred to nitrocellulose (Sartorius), and probed with anti-ERG30 antibodies using HRP-coupled secondary antibodies and ECL reagent (Amersham).
Immunoprecipitation
Rat brain membranes were isolated as described previously (
Fractionation by Triton X-114
Rat liver Golgi fractions were washed once with Tris-salt buffer (10 mM Tris-HCl, pH 7.6) and resuspended to a protein concentration of 4 mg/ml. Triton X-114 was added at 4°C to a final concentration of 2% (wt/vol). Solubilized membranes were incubated on ice for 4 min and then centrifuged for 10 min in a Beckman TLA100.1 rotor at 37,000 g. The supernatant was layered over a cushion of 0.25 M sucrose in a Tris-salt buffer containing 0.06% Triton X-114, and incubated at 30°C for 5 min. After centrifugation at 2,500 g in a benchtop centrifuge, the Triton X-114 phase was saved. The aqua phase was brought to 0.5% Triton X-114, and layered once more on a cushion, incubated at 30°C for 5 min, and centrifuged as described before. The detergent layers were combined and the supernatant kept separately. Before the SDS-PAGE separation the detergent was removed by Biobeads SM-2 (Bio-Rad).
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Results |
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Cloning cDNA Encoding ERG30
We have serendipitously cloned by the two-hybrid screen system a cDNA from a rat brain cDNA library that encodes a 30-kD protein. Because our analysis indicated that ERG30 is located in the ER and Golgi (see below), we have tentatively termed it ERG30. The nucleotide and deduced amino acid sequence of the cDNA are shown in Figure 1 A. The hydrophobicity profile of ERG30 suggests that its COOH-terminal region is highly hydrophobic [residues 224243 (
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ERG30 Is a Type II Integral Membrane Protein
The expression pattern of ERG30 in various rat tissues was examined by immunoblot analysis using affinity-purified anti-ERG30 antibodies. As evident from Figure 2 A, the 30-kD ERG30 protein is expressed in all tissues studied with a smaller 28-kD isoform, or a proteolytic product in the kidney, liver, and heart.
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Because the amino-acid sequence of ERG30 suggested that it might contain a COOH-terminal transmembrane domain (Figure 1 B), we examined its putative transmembrane topology by using anti-ERG30 antibodies for immunoblot analyses of cytosolic and membrane fractions. In rat brain, ERG30 was detected almost exclusively in the membrane fraction (Figure 2 B). The association of ERG30 with the membrane fraction was resistant to washing with 1 M KCl (data not shown) and the protein could be extracted entirely into a detergent phase upon solubilization with Triton X-114 (Figure 2 B). Using an in vitro translation system, we demonstrated that ERG30 was translocated into the microsomal membranes (Figure 2 C). To exclude the possibility of peripheral association with the microsomal membranes, the translated products were incubated with 100 mM Na2CO3, pH 11. Under these conditions ERG30 remained associated with the membrane fraction (Figure 2 C). We next addressed the topology of ERG30 with respect to the cytosol by using a protease protection assay. Treatment of the in vitro translated ERG30 (in the presence of microsomes) with proteinase K fully digested the translated protein, whereas the mature form of E. coli ß-lactamase, serving as a control protein translocated into the microsomes lumen, was fully protected (Figure 2 C). Taken together, these results indicate that ERG30 is a type II integral membrane protein, with an NH2-terminal domain facing the cytoplasm and a very short COOH-terminal hydrophobic domain located inside the membrane (Figure 2 D).
Self-oligomerization of ERG30
ERG30 shares with SNAREs a similar domain organization, including a predicted membrane-proximal coiled-coil domain, a motif common to various self-oligomerizing proteins involved in protein-protein interactions (NH2 terminus) containing residues 141243, and its complementary coiled-coil deletion mutant (
coiled-coil) containing residues 1140. Yeast cells were cotransfected with a construct of ERG30 fused to the Gal4 DNA binding domain, and ERG30 fused with pACT Gal4-activation domain. Only cells that contained both constructs were able to grow on a selective medium and exhibited significant ßGal activity. Furthermore, as shown in Figure 3, only the full length ERG30 was able to self-oligomerize in vivo, whereas the truncated forms, i.e., ERG30 (
coiled-coil) or ERG30 (
NH2 terminus), were inactive. Our results indicate that ERG30 can self-oligomerize and both the coiled-coil and the NH2-terminal motifs appear indispensable for oligomerization.
