Proc. Natl. Acad. Sci. USA Vol. 91, pp. 12487-12491, December 1994 Cell Biology Identification of synaptic proteins and their isoform mRNAs in compartments of pancreatic endocrine cells (/secretion/insulin/diabetes) GUNILLA JACOBSSON*, ANDREW J. BEANt, RICHARD H. SCHELLERt, LISA JUNTTI-BERGGRENt, JUDE T. DEENEYt, PER-OLOF BERGGRENt AND BJORN MEISTER*§ *Department of Neuroscience and tRolf Luft's Center for Diabetes Research, Department of Molecular Medicine, Karolinska Institute, S-171 77 Stockholm, Sweden; and tDepartment of Molecular and Cellular Physiology, Howard Hughes Medical Institute, Beckman Center, Stanford University, Stanford, CA 94305 Communicated by Tomas Hokfelt, August 30, 1994

ABSTRACT Several proteins that are of importance for clostridial neurotoxins, including tetanus toxin and botuli- membrane trafficking in the nerve terminal have recently been num neurotoxin B, whereas botulinum neurotoxins D and F characterized. We have used Western blot and immunohis- are capable of cleaving both forms of VAMP (10-12). tochemistry to show that , /VAMP VAMP-1 and VAMP-2 are encoded by two distinct genes (13) (vesicle-associated ), SNAP-25 (synaptosom- and are differentially expressed in the (14). al-associated protein of 25 kDa), and proteins are Cellubrevin is a homologue of VAMP, which is present in a present in cells of the islets of Langerhans in the endocrine wide variety of tissues and may be a membrane trafficking pancreas. Synaptotagmin-like immunoreactivity (-LI) was lo- protein of a constitutively recycling pathway (15). calized to granules within the cytoplasm of a few endocrine cells In contrast to synaptotagmin and VAMP, the synaptoso- located in the periphery of the islets, identified as somatostatin- mal-associated protein of 25 kDa (SNAP-25) is located at the containing cells, and in many nerve fibers within the islets. presynaptic plasma membrane (16, 17). It is a substrate for VAMP-LI was seen in granules of virtually all pancreatic islet palmitoylation, implying that fatty acylation may be involved cells and also in nerve fibers. SNAP-25-LI and syntaxin-LI were in membrane targeting ofthe protein. SNAP-25 is cleaved by predominantly present in the plasma membrane ofthe endocrine botulinum neurotoxins A and E (11, 12, 18). SNAP-25 is cells, including insulin-producing (I cells. In situ hybridization, proposed to be a component of a 20S multiprotein complex using isoform-specific oligonucleotide probes, detected that mediates vesicle docking and fusion (19, 20). VAMP-2, cellubrevin, SNAP-25, syntaxin 1A, 4, and 5, and The syntaxin family of vesicular transport receptors (21) munc-18 mRNAs in isolated pancreatic islets and in insulin- consists of proteins that have hydrophobic domains that producing cells. The results show the presence of several syn- anchor the proteins to the cytoplasmic surface of the plasma aptic proteins at protein and mRNA levels in pancreatic islet membrane. interact with synaptotagmin (22), cells, suggesting that they may have specific roles in the molec- N-ethylmaleimide-sensitive factor (NSF), soluble NSF- ular regulation of exocytosis also in insulin-secreting cells. attachment proteins (SNAPs) (19, 20), and the N-type ofCa2+ channels (22). Consequently, syntaxins have been suggested Release ofneurotransmitters from nerve terminals occurs via to be involved in the docking and/orfusion ofsynaptic vesicles regulated exocytosis of synaptic vesicles. At the presynaptic with the plasma membrane (21, 22). A brain protein of67 kDa nerve terminal, vesicles interact with the cytoskeleton and that stably binds to the N terminus of syntaxin was recently with soluble vesicle-binding proteins prior to docking at identified as a mammalian homologue of the Caenorhabditis specialized membrane sites known as the (1). elegans gene unc-18 and termed munc-18 (23). The protein After voltage-gated Ca2+ influx, the vesicle and the plasma munc-18 (also termed n-secl) has been suggested to represent membrane fuse, releasing the vesicle contents into the syn- a novel component of the VAMP/synaptotagmin/syntaxin aptic cleft. synaptic complex (23). A class of proteins that are involved in the targeted There is evidence suggesting that the molecular machinery movement and fusion reactions of the mem- for secretion is conserved in different cell types and in brane have recently been identified (see refs. 2 and 3). different species-i.e., the molecular components of secre- Synaptotagmin (p65) is an integral membrane protein of tion from endocrine cells of the pancreas may share