Nicole Calakos and Richard H. SchellerS. From the Department of Molecular and Cellular. Physiology, Howard Hughes Medical Institute, Stanford. University ...
Communication
THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 269, No. 40, Issue of October 7, pp. 24534-24537, 1994 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.
Vesicle-associatedMembrane Protein and SynaptophysinAre Associated on the Synaptic Vesicle”
these proteins associate with cytosolic proteins known to be important for vesicle fusion in Golgi transport assays: soluble NSF attachment proteins and N-ethylmaleimide-sensitive factor (1). These complexes are thoughtt o represent thebiochemical machinery responsible for vesicle docking and fusion. There is widespread support for the involvement of each of these proteins invesicle docking and fusion. Current issues crucial to of vesicle docking and fusion are (Received for publication, June 24, 1994, and in revised form, understanding the mechanism August 12, 1994) the temporalassembly and disassembly and physiologic modulators of this complex. In addition, there maybe other compoNicole Calakos and RichardH. SchellerS nents not yet identified, as well as distinct biochemical comFrom the Department of Molecular and Cellular plexes involved in other aspectsof synaptic vesicle trafficking. Physiology, Howard Hughes Medical Institute, Stanford Here we report the identification of a direct interaction beUniversity Medical Center, Stanford, California, 94305 tween two integral membrane proteinsof the synapticvesicle. The synaptic vesicle membrane protein VAMP (yesi- One of these proteins,VAMP, is a constituent of the 7 and 20 S cle-associatedmembrane protein or synaptobrevin) has complexes, while the other, synaptophysin, is not. been implicatedin synaptic vesicle docking and fusion. VAMPS comprise a family of proteins conserved among diSynaptophysin (p38),also a synaptic vesicle membrane verse organisms (2, 3). VAMP (also synaptobrevin) was origiprotein, has fourtransmembranedomainsandmay nally identified as an 18-kDa synaptic vesicle protein expressed function as a gap junction-like pore or channel. Herespecifically we within the nervous system (4, 5). Currently, there VAMP are multipleisoforms expressed in the nervous system(VAMP report evidence for a direct interaction between andsynaptophysin using chemicalcross-linkingfol1 and 2) (6, 71, isoforms with wide tissue expression (cellubrelowed by the identification of immunoreactive protein vin) (8), and VAMP-immunoreactive proteins identified in adicomplexes.A prominent complexof 56 kDa was foundto pocytes and pancreatic exocrine cells (9, 10). Homologs have consist of VAMP and synaptophysin. Furthermore, we been identified in organisms as divergent as yeast and humans demonstrate that this VAMP-synaptophysin complexis (3, 11, 12). Mutants of the yeast VAMP homologs, SNCl and enriched in the synaptic vesicle fraction of rat brain,is SNC2, are defective in secretion (11, 13),suggesting a n essenindependent of detergent solubilization, and is present tial role for VAMP in constitutive secretion. Selective removal in PC12 cells subjected to in vivo cross-linking. of VAMPS from synaptic vesicles by the zinc protease tetanus toxin blocks neurotransmitter release (14, 151, suggesting an important role for VAMP in the regulated secretion of neuroThe basis of chemical neurotransmission is the regulated transmitters. Recent experiments have shown that VAMP prosecretion of neurotransmitter by synaptic vesicle-mediated exo- teins interact specifically with distinct isoforms of syntaxin, a cytosis. Efforts to understand the molecular mechanism of this family of proteins residing on cellular membranes with which process have included the identification