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Assembly and Disassembly of a Ternary Complex of Synaptobrevin, Syntaxin, and SNAP-25 in the Membrane of Synaptic Vesicles

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Assembly and Disassembly of a Ternary Complex of Synaptobrevin, Syntaxin, and SNAP-25 in the Membrane of Synaptic Vesicles Proc. Natl. Acad. Sci. USA Vol. 94, pp. 6197–6201, June 1997 Cell Biology Assembly and disassembly of a ternary complex of synaptobrevin, syntaxin, and SNAP-25 in the membrane of synaptic vesicles HENNING OTTO*, PHYLLIS I. HANSON, AND REINHARD JAHN† Howard Hughes Medical Institute and Departments of Pharmacology and Cell Biology, Yale University School of Medicine, New Haven, CT 06510 Communicated by Vincent T. Marchesi, Yale University School of Medicine, New Haven, CT, April 7, 1997 (received for review February 15, 1997) ABSTRACT The synaptic membrane proteins synapto- we now report that a ternary complex containing syntaxin, brevin, syntaxin, and SNAP-25 form a ternary complex that synaptobrevin, and SNAP-25 can form in the membrane of can be disassembled by the ATPase N-ethylmaleimide- synaptic vesicles in the absence of plasma membranes and that sensitive factor (NSF) in the presence of soluble cofactors this complex is disassembled by NSF and SNAP proteins in an (SNAP proteins). These steps are thought to represent mo- ATP-dependent manner. lecular events involved in docking and subsequent exocytosis of synaptic vesicles. Using two independent and complemen- tary approaches, we now report that such ternary complexes MATERIALS AND METHODS form in the membrane of highly purified and monodisperse Materials. cDNAs for a-SNAP, g-SNAP, and NSF were synaptic vesicles in the absence of the plasma membrane. kindly provided by S. Whiteheart and J. E. Rothman (New Furthermore, the complexes are reversibly dissociated by NSF York). The proteins were expressed in Escherichia coli as and SNAP proteins. Thus, ternary complexes can be assem- His -tagged fusion proteins and were purified on Ni21–NTA– bled and disassembled while all three proteins are anchored 6 agarose columns as described earlier (12). Recombinant L as neighbors in the same membrane, suggesting that NSF is chains of tetanus toxin (TeNT), botulinum neurotoxin (BoNT involved in priming synaptic vesicles for exocytosis. y A), and BoNTyC1 were generous gifts of H. Niemann (Tu¨- bingen, Germany). Synaptobrevin (also referred to as VAMP), SNAP-25, and Disassembly of the Ternary Complex. Synaptic vesicles were syntaxin are crucial components of the exocytotic apparatus in prepared from synaptosomes using chromatography on con- neurons (1–4). Synaptobrevin is exclusively localized to syn- trolled pore glass beads as the last purification step (14). For aptic vesicles whereas syntaxin and SNAP-25 are mainly localized to the neuronal plasma membrane. Any interference a typical disassembly reaction, synaptic vesicle protein (5–25 with the function of these proteins, e.g., proteolysis by clos- mg unless indicated otherwise) was incubated in a 50-ml volume tridial neurotoxins (5, 6) or genetic deletion (7), inhibits containing Hepes–NaOH (pH 7.8; 20 mM), ouabain (1 mM), exocytotic neurotransmitter release. Relatives of these pro- KCl (100 mM), glycerol (1% volyvol), DTT (1 mM), phenyl- teins participate in many intracellular membrane traffic steps methylsulfonyl fluoride (1 mM), a-SNAP (8 mM), g-SNAP (1 in all eukaryotic cells (3), suggesting that membrane fusion is mM), and NSF (0.6 mM) and an ATP regenerating system mediated by a common and conserved mechanism. consisting of ATP (2.5 mM), MgCl2 (2 mM), creatine phos- Despite compelling evidence linking syntaxin, SNAP-25, phate (20 mM), and creatine kinase (0.1 mgyml). In control and synaptobrevin to exocytosis, it is not understood how these and reassembly reactions, 10 mM EDTA was added to chelate proteins operate in the sequence of events that leads to vesicle the Mg21 and thus prevent ATP hydrolysis by NSF. Unless docking and membrane fusion. In detergent extracts, the three indicated otherwise, reactions were carried out for 30 min at proteins form a stable complex that binds the soluble proteins 30°C and terminated by addition of SDS sample buffer con- aybyg-SNAP and N-ethylmaleimide-sensitive factor (NSF), taining 62.5 mM TriszHCl, pH 6.8, 4% (wtyvol) SDS, 10% leading to their designation as v-SNAREs (synaptobrevin, (wtyvol) sucrose, 5% (volyvol) b-mercaptoethanol, and 0.01% vesicular SNAp-REceptors) and t-SNAREs (syntaxin and (wtyvol) bromphenol blue. The samples were then incubated SNAP-25, target membrane SNAp REceptor) (8, 9). ATP- for an additional 30 min at 30°C before separation by SDSy hydrolysis by NSF causes disassembly of the complex. A PAGE and immunoblotting. complex with very similar properties also can be formed from Electrophoretic Procedures. SDSyPAGE and immunoblot- recombinant proteins lacking their transmembrane domains ting were carried out using standard protocols (15, 16). For (10–12). Based on these, observations it has been suggested reelectrophoresis in a second dimension, synaptic vesicle pro- that v-SNARE–t-SNARE interactions are responsible for teins were prepared for SDS PAGE as described above. After docking of vesicles at their target membrane and that the y