many synaptic vesicles, which has a cytoplasmic Ca2+-binding attributes in common with those identified in and domain. Ca2+ binding increases the ability of synaptotagmin there are several similarities between secretory mechanisms to bind phospholipids, suggesting a role in vesicle-plasma in yeast and mammalian cells (2, 3, 24). To identify synaptic membrane fusion (4). Introduction of anti-synaptotagmin proteins in cells of the endocrine pancreas-i.e., islets of antibodies orfragments ofsynaptotagmin fusion proteins into Langerhans-we have used Western blot, immunohis- cultured cells impairs exocytosis (5). In vitro, synaptotagmin tochemistry, and in situ hybridization. binds to several plasma membrane proteins, including syn- taxins (see below), neurexins, and Ca2+ channels (6). MATERIALS AND METHODS The vesicle-associated membrane proteins VAMP-1 (syn- Western Blot. Previously dissected tissues or cultured cells aptobrevin 1) and VAMP-2 (synaptobrevin 2) are homolo- were homogenized in Hepes buffer (10 mM Hepes-KOH, pH gous proteins of 18 and 17 kDa, respectively, which are 7.5/2.5 mM KOAc/1 mM MgCl2/0.1 mM EGTA/0.03 M anchored to the cytoplasmic surface of the synaptic vesicles by a carboxyl-terminal transmembrane domain (7-9). Abbreviations: VAMP, vesicle-associated membrane protein; -LI, VAMP-2 has been identified as a selective substrate for -like immunoreactivity; NSF, N-ethylmalemide-sensitive factor; SNAP, soluble NSF-attachment protein; SNAP-25, synaptosomal- associated protein of 25 kDa. The publication costs of this article were defrayed in part by page charge §To whom reprint requests should be addressed at: Department of payment. This article must therefore be hereby marked "advertisement" Neuroscience, The Berzelius Laboratory, Karolinska Institute, 171 in accordance with 18 U.S.C. §1734 solely to indicate this fact. 77 Stockholm, Sweden.

12487 Downloaded by guest on September 30, 2021 12488 Cell Biology: Jacobsson et al. Proc. Natl. Acad. Sci. USA 91 (1994) phenylmethylsulfonyl fluoride) with a ground glass Duvall. Triton X-100 was added to the homogenates to a concentra- tion of 2% and they were incubated at 4°C for 60 min. Insoluble material was removed by centrifugation at 15,000 CL rpm for 15 min, and protein concentrations were estimated 0 0 (Bio-Rad protein assay). Proteins were resolved by using U SDS/PAGE and electrophoretically transferred to nitrocel- lulose in 384 mM glycine/50 mM Tris/10% MeOH. Nitro- C cellulose fiters were cut into strips based on the movement in 3O o z of prestained size markers run in lanes adjacent to the i0 0 M samples. The filters were incubated with blocking buffer [5% synaptotagmin dry milk in phosphate-buffered saline (PBS)] for 60 min and then the appropriate filter strip was incubated with either syntaxin non-isoform-specific antiserum to VAMP (1:1000; kind gift of W. S. Trimble), SNAP-25 (1:1000; SMI-81, Sternberger _ S~~~NAP-25 Monoclonals, Baltimore), syntaxin (1:1000) (25), or synap- totagmin (1:4000) (26) for 18 hr at 40C. Filters were washed VAMP (10 mM Tris/150 mM NaCl/0.5% Tween 20) and incubated with horseradish peroxidase-conjugated anti-rabbit or anti- FIG. 1. Western blot analysis of 10 ug of protein from dissected mouse secondary antisera (4°C for 60 min). After washing as whole rat pancreas, ob/ob mouse pancreas, ob/ob mouse cells, or before, bands were visualized using chemiluminescence RIN m5F cells was separated by SDS/PAGE and transferred to (Amersham ECL). Filters were exposed for 0.5-3 min. nitrocellulose. The blot was probed with antisera to synaptotagmin, Immunohistochemistry. Male Sprague-Dawley rats (150- syntaxin, SNAP-25, and VAMP and detected using ECL (Amer- 200 g; B & K Universal, Stockholm) and ob/ob were anes- sham). Bands representing 65 kDa (synaptotagmin), 35 kDa (syn- taxin), 25 kDa (SNAP-25), and 18 kDa (VAMP) are seen. Low levels thetized with sodium pentobarbital (Mebumal; 40 mg/kg i.p.) of synaptotagmin are found in pancreas and cells from ob/ob mice, and perfused via the ascending aorta with 50 ml of Ca2+-free and even lower levels are detected in RIN m5F cells. No synap- Tyrode's solution followed by a formalin/picric acid fixative totagmin protein could be detected in rat pancreas. Syntaxin and (4% paraformaldehyde/0.4% picric acid in 0.16 M phosphate SNAP-25 are demonstrated in all tissues, with particularly high levels buffer, pH 6.9) for 6 min. The pancreas was rapidly dissected in RIN m5F cells. VAMP is detected in all tissues. out, postfixed in the same fixative for 90 min, and rinsed for at