and characterizationof transport vesicles fuse (16). The interactionbetween these two the protein constituents of the synaptic vesicle membrane. proteins mayprovide a mechanism to ensurespecific targeting These proteins are likely to be central mediators of the func- of transport vesicles to their appropriate target membranes tions of synaptic vesicles, including biogenesis, docking, fusion, (16, 17). and recycling within nerve terminals.Additional synaptic vesSynaptophysin is a synaptic vesicle membrane protein with icle proteins have served as tools to identify interacting pro- homologs identified in higher vertebrates (18-21). A synaptoteins in the cytosol and nerve terminal membrane. It has re- physin homolog with non-neuronal tissueexpression has been cently become apparent that many of the proteinsinvolved in described recently (22). Synaptophysin has four transmemsynaptic vesicle trafficking have homologs involved in intracel- brane domains and forms a homo-oligomer (23-25) that may lular vesicular trafficking in a variety of systems. While syn- function as a channel (24). A role for synaptophysin in neuroaptic vesicle trafficking utilizes components necessary for the transmitter secretion has been suggested by experiments using intracellular vesicular trafficking of a wide variety of vesicles, antibodies or antisenseoligonucleotides in Xenopus oocytes or it must also have specialized components that allow neurose- embryos (26, 27). cretion to occur in a regulated fashion with extreme rapidity VAMP, synaptophysin, and two other synaptic vesicle proand calcium dependence. A 7 S complex of proteins has been teins (synaptotagmin andSV2) have been shown tocoimmunoisolatedwhichincludes 1) thesynaptic vesicle proteins, 2 ) precipitate from detergent-solubilized rat brain synaptic VAMP,’ synaptotagmin, and the plasma membrane proteins, vesicles (28). In this study we have used homobifunctional, and 3) syntaxin and synapse-associated protein 25. Three of primary amine-reactive chemical cross-linkers to identify pro* This workwas supported by the National Institute of Mental Health teins that are directly interacting. and NIGMS, National Institutes of Health. The costs of publication of EXPERIMENTAL PROCEDURES this article were defrayed in part by the payment of page charges. This Materials-Octyl glucoside, phenylmethylsulfonyl fluoride, and article must therefore be hereby marked “aduertisement”in accordance SY38 monoclonal antibody were obtained from Boehringer Mannheim. with 18 U.S.C. Section 1734 solely to indicate this fact. (PAGE) molecular $ To whom correspondence should be addressed. Tel.: 415-723-9075; Prestained polyacrylamide gel electrophoresis weight standards were purchased from Life Technologies, Inc. All cenFax: 415-725-4436. The abbreviations used are: VAMP, vesicle-associated membrane trifuge rotors were from Beckman Instruments. Reagent-grade chemiprotein; p38, synaptophysin; PAGE, polyacrylamidegel electrophoresis; cals were obtained from Sigma TritonX-100 and electrophoresis-grade DSS, disuccinimidyl suberate;DTSSP, 3,3‘-dithiobis(su1fosuccinimidy1- reagents were purchased fromBio-Rad.Nitrocellulose was obtained from Schleicher & Schuell. propionate).
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VAMP and Synaptophysin Association
