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Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic... The EMBO Journal vol.14 no.2 pp.224-231, 1995 Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine Rothman and to the current Lambert Edelmann, Phyllis lHanson, Warren, 1994). According conserved membrane form model, a set of highly proteins Reinhard Jahn' Edwin R.Chapman and fusion machine. In neuronal the core of an exocytotic Howard Hughes Medical Institute and Departments of Pharmacology these include the vesicle exocytosis proteins synaptic and Cell Biology, Yale University School of Medicine, New Haven, Trimble et 1988; protein synaptobrevin (VAMP; al., CT 06510, USA et and the membrane Baumert al., 1989) synaptic proteins ICorresponding author et al., synaptosome-associated protein (SNAP)-25 (Oyler Communicated P.De Camilli 1989) and syntaxin I (Bennett et al., 1992a; Inoue and by lines of evidence Akagawa, 1992). Several independent The synaptic vesicle protein synaptobrevin (VAMP) have been support this model. First, all three proteins has recently been implicated as one of the key proteins identified recently as sole targets of clostridial neurotoxins. involved in exocytotic membrane fusion. It interacts a of that These neurotoxins include group proteins with the synaptic membrane proteins syntaxin I and irreversibly block exocytosis in neurons and which are synaptosome-associated protein (SNAP)-25 to form a responsible for tetanus and botulism. The light chains of complex which precedes exocytosis [Sollner et al. these toxins are metallo-endoproteases with high (1993b) Cell, 75, 409-418]. Here we demonstrate that selectivity for their respective substrates (reviewed by to vesicle the majority of synaptobrevin is bound the Niemann et al., 1994). Second, all three proteins are in No protein synaptophysin detergent extracts. syn- significantly homologous to proteins identified in yeast taxin I was found in this complex when synaptophysin- by genetic approaches. These proteins are essential for specific antibodies were used for immunoprecipitation. intracellular membrane traffic including exocytosis with the Conversely, no synaptophysin was associated (reviewed by Bennett and Scheller, 1993; Ferro-Novick I when synaptobrevin-syntaxin complex syntaxin- and I and Jahn, 1994). Third, synaptobrevin, syntaxin specific antibodies were used for immunoprecipitation. have been shown to function as mem- SNAP-25 recently bound to Thus, the synaptobrevin pool synaptophysin for a set of soluble that are brane receptors proteins I and is not available for binding to syntaxin SNAP-25, membrane fusion in cell-free fusion required for assays and vice versa. Synaptobrevin-synaptophysin binding These include N- (Sollner et al., 1993a). proteins chemical in was also demonstrated by cross-linking sensitive factor a-SNAP and ethylmaleimide (NSF), y- recombinant isolated nerve terminals. Furthermore, SNAP here the for soluble NSF (SNAP being acronym II bound and synaptobrevin efficiently synaptophysin Both NSF and a-SNAP are attachment proteins). highly its isoform but not the more synaptoporin, distantly conserved in evolution and correspond to the yeast proteins related synaptic vesicle protein p29. Recombinant in Sudhof et Seci 8 and Sec 17, respectively (reviewed al., synaptobrevin I bound with similar efficiency, whereas and 1993; Ferro-Novick and Jahn, 1994; Rothman the non-neuronal isoform cellubrevin a lower displayed Warren, 1994). affinity towards synaptophysin. Treatment with high The interactions between these soluble factors and in NaCl concentrations resulted a dissociation of the in membrane proteins have been studied detergent extracts, synaptobrevin-synaptophysin complex. In addition, resulting in a model describing the sequence of events in the interaction of synaptobrevin with synaptophysin membrane fusion (Sollner et al., 1993b). According to was abolished low amounts of irreversibly by SDS, this study, the vesicle protein synaptobrevin binds to while the interaction with syntaxin was enhanced. I and SNAP-25 in the membrane, syntaxin synaptic plasma interacts We conclude that synaptophysin selectively resulting in the docking of synaptic vesicles at the site of in which excludes the with synaptobrevin a complex release. This complex also contains small amounts of t-SNAP receptors