least 24 hr in 0.1 M phosphate buffer (pH 7.4) containing and minimal homology with GenBank-entered nucleotide 10% sucrose. Sections were cut (10 ,um) in a cryostat and sequences and were synthesized (Scandinavian Gene Syn- incubated at 4°C overnight with rabbit non-isoform-specific thesis, Koping, Sweden) complementary to nucleotides antiserum to synaptotagmin (1:200), rabbit antiserum to 2774-2823 of synaptotagmin A (29) and 1783-1830 of synap- VAMP (1:400), mouse monoclonal antibodies to SNAP-25 totagmin B (30), 271-318 of VAMP-1 and 1279-1326 of (1:200), and rabbit antiserum to syntaxin (1:200). Sections VAMP-2 (8), 1570-1617 of cellubrevin (15), 61-106 of were also double-labeled by combining antisera against syn- SNAP-25 (16), 286-333 of syntaxin 1A, 1036-1084 of syn- aptotagmin, VAMP, SNAP-25, or syntaxin with guinea-pig taxin 1B, 425-477 of syntaxin 2, 581-629 of , 25-72 antiserum against insulin (1:200, UCB-Bioproducts) or of syntaxin 4, and 1108-1167 of syntaxin 5 (21), and 958-1005 mouse monoclonal antibodies to somatostatin (1:250; Mab of munc-18 (23) mRNAs. 10; kindly provided by J. Brown, University of British The probes were 3' end-labeled with [a-[35S]thio]dATP Columbia, Vancouver). After rinsing in PBS, the sections (NEN), using terminal deoxynucleotidyltransferase (Am- were incubated for 30 min at 37°C with fluorescein isothio- ersham) and purified using Nensorb 20 columns (NEN). cyanate (FITC)-conjugated donkey anti-rabbit (1:40) or FITC-conjugated sheep anti-mouse (1:20) secondary anti- bodies. For the double-stained sections, lissamine/ rhodamine (LRSC)-conjugated goat anti-guinea pig (1:40) or goat anti-mouse (1:40) was used. After mounting in a mixture or PBS and glycerol (1:3) containing 0.1% p-phenylenedi- amine, the sections were examined in a Nikon Microphot epifluorescence microscope. Sections were also examined in a Bio-Rad MRC-600 laser scanning confocal imaging system equipped with a krypton/argon mixed gas laser and a Nikon Optiphot II microscope. The standard blue excitation high sensitivity (BHS) single channel filter block with an excita- tion filter (488 DF 10 for FITC-induced fluorescence and a 568 DF 10 for LRSC-induced fluorescence) was used to examine the immunoreactivity. The images were printed using a Tektronix Phaser IIsd printer. In Situ Hybridization. Pancreatic islets from 12-month-old ob/ob mice (C57BL/6J) of both sexes were isolated by a collagenase technique (27). RIN m5F cells, a clonal insulin- producing cell line, were established from a transplantable rat islet tumor (28). The tissues were fixed in 4% paraformalde- hyde for 1 hr, washed with PBS, and frozen on dry ice. Islets and RIN m5F cells were sectioned in Microfuge tubes at 14 FIG. 2. Immunofluorescence photographs of a section of a pan- ,am in a cryostat and thaw-mounted onto precleaned ProbeOn creatic islet after incubation with antiserum to synaptotagmin. Syn- microscope slides (Fisher Scientific). Rat brain and newborn aptotagmin-LI is mainly present in nerve fibers throughout the islet mice were frozen and sectioned for control purpose. Oligo- but is also in some peripherally located cell bodies (arrows). (Bar = nucleotide probes were designed for an optimal G-C content 50 Am). Downloaded by guest on September 30, 2021 Cell Biology: Jacobsson et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12489 Tissue sections were air-dried and incubated for 16 hr at apposed to a-max autoradiography film (Amersham) at 42°C with 106 cpm of the labeled probe in a hybridization -20°C. After 1-6 weeks of exposure, the films were devel- solution containing 50% deionized formamide, 4 x standard oped with Kodak LX 24 for 4 min and fixed for 15 min with saline citrate (SSC) (lx SSC = 0.15 M NaCl/0.015 M Kodak AL 4. In addition to film autoradiography, the sodium citrate), lx Denhardt's solution (0.02% bovine sections were dipped in Kodak NTB2 autoradiography serum albumin/0.02% Ficoll/0.02% polyvinylpyrrolidone), emulsion in distilled water, exposed for 3-10 weeks at 40C, 1% N-lauroylsarcosine, 0.02 M NaPO4 (pH 7.0), 10% dex- developed in Kodak D19 (3 min), and fixed in Kodak 3000 tran sulfate, 500 ,g of denatured salmon sperm DNA per (6-7 min). Sections were rinsed in distilled water and ml, and 200 mM dithiothreitol. After hybridization, the counterstained with hematoxylin/eosin. All sections were sections were rinsed in lx SSC at 55°C for 60 min, rinsed examined under bright- or dark-field illumination, using a in distilled water, dehydrated in 60% and 95% ethanol, and Nikon Microphot-FX microscope.