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Rat Brain Preparations-Brain homogenates were prepared from A MW B MW freshly sacrificed Sprague-Dawley rats. Tissue was homogenized by 8 1 2 3 4 ( k W 1 2 3 4 5 6 7 8 (kDa) up and down strokes with a Teflon Dounce tissue homogenizer in su-200 200 crose buffer (0.32 M sucrose, 10 mM Hepes-KOH (pH 7.6), 1 mM EGTA, 0.1 mM EDTA, 0.3 mM phenylmethylsulfonyl fluoride). The homogenate -97 -97 was subjected to centrifugation for 10 min at 2,500 rpm (1,000 x g) in a -68 v+s> JS-13.1 rotor. The supernatant (5 mg/ml protein concentration) was v+s> -43 -43 S>( solubilized in 2% Triton X-100 (v/v) for 1 h a t 4 "C. For Fig. lB, the rat brain homogenate was stored at -20 "C for 3 days before use. Upon -29 29 thawing the homogenate was solubilized in 2% Triton X-100 (v/v) a t 5 mg/ml protein concentration. Rat brain fractions were isolated as described previously from steps FIG.1.A cross-linked56-kDa complexcontains both VAMP and of a modified synaptic vesicle preparation (28, 29) using freshly sacri- synaptophysin (p38) immunoreactivity. Triton X-100-solubilized ficed Sprague-Dawley rats. The cytosol-enriched fraction 6 2 ) is the crude rat brain homogenates were cross-linked with DTSSP. Panel A, supernatant obtained following centrifugation of the postnuclear super- samples were incubated with 0, 0.5, 1, or 2 mhl DTSSP (lanes 1 4 , natant at 12,500 rpm (25,000 x g) for 10 min in a JS-13.1 rotor. The respectively). 100 pgof protein was electrophoresed on 10% nonreducsynaptosomal membrane fraction (LP1) is the pellet obtained following ing polyacrylamide gel followed by anti-VAMP immunoblotting. Panel centrifugation of the hypotonically-lysed synaptosomes a t 15,000 rpm B , lanes 1 and 2, anti-synaptophysin immunoreactivity of 0 nd 2 mhf 3 and 4, (42,000 x g) for 16 min inan SW28 rotor. The synaptic vesicle fraction DTSSP incubations, respectively (nonreduced samples). Lanes (LP2) is the pellet recovered from the post-LP1 supernatant by centrifu- anti-VAMP immunoreactivity of 0 and 2 mM DTSSP incubations, respectively (nonreduced samples). Lanes 5-8, same a s lanes 1-4, respecgation at 28,000 rpm (141,000x g) for 4 h in anSW28 rotor. tively, but reduced with 100 mM Dl". On the nonreducing gel, a dimer Chemical Cross-linking-250 pl of 5 mg/ml protein extracts were 0 and 2 mM DTSSP samplesupon longer incubated with the indicated amounts of DTSSP (Pierce)at 4 "C for 2 h. of p38 could be seen in both the At the end of the reaction period, glycine was added to a final concen- exposure. V, VAMP; *, 30-kDa VAMP complex; S , synaptophysin; V+S, tration of 100 mM. For the in vivo cross-linking, the cross-linker DSS 56-kDa complex of VAMP and synaptophysin. (Pierce Chemicals) was used. PC12 cells on 15 cm Petri dishes were rinsed twice in phosphate-buffered saline (137 mM NaCI, 2.7 mM KCI, 10 cross-linked to anotherlow molecular mass protein of approximM Na,HPO,, 1.8mM KH,PO,) a t room temperature and then incubated mately 12 kDa. Preliminary in vitro experiments usingrecomin 5 ml of 10% Me,SO (v/v) in phosphate-buffered salinein thepresence binant fusion proteins of the cytoplasmic domain of VAMP did or absence of 2 mM DSS for 30 min at room temperature. Tris (pH7.5) not support a VAMP homodimeric interaction. Further experiand glycine were added at a final concentration of 20 and 50 mM, ments with VAMP proteins that include the membrane dorespectively, to quench thecross-linker. The PC12 cells were harvested by trituration, centrifugation at 3,000 x g for 5 min in a Beckman GP mains are required to resolve this issue. Because VAMP and centrifuge, and solubilization in 10 mM Hepes-KOH (pH 7.5), 140 mM synaptophysin (p38) have been shown to coimmunoprecipitate potassium acetate, 1 mM MgCl,, 0.1 mM EGTA (HKA buffer) and 4% from Triton X-100-solubilized synaptic vesicles (28) and the octyl glucoside (w/v) for 1 h a t 4 "C. 56-kDa cross-linked complex is an increase of 38 kDa above the PAGE a n d Western Analysis-Samples (100 pgof proteidane) were migration of VAMP monomers, an immunoblot was performed electrophoresed on each laneof a 4.8% stacking, 10% resolving polyac- to determine if synaptophysin migrated with anidentical shift rylamide