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Identification of Synaptic Proteins and Their Isoform Mrnas In 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 (exocytosis/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 synaptotagmin, synaptobrevin/VAMP VAMP-1 and VAMP-2 are encoded by two distinct genes (13) (vesicle-associated membrane protein), SNAP-25 (synaptosom- and are differentially expressed in the nervous system (14). al-associated protein of 25 kDa), and syntaxin 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. Syntaxins 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 active zone (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 vesicle fusion 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 synaptic vesicle 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 neurons 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,
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