FIG. 3. Immunofluorescence photo- graphs obtained by confocal laser mi- croscopy of sections of pancreatic islets after double-labeling. Rabbit antiserum to synaptotagmin (A) was combined with mouse monoclonal antibodies to somato- statin (B). Rabbit antiserum to VAMP (C), mouse monoclonal antibodies to SNAP-25 (E), and rabbit antiserum to syntaxin (G) were combined with guinea pig antiserum to insulin (D, F, and H). Synaptotagmin-LI is present in the cyto- plasm of cell bodies located in the pe- riphery of the islets and they all contain somatostatin-LI (cf. arrows in A and B). VAMP-LI is present in most islet cells and many contain insulin-LI (cf. C and D); however, there are also VAMP- positive/insulin-negative cells (cf. small curved arrows in C and D). SNAP-25-LI (E) and syntaxin-LI (G) are seen in the plasma membrane of many cells in the islet and most ofthem contain insulin (cf. arrows in E and G and F and H). (Bar = 10 ,um). Downloaded by guest on September 30, 2021 12490 Cell Biology: Jacobsson et al. Proc. Natl. Acad Sci. USA 91 (1994) RESULTS 3G). Syntaxin-LI was demonstrated in many 8 cells (cf. Fig. 3G with 3H) and also in 8 cells (data not shown). Western Blot. Western blot analysis oftissue material from In Situ Hybridization. Hybridization oftissue material from whole rat and mouse pancreas, isolated pancreatic islets from islets isolated from ob/ob mice and from RIN m5F cells ob/ob mice, and RIN m5F cells showed bands of 65, 35, 25, revealed hybridization signal using probes to VAMP-2 (Fig. and 18 kDa corresponding to synaptotagmin, syntaxin, 4A), cellubrevin (Fig. 4B), SNAP-25 (Fig. 4C), syntaxin 1A SNAP-25, and VAMP, respectively (Fig. 1). There was only (Fig. 4D), syntaxin 4 (Fig. 4E), syntaxin 5 (Fig. 4F), and a weak band for synaptotagmin in whole pancreas from munc-18 (Fig. 4G) (see Table 1). No detectable hybridization ob/ob mice and very weak bands for synaptotagmin in signal could be seen in islet cells from ob/ob mouse or in RIN isolated islets from ob/ob mice and RIN m5F cells (Fig. 1). m5F cells after hybridization with probes to synaptotagmin There was no detectable band for synaptotagmin in whole rat A, synaptotagmin B, VAMP-1, and syntaxin 1B, 2, and 3 pancreas (Fig. 1). (Table 1), which in parallel experiments all gave strong Immunohistochemistry. Within the endocrine pancreas of labeling in coronal sections of rat brain, spinal cord, and rat and ob/ob and normal mouse, synaptotagmin-like reac- sagittal sections of whole neonatal mouse (data not shown). tivity (-LI) was demonstrated in a few cells located in the No hybridization signal could be observed after addition of periphery of the islets (Figs. 2 and 3A), identified as - tostatin-containing 8 cells by double-labeling (cf. Fig. 3A with an excess (100 times) ofthe respective unlabeled probe to the 3B). There was no evidence for the presence of synaptotag- radiolabeled probe (data not shown). min in insulin-containing cells. Many synaptotagmim- immunoreactive nerve fibers and terminals innervating pan- DISCUSSION creatic islets were also seen (Fig. 2). With the antiserum to VAMP, immunoreactivity was demonstrated in virtually all The present study shows that several synaptic proteins that endocrine cells but also in nerve fibers surrounding and function in neuronal vesicle targeting and fusion are also extending into the islet (Fig. 3C). Double-labeling showed present in compartments of pancreatic endocrine cells. This colocalization ofVAMP with insulin (cf. Fig. 3C with 3D) and is in agreement with the demonstration that regulated secre- somatostatin (data not shown). Synaptotagmin-LI (Fig. 3A) tory pathways of neurons and endocrine