SDS-PAGE gel (30). Western immunoblot transfer to nitrocela shift lulose was done in 10% methanol, 50 mM Tris base, 384 mM glycine for in electrophoretic mobility. As shown in Fig. lB, there is 1.5 h at 80 V (31). Affinity-purified VAMP antibodies (32) and SY38 in synaptophysin immunoreactivity to a band of 56 kDa identical to thatof VAMP (lanes 2 and 4 ) . Reduction of the samples monoclonal antibodies(BoehringerMannheim)wereusedtolabel VAMP and synaptophysin,respectively. Primary antibodies were visu- with dithiothreitol after cross-linking eliminated the complex alized with'2sI-conjugated secondary anti-Ig antibodies (DuPont NEN). (Fig. ll3, lanes 6 and 8), indicating that it is the product of the In Fig. 3, enhanced chemiluminescence visualization (Amersham Corp.) DTSSP cross-linker. Because of the direct interaction between using horseradish peroxidase-conjugated secondary anti-Ig antibodies (Zymed) was used. Densitometric quantitation of bands was done using VAMP and syntaxin identified in vitro (16) and syntaxin is 35 kDa, we probed an immunoblot of the cross-linked rat brain a model 300A MolecularDynamicsdensitometerandImageQuant preparation with syntaxin antiserum. We did not detect a comsoftware. %sue Culture Methods-PC12 cells were g r o w n on uncoated tissue plex in a molecular mass range thatwould indicate that VAMP culture Petri dishes a t 5% CO,, 37 "C in Dulbecco's modified Eagle's and syntaxin were cross-linked (data not shown). medium, including 4 g of glucosefliter, 10% (v/v) fetal bovine sera, 5% We next fractionated rat brain to determine thesubcellular (v/v) heat-inactivated horse sera, 100 units/ml penicillin, andpg/ml 100 localization of the complex. As shown in Fig. 2, the complex was streptomycin (33). enriched in the synaptic vesicle preparation. Furthermore, in Immunoprecipitation-The solubilizedPC12sampleswerecentrifuged 15 min a t 18,000 x g. The supernatant was incubated with 67 pl Fig. 2 the cross-linking was performed in theabsence of deterof affinity-purified rabbit anti-VAMP sera (32). 3 pl of rabbit antigents, indicating that the interaction was not an artifact of Na+K+-ATPasea subunit sera (giftof Dr. James Nelson, Stanford Uni- detergent solubilization. versity), or no antibody prebound to protein A-Sepharose beads (PharUsing a membrane-permeant cross-linker, DSS, similar to macia Biotech Inc.) for 1.5 h at 4 "C. Samples were washed three times DTSSP, we investigated the VAMP-synaptophysin interaction with the solubilization buffer and recovered by brief microcentrifugai n vivo. The neuroendocrine cell line PC12 was incubated with tion. Immunoprecipitates were eluted from the beads with SDS gel cross-linker in phosphate-buffered saline. Cells were lysed afsample buffer.
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RESULTS
In order toidentify proteins interactingdirectly with VAMP, we chemically cross-linked a crude rat brainhomogenate using the homobifunctional, amine-reactive, thiol-cleavable crosslinker DTSSP, which has a spacer arm lengthof 12 A. Fig. LA shows the shift inelectrophoretic mobility of VAMP immunoreactivity upon cross-linkingwith increasingconcentrations of DTSSP (lanes 2 4 ) . There aretwo prominent cross-linked complexes that appear: one with a molecular mass of approximately 30 kDa and another of 56 kDa. The 30-kDa complex is often less abundant than the 56-kDa complex. This complex may be a VAMP homodimer or, alternatively, may be VAMP