syntaxin I and SNAP-25, suggesting synaptotagmin, a synaptic vesicle membrane protein a role for synaptophysin in the control of exocytosis. to mediate Ca21 and thought signaling (Popov Poo, Key words: exocytosis/SNAREs/synaptobrevin-VAMP/ 1993). Once this complex is formed, a-SNAP binds and synaptophysin/syntaxin synaptotagmin is displaced, followed by the binding of NSF. When ATP is hydrolyzed by NSF, the complex This disassembles. event may reflect the changes in protein-protein interactions that underlie membrane Introduction fusion. Synaptobrevin, SNAP-25 and syntaxin I have Neurotransmitters are released from neurons by Ca2+- therefore also been named SNAP receptors (SNAREs), dependent exocytosis of synaptic vesicles. In recent years, with the vesicle protein synaptobrevin designated v- major progress has been made in our understanding of the SNARE, and syntaxin I and SNAP-25 which reside on molecular mechanisms underlying exocytotic membrane the target membrane designated t-SNAREs (Sollner et al., fusion (reviewed by Ferro-Novick and Jahn, 1994; 1993b). Each intracellular fusion event appears to have 224 to synaptophysin Synaptobrevin binding its specific set of v-SNAREs and t-SNAREs, while NSF, synaptobrevin recombinant function as general fusion factors a-SNAP and y-SNAP isoform in all of these events (Rothman and Warren, 1994). 11 11 I 11 suggests that the membrane proteins Recent evidence syntaxin I, SNAP-25 and synaptobrevin have the ability to bind to each other without any additional factors or 48 - Using recombinant proteins, it has been shown energy. CO) can interact directly with syntaxin and o that synaptobrevin 33 .. that SNAP-25 enhances this binding (Calakos et al., 1994; 29 - Pevsner et al., 1994b). The binding is mediated by distinct domains, involving the cytoplasmic portion of synaptobrevin (residues 1-94) and a confined domain of which is adjacent to the transmembrane domain syntaxin (residues 194-267; Calakos et al., 1994). Furthermore, we have recently demonstrated that the N-terminal portion of SNAP-25 binds with high affinity to the same region of syntaxin I. In addition, synaptobrevin binds directly to SNAP-25 involving both C- and N-terminal portions of SNAP-25 (Chapman et al., 1994; Pevsner et al., 1994b). Fig. 1. Isoform specificity of synaptobrevin antibodies. Recombinant Structural analysis has shown that the domains of all three synaptobrevins I and II were expressed as fusion proteins linked to proteins participating in these interactions have a high by glutathione-Sepharose affinity chromatography and GST, purified propensity to form coiled-coils, suggesting that the cleaved by thrombin prior to SDS-PAGE and immunoblotting (-5 ,ug/ SNARE-protein complexes are primarily stabilized by lane). The following antibodies were examined: a polyclonal rabbit serum, raised against a peptide (CSAPAQPPAEGTEGA) such interactions (Chapman et al., 1994). corresponding to the N-terminal portion of synaptobrevin I; Based on these findings, one would expect that mem- monoclonal antibody Cl 69.1, raised against an N-terminal peptide branes bind to each other and fuse whenever the correct corresponding to synaptobrevin II; and monoclonal antibody Cl 10.1, t-SNAREs and v-SNAREs are paired. However, it is raised against gel-purified synaptobrevin (Baumert et aL, 1989). The bands with an Mr of -30 000 correspond to the positions of the difficult to imagine that these membrane 'glues' are uncleaved GST fusion proteins. constitutively active during membrane recycling or bio- synthesis and degradation. In fact, recent observations show that in neurons the t-SNAREs are widely distributed Results over the axonal plasmalemma despite the fact that vesicle docking and release only occurs at specialized sites with Synaptophysin copurifies with synaptobrevin upon (E.P.Garcia, P.S.McPherson, high topological precision immunoprecipitation P.DeCamilli, manuscript submit- interact with synaptobrevin, K.Takei, T.Chilcote and To identify proteins which P.De communication). Therefore it (clone Cl 69.1) ted; Camilli, personal we generated a new monoclonal antibody SNAREs are controlled by other proteins to the N-terminal domain is likely that the using a peptide corresponding ability to enter the SNARE complex. II To test its specificity, which regulate their of synaptobrevin as the antigen. a