cells show similar- and VAMP-LI (Fig. 3C) had a granular appearance, as ities (24) and that many secretory proteins are well conserved revealed by confocal laser microscopy. SNAP-25-LI was from yeast to man (2, 3). demonstrated in virtually all endocrine cells (Fig. 3C) and in Synaptotagmin has previously been reported to be present nerve fibers surrounding vessels and going into the islets in cells of the endocrine pancreas, identified as presumable (data not shown). Within the cells, the immunoreactivity was insulin-secreting cells (26). In this study we did not detect localized mainly to the plasma membrane but also to a few synaptotagmin-LI in insulin-producing cells. Instead, the granules located in a region located close to the cell nucleus, immunoreactivity was demonstrated in the somatostatin- presumably representing the (Fig. 3E). containing 8 cells. VAMP has also been shown to be present SNAP-25-LI was demonstrated in many insulin-containing , in the endocrine pancreas; however, its colocalization with cells (cf. Fig. 3E and 3F) but also in somatostatin-containing other pancreatic hormones has not been clarified (9). 8 cells (data not shown). Syntaxin-LI was seen in virtually all Our results from Western blot and immunohistochemistry endocrine cells as well as in nerve fibers surrounding vessels show that insulin-producing cells contain VAMP, SNAP-25, and going into islets (Fig. 3G). Within the cells, syntaxin-LI and syntaxin but lack synaptotagmin protein. With the anti- was localized to the plasma membrane and in the perinuclear bodies used, it was not possible to discriminate between region, presumably representing the Golgi complex (Fig. different forms ofthe synaptic proteins. However, the in situ

4. Bright-field photomi- crographs (A-D a'nd G) and film autoradiograms (E and F) after in situ hybridization of sections of RIN mSF cells (A, B, D, and G) and isolated pancreatic islets from ob/ob mice .(C, E, and F) with oligonucleotide probes to VAMP- 2 (A), celiubrevin (B), SNAP-25 (C), syntaxin lA (D), syntaxin 4 (E), syntaxin 5 (F), and munc-18 (G). Note the presence of silver A 09 aa- ~grains overlying individual cells in emulsion-dipped and hematoxy- lin/eosin-counterstained sections (A-D and G) and the presence of hybridization signal in autoradio- grams (E and F) (Bars in A-D and G 50 pm and in E and F = 1 se...... e. Wmm.) Downloaded by guest on September 30, 2021 Cell Biology: Jacobsson et al. Proc. Natl. Acad. Sci. USA 91 (1994) 12491 Table 1. Summary of the presence (+) or absence (-) of detectable insulin release and perhaps be involved in the pathogenesis of mRNA for synaptic proteins in pancreatic endocrine cells as non-insulin-dependent diabetes mellitus. demonstrated by in situ hybridization This research was supported by grants from the Swedish Medical ob/ob Research Council (04X-10358, 03X-09890, 19X-00034), Fredrik and mouse RIN Ingrid Thurings Stiftelse, Ake Wibergs Stiftelse, Magnus Bergvalls mRNA ,B cells m5F Stiftelse, U.S. Public Health Service Grants DK-35914 and DK- Synaptotagmin A 46200, the Bank of Sweden Tercentenary Foundation, the Swedish Diabetes Foundation, the Novo Nordisk Foundation, and funds from Synaptotagmin B the Karolinska Institute. VAMP-1 VAMP-2 + 1. Trimble, W. S., Linial, M. & Scheller, R. H. (1991) Annu. Rev. Cellubrevin + Neurosci. 14, 93-122. SNAP-25 2. Bennett, M. K. & Scheller, R. H. (1993) Proc. Natl. Acad. Sci. Syntaxin 1A USA 90, 2559-2563. Syntaxin 1B 3. Ferro-Novick, S. & Jahn, R. (1994) Nature (London) 370, Syntaxin 2 191-193. 4. Brose, N., Petrenko, A. G., Sudhof, T. C. & Jahn, R. (1992) Syntaxin 3 Science 256, 1021-1025. Syntaxin 4 + + 5. Elferink, L. A., Peterson, M. R. & Scheller, R. H. (1993) Cell Syntaxin 5 72, 153-159. munc-18 6. Popov, S. V. & Poo, M.