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ter quenching agents were added to stop the cross-linking. Unlike the crude rat brainhomogenate, it was difficult to detect the cross-linked complex byexamination of the PC12 whole cell lysates. Therefore, we immunoprecipitated octyl glucosidesolubilized PC12 cell lysates with VAMP antibodies. We have shown previously that this detergent disrupts thecoimmunoprecipitation of synaptophysin and VAMP (28). Therefore, only synaptophysin, which is covalently bound to VAMP, should immunoprecipitate. No monomeric synaptophysin coimmunoprecipitated in the three VAMP immunoprecipitations (Fig. 3, lanes 4-6). The 56-kDa complex coimmunoprecipitated only in the sample exposed to cross-linker (Fig. 3, lane 6 ) . Much less cross-linked product was observed in thecontrol sample, which
Synaptophysin Association VAMP and
24536
VAMP immunoreactivity was present as the 56-kDa complex and approximately 15% of the total synaptophysin immunoreactivity was present as thisspecies. -200 Prior investigations haveobserved a 56-kDa synaptophysin immunoreactive complex upon cross-linking Triton X-100-soh-97 2s: -68 bilized synaptic vesicles (25, 34). However, the identity of the v+s: , -43 protein to which synaptophysin was cross-linked was not deS: termined. Our results suggest that this protein is VAMP.We -29 have further shown that synaptophysin and VAMP are crossV>" linked in the absence of detergentsin rat brain synaptic -18 vesicles and in PC12 cells. The in vivo cross-linking of VAMP FIG.2. The 66-kDa VAMP-synaptophysin complex is enriched in synaptic vesiclefraction. Panel A, VAMP immunoblot; panel B, and synaptophysin in PC12 cells suggests that this is a physisynaptophysin immunoblot. Lanes 1 and 2, synaptic vesicle fraction ologically significant interaction. The 95-kDa synaptophysin (LP2); lanes 3 and 4 , synaptosomal-membrane enriched fraction (LP1); lanes 5 and 6, cytosolic fraction (S2).Lanes 1 , 3 , and 5 are incubated in immunoreactive band seen in theVAMP immunoprecipitation has also beenobserved in previous synaptophysin cross-linking the absence of cross-linker. Lanes 2, 4 , and 6 are incubated in the presence of 2 mM DTSSP. Samples were frozen a t -20 "C overnight in experiments (25, 34). The authors describe this complex as a gel loading buffer before electrophoresis on a 10% nonreducing polyac- dimer of synaptophysin to which the 18-kDa protein is crossrylamide gel. Each sample was loaded on two lanes of the same gel. linked. It is interesting tonote that synaptoporin (synaptophyAfter transfer to nitrocellulose, the filter was cut lengthwise in half in order to probe each sample with VAMP and p38 antibodies.V,VAMP; *, sin 11)has also been identifiedin a 56-kDa complex upon cross30-kDa VAMP complex; S, synaptophysin, V+S, 56-kDa complex of linking synapticvesicles (34). Given the identical migration of VAMP and synaptophysin; 2S, synaptophysin dimer. this complex it islikely that synaptoporin is also cross-linked to VAMP. When a variety of detergents (CHAPS, octyl glucoside, and Triton X-100) were used, the 56-kDa cross-linked species was -200 most prominent inTriton X-100-solubilized preparations (data -97 not shown). TritonX-100 is the detergent inwhich VAMP and -68 synaptophysin have been shown previously to coimmunopre-43 cipitate in theabsence of three other synapticvesicle proteins (synaptotagmin, Rab 3A, and SV2) (28). Furthermore, just as the coimmunoprecipitation ofVAMP and synaptophysin was -29 disrupted by solubilization in octyl glucoside (281, we were un-18 able to detect the 56-kDa cross-linked complex in octyl glucoFIG.3. In vivo cross-linking of VAMP and synaptophysin in side-solubilized preparations. Whereas Bennettet al. (28) have PC12 cells. Protein of PC12 cells in culture was cross-linked with mM 2 previously characterized a high molecularmass complex of synDSS, immunoprecipitated withVAMP antibodies, electrophoresedon a aptic vesicle proteins (synaptotagmin, SV2, VAMP, and synap10% nonreducing polyacrylamide gel, and then immunoblotted with synaptophysin