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ERG30 Is Localized to the ER and the Pre-Golgi Intermediates
Intracellular targeting of a protein to a particular organelle often provides a valuable insight into its specific biological role. To examine the subcellular localization of ERG30 in NRK cells, we performed indirect immunofluorescence analysis using affinity-purified polyclonal anti-ERG30 antibodies. As illustrated in Figure 4 A (panels 2 and 4), ERG30 is localized in a juxta-nuclear crescent resembling the Golgi complex and possibly in parts of the ER. No labeling was observed when anti-ERG30 antibodies were incubated with excess recombinant ERG30 in the form of a MBP fusion protein (data not shown). To identify the subcellular localization of ERG30, we performed a double labeling experiment using monoclonal antibodies directed against ßCOP, a commonly used marker for the Golgi, together with anti-ERG30 antibodies. Using confocal microscopy, we found that labeling with the anti-ERG30 antibodies partially coincided with that of ßCOP, indicating that in NRK cells ERG30 is localized in the vicinity of the Golgi complex (Figure 4 A, panels 3 and 4). Treating cells with the drug BFA was shown previously to specifically disrupt the Golgi complex in vivo (
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The intracellular localization of ERG30 was also determined by subcellular fractionation of bovine liver postnuclear supernatant on equilibrium density sucrose gradients (Figure 4 B). Fractions were analyzed by immunoblotting with affinity-purified anti-ERG30 antibodies and with antibodies that recognize either Gos28, a marker of the Golgi apparatus, or PDI, an ER marker. Membranes that were concentrated at the 0.86/1.25 interface of the first gradient were harvested, adjusted to 1.6 M sucrose, and loaded onto the bottom of a second gradient (Figure 4 B). Immunoblot analysis of the second gradient showed that ERG30 predominantly colocalizes with PDI and to some lesser extent with Gos28, indicating that it associates with both Golgi and ER membranes.
ERG30 subcellular localization was further analyzed by immunoelectron microscopy. Silver grains representing sites of immunoreactivity for ERG30 were localized predominantly on the cytoplasmic faces of the RER and on the outer leaflet of the nuclear membraneboth sites of synthesis for membrane and secreted proteins (Figure 4 C, panel 1). Labeled cisternae of RER were found throughout the cytoplasm. Little immunoreactivity was localized to the Golgi apparatus. However, silver grains often were observed on RER cisternae that were very close to the Golgi, and on cisternae that appeared to be transitional between RER and the cis-Golgi (Figure 4 C, panel 2 and inset). Very few silver grains were seen on other structures such as the plasma membrane, mitochondria, or the nucleus. These represented nonspecific labeling since they were not eliminated by absorption of ERG30 antibodies with excess antigen, whereas labeling of the RER, nuclear envelope, and RER-Golgi transitional structures was virtually eliminated (data not shown). Taken together, our results indicate that ERG30 is localized on the cytosolic surface of the ER and on the pre-Golgi intermediates.
ERG30 Is Not Part of the Synaptic SNARE Complex
It has been demonstrated in Aplysia that VAP-33 directly interacts with the synaptic v-SNARE, VAMP, suggesting its involvement in docking and fusion of synaptic vesicles (S. The 20S fusion particle assembly reaction can therefore be used as a cell-free read-out system to test whether candidate proteins are SNAREs, or specifically interact with a SNARE. The results of such a 20S particle experiment, using rat brain membrane extract as a source for SNAREs, are presented in Figure 5 B. Specific (Mg-ATP) and nonspecific elutions (Mg-ATP
S) were analyzed by Western blots. As expected, the known synaptic membrane proteins VAMP, syntaxin, and SNAP-25 assembled into the 20S particle derived from brain membrane extract, but ERG30 did not. These results indicate that ERG30 is not part of the synaptic SNARE complex.
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ERG30 Is Involved in Intra-Golgi Transport
The subcellular distribution of ERG30 indicates that it might play a role early in the secretory pathway. We used the cell-free intra-Golgi transport system (
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The involvement of ERG30 in intra-Golgi transport was also demonstrated by using soluble recombinant MBP-ERG30. The effect of recombinant full-length MBP-ERG30, MBP-ERG30 (coiled-coil) or MBP-ERG30 (
NH2 terminus) mutants on VSV-G transport in vitro was therefore examined. As shown in Figure 6 D, addition of increasing amounts of recombinant MBP-ERG30 fusion protein inhibited transport in vitro by nearly 90%. No inhibition was detected in the presence of equivalent concentrations of MBP-ERG30-
coiled-coil, MBP-ERG30-
NH2 terminus, or MBP-control. This result is in agreement with the fact that only the wild-type ERG30 can interact with the endogenous ERG30, whereas the truncated proteins could not (Figure 3 B). We therefore concluded that inhibition observed in the presence of the soluble MBP-ERG30 results from blocking the function of the endogenous membranal ERG30.