conformational change caused by NSF contributes to mem- completion of the first dimension, the lane containing the brane fusion (1, 9). The specificity of v-SNARE–t-SNARE separated proteins was excised, soaked for 20 min in 10% pairing would ensure that trafficking vesicles only dock at an (volyvol) acetic acid and 25% (volyvol) isopropanol, briefly appropriate target membrane. These proposals are referred to washed with H2O, and incubated for 20 min in SDS sample as the SNARE hypothesis (1) and have gained wide accep- buffer. After heating for 2 min to 100°C in a microwave oven, tance in the field (1–3). the strip was mounted on top of a 12.5% gel, reelectropho- Recently, we have observed that, unlike other residents of resed, and analyzed by immunoblotting. the presynaptic plasma membrane, significant amounts of syntaxin and SNAP-25 are also localized to synaptic vesicles Abbreviations: BoNT, botulinum neurotoxin; NSF, N-ethylmaleimide- (13). Using two independent and complementary approaches, sensitive factor; SNAP, soluble NSF attachment protein; SNAP-25, synaptosomal-associated protein of 25 kDa; SNARE, SNAP receptor; The publication costs of this article were defrayed in part by page charge TeNT, tetanus toxin; LC, light chain. *Present address: Institut fu¨r Biochemie, Freie Universita¨t Berlin, payment. This article must therefore be hereby marked ‘‘advertisement’’ in Thielallee 63, D-14195 Berlin, Germany. accordance with 18 U.S.C. §1734 solely to indicate this fact. †To whom reprint requests should be addressed. e-mail: © 1997 by The National Academy of Sciences 0027-8424y97y946197-5$2.00y0 [email protected]. 6197 Downloaded by guest on September 28, 2021 6198 Cell Biology: Otto et al. Proc. Natl. Acad. Sci. USA 94 (1997) Proteolysis by Light Chains of Clostridial Neurotoxins. several distinct bands, with Mrs ranging from 60 to '300 that Proteolysis of syntaxin, SNAP-25, or synaptobrevin was initi- reacted with a mix of antibodies directed against synaptobre- ated by adding 2 mM of the appropriate toxin light chain to vin, syntaxin, and SNAP-25 (Fig. 1a). When the samples were each disassembly reaction immediately after adding ayg- incubated at 100°C before electrophoresis, these bands largely SNAP and NSF and before starting the reaction with Mg-ATP. disappeared, and only the monomeric proteins were detected The samples then were treated as described for the disassembly (Fig. 1a). To ensure that these complexes are associated with reaction. Unless otherwise stated, samples were heated for 3 synaptic vesicles, we monitored their distribution in interme- min to 100°C before SDSyPAGE and immunoblotting. diate fractions obtained during vesicle purification. Fig. 1b shows that the complexes coenriched with synaptic vesicles. To RESULTS examine whether each of the high Mr bands contained all three proteins, a gel lane was excised after electrophoresis, heated to SDS-Resistant Ternary Complexes Are Present in Synaptic 100°C to disrupt the complexes, and electrophoresed under Vesicles and Are Disassembled by a-SNAP and NSF. To study identical conditions in a second dimension. Fig. 1c shows that membrane protein complexes, it is customary to solubilize all high Mr complexes contained comparable amounts of membranes in nondenaturing detergents that allow for bio- syntaxin, synaptobrevin, and SNAP-25, confirming observa- chemical analysis of protein complexes. However, in prelimi- tions by us and others that only ternary complexes containing nary experiments, we found that stable ternary complexes of all three proteins are resistant to SDS treatment. To ensure synaptobrevin, syntaxin, and SNAP-25 assemble after solubi- that the high Mr complexes did not form during or after SDS lization of brain membranes in nonionic detergent and thus do treatment, radioactively labeled syntaxin, generated by in vitro not report the status of the proteins before solubilization translation, was added together with SDS to the sample before (unpublished observations). To avoid assembly after solubili- electrophoresis. The radioactively labeled syntaxin was not zation, we took advantage of the recent observation made by incorporated into the high Mr complexes (not shown). Niemann and coworkers that ternary SNARE complexes To examine whether NSF can disassemble the vesicular partially resist treatment with SDS (10). Large forms of these ternary complex, synaptic vesicles were incubated with recom- complexes appear as heat-sensitive, distinct bands of high binant NSF, a-SNAP, g-SNAP, and ATP in the absence or molecular mass when the samples are separated by SDSy presence of EDTA, which complexes Mg21 and thus prevents PAGE (10). These SDS-resistant forms cannot assemble after ATP cleavage. As shown in Fig. 2 (control 2), NSF and addition of SDS (see below) and thus represent ternary ayg-SNAP had no influence on the SDS-resistant complexes complex preexisting in the membrane before solubilization. when ATP hydrolysis was prevented. In the presence of Mg21, Synaptic vesicles were purified according to established however, the amount of these complexes was markedly re- procedures using chromatography on controlled pore glass duced (Fig. 2, disassembly). Subsequent addition of either beads as the last purification step.
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