antibodies.Lane 1 , 10% of the solubilized material used tophysin) in CHAPS-solubilized synaptic vesicles, we found for the immunoprecipitations. Lane 2,mock immunoprecipitate-PC12 that, upon cross-linking, VAMP and synaptophysin were the cell lysate was incubated without antibody. Lane 3 , anti-VAMP antibod- only two proteins that formed a distinct complex. Although we ies incubated without PC12 cell lysate. There are three bands due to the cross-reactivity of the secondary anti-mouse immunoglobulin antibody did not observe cross-linking of the other synapticvesicle proa variety of conditions used for immunoblotting and the immunoprecipitating rabbit antibody. teins or of VAMPand syntaxin, there are Lane 4 , anti-VAMP immunoprecipitate of uncross-linked lysate.Lane 5, that could prevent interactingproteins from being cross-linked anti-VAMP immunoprecipitate of PC12 cell lysate prepared from cells such as the chemical groups with which the cross-linker reacts, reacted with quenching agent and cross-linker simultaneously immereaction diately before detergent solubilization.Lane 6, anti-VAMP immunopre- the lengthof the cross-linker spacer arm, and general cipitate of cross-linked lysate.V, VAMP S, synaptophysin; V+S,56-kDa conditions such as buffer and temperature. complex of VAMP and synaptophysin;2s. synaptophysin dimer;V+2S, Synaptophysin does not appear tobe a constituent of the 20 95-kDa complex of VAMP monomer and synaptophysindimer. S complex of nerve terminal proteins that implicated is in vesicle docking and fusion (2). Although VAMP is a constituent of was exposed to cross-linker and quenching agents simultathe 20 S complex, there isa bimodal distribution of VAMP in a neously just prior to cell lysis and detergentsolubilization (Fig. glycerol density gradient (2). One peak sediments in the20 S also found region, while another peak remains near top 3, lane 5 ) .Afaint bandof approximately 95 kDa was theof the gradient only in the cross-linked sample. This species is likely to be a partially overlapping with the distribution of synaptophysin. heterotrimeric complex of a synaptophysin dimer anda VAMP Either the VAMP and synaptophysin interaction hasnot been monomer, since in non-reducing preparations synaptophysin preserved during the preparation of these complexes or the dimers areoften observed (see Fig. 2 B , lanes 1 and 2 and Refs. VAMP and synaptophysin interaction has import a t either a 25 and 34). spatially or temporally distinct stage of synaptic vesicle trafficking. Other aspects of synaptic vesicle trafficking for which DISCUSSION the VAMP and synaptophysin interaction could be important In this report we have shown that two distinct VAMP-immu- include synaptic vesicle recycling after neurotransmitter renoreactive complexes (30 and 56 kDa) arerecovered following lease and synapticvesicle biogenesis. Alternatively, this interchemical cross-linking. The complexes are detectable in a crude action could be involved in vesicle docking and fusion but asa detergent-solublized rat brain preparation, as well as a non- biochemical complex distinct from the 20 S complex. For exsolublized synaptic vesicle preparation. After cross-linking, ample, the VAMP and synaptophysin interaction could inhibit synaptophysin immunoreactivity comigrateswith VAMP at the synaptophysin from interacting with its proposed partner on 56-kDa position, suggesting that this is the covalent cross- the target membranewith which it may form a fusion pore. If linking product of these two proteins. Under the cross-linking this is true, when vesicle fusion is triggered, the VAMP and conditions used in this study, a range of 2 5 4 5 % of the total synaptophysin interaction may be physiologically disrupted so