Anti-ERG30 Antibodies Cause an Accumulation of COPI-coated Vesicles
Anti-ERG30 antibodies were used to establish the stage in the transport pathway at which ERG30 is required. For that purpose we used conditions that promote budding of COPI vesicles from Golgi membranes. Under such budding reactions, Golgi membranes were incubated with crude cytosol in the presence or absence of either GTPS or anti-ERG30 antibodies. The Golgi cisternae were then pelleted at 14,000 rpm and the supernatant was fractionated on a sucrose gradient to isolate the COPI-coated vesicles. As shown in Figure 7, incubation of Golgi membranes in the presence of GTP
S resulted in a significant accumulation of COPI-coated vesicles. A significant quantity of COPI vesicles also accumulated in the presence of affinity-purified anti-ERG30 antibodies. When both GTP
S and anti-ERG30 were present in the budding reaction, even more COPI vesicles accumulated (data not shown). Notably, the vesicles accumulated in the presence of anti-ERG30 antibodies appeared more homogeneous in comparison to those accumulated in the presence of GTP
S. These results show that ERG30 is not involved in the budding process but rather in the consumption of these vesicles. Most of the ERG30 remained associated with the Golgi cisternae and did not migrate with the accumulated COPI vesicles (data not shown).
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Discussion |
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We show here that ERG30, the rat homologue of aVAP-33, is ubiquitously expressed and is localized primarily within the ER and the pre-Golgi intermediates. The localization of ERG30 suggests that it functions early in the secretory pathway rather than in the plasma membrane. This is further supported by the observation that ERG30 plays a role in intra-Golgi transport in vitro. We propose that ERG30 represents a novel integral membrane protein family which is involved in the process of vesicle fusion with target membranes.
Previous studies demonstrated that A. californica VAP-33 can interact with VAMP (synaptobrevin) and that it participates in the process of synaptic release (
The accumulation of COPI vesicles caused by anti-ERG30 antibodies coupled with the localization of ERG30 both in the Golgi and ER suggest that this protein is involved in transport between these organelles, possibly in the retrograde direction. The accumulation of COPI vesicles may also indicate that ERG30 is involved in regulating the uncoating of these vesicles. It is not clear why the accumulated vesicles did not uncoat in the presence of the anti-ERG30 antibodies. ERG30 might be involved in triggering ADP-ribosylation factor (ARF)-GTPase activating protein (GAP), an activity which, in turn, stimulates the uncoating process. Additional experiments are needed to determine whether uncoating of vesicles is affected by ERG30 directly, or is coupled to the vesicles' docking; the latter would implicate a role for ERG30 in targeting vesicles to the appropriate organelle.
ERG30 is a type II integral membrane protein, most of which is presented to the cytosol. Examining the protein sequence by the Paircoil program revealed a strong probability for a coiled-coil domain at positions 161194 followed by several basic residues. This structure resembles that of the SNARE family (
We found that ERG30 functions in the early secretory pathway, predominantly within the ER and the Golgi complex. Our immunoelectron microscopy studies indicated that it is found mainly in the ER and in transitional elements found between this organelle and the cis-Golgi. These structures might represent the ER-Golgi intermediate compartment, known also as pre-Golgi intermediates (PGIs). It has been suggested by Balch and co-workers that ER-derived transport vesicles fuse to form vesicular-tubular clusters (VTCs) (
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Acknowledgements |
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We thank Ms. Frida Shimron for her excellent technical assistance. We also thank Drs. Evelyn Ralston and Jung-Hwa Tao Cheng for their advice on immunogold labeling and Ms. Virginia Tanner for the preparation of sections. We are grateful to the members of Dr. Elazar's lab for stimulating discussions and practical help. Z. Elazar is an incumbent of the Shloimo and Michla Tomarin Career Development Chair of Membrane Physiology.
This work was supported in part by the German-Israeli Foundation and the Weizmann Institute Minerva Center (to Z. Elazar), and in part by a grant from the National Institutes of Health (grant CA 72981 to Y. Yarden).
Submitted: January 26, 1999; Revised: May 25, 1999; Accepted: June 3, 1999.
1.used in this paper: ßCOP, ß subunit of COPI; BFA, brefeldin A; COP, cytosolic coat protein; ERG30, ER and Golgi 30-kD protein; MBP, maltose binding protein; NSF, NEM-sensitive fusion protein; PDI, protein disulfide isomerase; SNAP, soluble NSF attachment protein; VAMP, vesicle-associated membrane protein; VAP-33, VAMP-associated protein of 33 kD; VSVG, vesicular stomatitis virus G protein
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