-M. (1993) Cell 73, 1247-1249. 7. Trimble, W. S., Cowan, D. M. & Scheller, R. H. (1988) Proc. RIN m5F, rat insulinoma cell line mSF. Natl. Acad. Sci. USA 85, 4538-4542. 8. Elferink, L. A., Trimble, W. S. & Scheller, R. H. (1989w J. hybridization experiments, using isoform-specific probes and Biol. Chem. 264, 11061-11064. isolated -cells, revealed that insulin-secreting cells contain 9. Baumert, M., Maycox, P. R., Navone, F., De Camilli, P. & VAMP-2, cellubrevin, SNAP-25, syntaxin 1A, 4, and 5, and Jahn, R. (1989) EMBO J. 8, 379-384. munc-18 mRNA. 10. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polv- erino de Laureto, P. & DasGupta, B. R. (1992) Nature (Lon- Using confocal microscopy it was evident that synaptotag- don) 359, 832-835. min-LI and VAMP-LI were granular and that SNAP-25-LI 11. Schiavo, G., Rossetto, O., Catsicas, S., Polverino de Laureto, and syntaxin-LI were located mainly to the plasma mem- P., DasGupta, B. R., Benfenati, F. & Montecucco, C. (1993) J. brane. These findings are in congruence with the anticipated Biol. Chem. 268, 23784-23787. location sites for each individual protein. Although VAMP 12. Huttner, W. B. (1993) Nature (London) 365, 104-105. 13. Archer, B. T., Ozcelik, T., Jahn, R., Francke, U. & Suidhof, was demonstrated in insulin-containing cells, it remains to be T. C. (1990) J. Biol. Chem. 265, 17267-17273. shown by ultrastructural techniques whether VAMP is pre- 14. Trimble, W. S., Gray, T. S., Elferink, L. A., Wilson, M. C. & sent in the insulin-containing vesicles. Scheller, R. H. (1990) J. Neurosci. 10, 1380-1387. According to the "SNARE hypothesis" (20), in neurons 15. McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., the soluble ATPase NSF binds to membranes when one or Binz, T., Niemann, H., Jahn, R. & Sudhof, T. C. (1993) Nature (London) 364, 346-349. more SNAPs are bound to their receptors (SNAP receptors, 16. Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., SNAREs). This interaction produces a 20S multi-subunit Billingsley, M., Bloom, F. E. & Wilson, W. C. (1989) J. Cell particle that contains VAMP, syntaxin, and SNAP-25 (19). It Biol. 109, 3039-3052. has been proposed that an integral membrane protein resident 17. Hess, D. T., Slater, T. M., Wilson, M. C. & Skene, J. H. P. on the transport vesicle (v-SNARE) pairs with the receptor (1992) J. Neurosci. 12, 4634-4641. on the to initiate In 18. Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De target membrane (t-SNARE) fusion (19). Camilli, P., Sudhof, T. C., Niemann, H. & Jahn, R. (1993) neurons, the v-SNARE and t-SNARE correspond to VAMP Nature (London) 365, 160-163. and syntaxin, respectively. The t-SNARE, which is probably 19. Sollner, T., Whitehart, S. W., Brunner, M., Erdjument-Brom- complexed with another membrane-bound protein (t- age, H., Geromanos, S., Tempst, P. & Rothman, J. E. (1993) SNARE "assistant"), which in the nerve terminal is repre- Nature (London) 362, 318-323. 20. Sollner, T., Bennett, M. K., Whitehart, S. W., Scheller, R. H. sented by SNAP-25, interacts only with its matching & Rothman, J. E. (1993) Cell 75, 409-418. v-SNARE, which ensures that the transport vesicle fuses 21. Bennet, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peter- with the correct acceptor compartment. son, K., Fleming, A. M., Hazuka, C. D. & Scheller, R. H. In the present study we have identified a number of (1993) Cell 74, 863-873. exocytotic synaptic proteins in compartments of pancreatic 22. Bennett, M. K., Calakos, N. & Scheller, R. H. 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