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Synaptophysin Association VAMP and that synaptophysin can now bind to its plasma membrane partner and create a fusion pore. Further studies are required to
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Commun. 189,1017-1023 16. Calakos, N., Bennett, M. K., Peterson, K. E., and Schelltr, R. H. (1994)Science 263, 1146-1149 understand these issues. 17. Sollner. T.. Whiteheart. S. W.. Brunner. M.. Erdiument-Bromaae. H.. Geromanos,'S.,'Tempst, P., and R&hman, J: E..(19&) Nature 362,318-324 18. Siidhof, T. C., Lottspeich, F., Greengard, P., Mehl, E., and Jahn, R. (1987) REFERENCES Science 238,1142-1144 1. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman,J. 19. Buckley, K. M., Floor, E., Kelly, R. B. (1987) J. Cell Biol. 106, 2447-2456 E. (1993) Cell 76,409A18 20. Leube, R. E., Kaiser, P., Seiter, A., Zimbelmann, R., Franke, W.W., Rehm, H., 2. Bennett, M. K., and Scheller, R. H. (1994)Annu. Reu. Biochem. 63,63-100 Knaus, P., Prior, P, Betz, H., Reinke, H., Beyreuther, K., and Wiedenmann, 3. Bennett, M. K., and Scheller, R. H. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, B. (1987) EMBO J . 6, 3261-3268 2559-2563 21. Sudhof, T. C., Baumert, M., Perin, M. S., and Jahn, R. (1987) Neuron 2, 4. Trimble, W. S., Cowan, D. M., and Scheller, R. H. (1988) Proc. Natl. Acad. Sci. 1475-1481 U. S. A. 85,45384542 22. Zhong, C . , Hayzer, D. J., andRunge, M. S. (1992)Biochim. Biophys. Acta 1129, 5. Baumert, M.. Mavcox. P. R., Navone, F., DeCamilli. P., and Jahn. R. (1989) 235-238 EMBO J. a, 379-384 23. Rehm, H., Wiedenmann, B., and Betz, H. (1986) EMBO J. 6, 535-541 6. Elferink, L. A,, Trimble, W. S., and Scheller, R. H. (1989) J . Bid. Chem. 264, 24. Thomas, L., Hartung, K., Langosch, D., Rehm, H., Bamberg, E., Franke, W. W., 11061-11064 and Betz, H. (1988) Science 242, 1050-1053 7. Trimhle, W. S., Gray, T. S., Elferink, L. A,, Wilson, M. C., and Scheller, R. H. 25. Johnston, P. A,, and Sudhof, T. C. (1990) J. B i d . Chem. 266, 88694873 (1990) J. Neurosci. 10, 1380-1387 26. Alder, J., Lu, B., Valtorta, F., Greengard, P., and Poo, M.(1992) Science 267, 8. McMahon, H. T., Ushkaryov, Y. A,, Edelmann, L., Link, E.,Binz, T., Niemann, 657-661 H., and Jahn, R. (1993) Nature 364,346-349 27. Alder, J., Xie, Z., Valtorta, E , Greengard, P., and Poo, M. (1992) Neuron 9, 9. Cain, C. C., Trimble, W. S., and Lienhard, G. E. (1992) J. Biol. Chem. 267, 759-768 11681-11684 28. Bennett, M. K , Calakos, N., Kreiner, T., and Scheller, R. H. (1992) J . Cell Biol. 10. Braun, J. E., Fritz, B. A,, Wong, S. M., and Lowe, A. W. (1994)J. Biol. Chem. 116, 761-775 269,5328-5335 29. Huttner, W. B., Schiehler, W., Greengard, P., and DeCamilli, P.(1983) J. Cell 11. Gerst, J. E., Rodgers, L., Riggs, M., and Wigler, M.(1992)Proc. Natl. Acad. Sci. Biol. 96, 1373-1388 U. S. A. 89,43384342 30. Laemmli, U. K. (1970) Nature 227,68&685 12. Archer, B. T., Ozcelik, T., Jahn, R., Francke, U.. and Siidhof, T. C. (1990) J. Biol. Chem. 266, 17267-17273 31. Towbin, H., Staehelin, T., and Gordon, J. (1979) Proc. Natl. Acad. Sci. U. S. A. 13. Protopopov, V., Govindan, B.,Novick, P., and Gerst, J. E. (1993) Cell 74, 76,4350-4354 855461 32. Trimble, W. S., Gray, T. S., Elferink, L. A,, Wilson, M. C., and Scheller, R. H. 14. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., (1990) J . Neurosci. 10, 1380-1387 DasGupta, B. R., and Montecucco, C. (1992) Nature 369, 832-835 33. Greene, L. A., and Tischler, A. S. (1982)Ada Cell. Neurobiol. 3, 373A14 15. Link, E., Edelmann, L., Chou, J. H., Binz, T., Yamasaki, S.,Eisel, U., Baumert, 34. Fykse, E. M.,Takei, K., Walch-Solimena, C., Geppert, M., Jahn, R.,DeCamilli, M., Siidhof, T. C., Niemann,H., and Jahn,R. (1992) Biochem. Biophys. Res. P., and Siidhof, T. C. (1993) J. Neurosci. 13,4997-5007