candidate for such a regulatory protein has I and II were separated Recently, recombinant rat synaptobrevin been characterized. This was named rb-Secl, m- and transferred to nitrocellulose. The protein by SDS-PAGE 1 is for II, with no cross- Sec or munc- 18 due to its homology to the corresponding antibody specific synaptobrevin in elegans towards synaptobrevin I (Figure 1), cellubrevin proteins yeast and the nematode Caenorhabditis reactivity et al., 1994; Pevsner et al., other in brain extracts (results not shown). (Hata et al., 1993; Garcia or any protein in or unc-18 gene result in monoclonal synaptobrevin II was 1994a). Deficiencies the SEC] Using this antibody, that the from Triton X-100 extracts of a loss of exocytosis, demonstrating corresponding immunoprecipitated for membrane fusion (Novick The immunoprecipitate was separated by gene products are essential synaptosomes. silver et Munc-18 interacts SDS-PAGE and the were visualized by et al., 1980; Hosono al., 1992). proteins revealed that with that this protein is An of the protein pattern directly syntaxin I, suggesting staining. analysis an control element regulating the function of in addition to antibody-derived bands, single major important an of 38 000 co- I et Garcia et al., 1994; Pevsner protein band with approximate syntaxin (Hata al., 1993; Mr with 2A). We tested et the function of munc-18 precipitated synaptobrevin (Figure al., 1994a). Although precise to synaptophysin, a is not it is possible that this protein whether this protein corresponded yet established, controls the membrane of Mr 38 000 present in functions as a molecular switch which ability major integral protein understood vesicles whose function is still of I to enter the SNARE complex. synaptic poorly syntaxin a review see Jahn and Immunoblotting For the vesicle protein synaptobrevin, corresponding (for Sudhof, 1994). revealed that a of We have now for major proportion have not been identified. analyzed synaptophysin regulators with II inter- coprecipitated synaptobrevin (Figure whether synaptobrevin undergoes protein-protein synaptophysin we immuno- To confirm this other than the of the 2B, left lanes). interaction, actions with proteins components a monoclonal Here we that binds precipitated synaptophysin directly using SNARE report synaptobrevin complex. Jahn et and the with C analyzed antibody (clone 7.3; al., 1985) to the vesicle synaptophysin high specificity. protein for the of in nerve terminal extracts synaptobrevin. immunoprecipitate presence This interaction predominates that a at the of to the 2A shows major protein migrating to the Figure and is alternative binding synaptobrevin with of synapto- SNARE position synaptobrevin coprecipitated complex. 225 L.Edelmann et aL for antibody mmuJnoprecipitation/ _. }- _om. synaptotagmin _W _ _ T + synaptosomal h.c. +l+ -] extra ct __ _ _ synaptophysin 66- _* . piII_ _I synaptobrevin h. c. r_ _ w~~~- synapto- 29 - physin CO I a/b __g~dEI~4I4II) syntaxin 0 _~uI SV2 2 0- synapto- brevin - p29 14 - -W qP0 42MVW NM DA-receptor Fig. 2. of from Triton extracts of Immunoprecipitation synaptobrevin-containing X-100 Protein protein complexes synaptosomes. (A) pattern obtained after of the electrophoretic separation precipitates 15% obtained with H and (SDS-PAGE, gel) synaptobrevin (syb)- synaptophysin antibodies h.c. and indicate the of the (syp)-specific (silver staining). l.c. and Immunoblot positions IgG heavy light chain, respectively. (B) analysis of the extract used as the synaptosomal detergent (synapt. extr.) and the starting material, immunoprecipitate (IP) remaining supernatant (S) for synaptotagmin II and chain of the antibodies I, synaptophysin, synaptobrevin syntaxin. that reacted with h.c., IgG heavy immunoprecipitating the detection Note that no detectable of the vesicle I was observed. system. coprecipitation protein synaptotagmin syx, syntaxin Ia/b. The of this with physin. identity band synaptobrevin was Cross-linking of synaptophysin and synaptobrevin The confirmed by middle results described so far indicate that in the immunoblotting (Figure 2B, lanes). synapse Virtually identical results were obtained when major proportion of synaptobrevin is octylgluco- complexed to side or CHAPS instead of Triton X-100 were used for synaptophysin, probably on the surface of synaptic vesicles. membrane solubilization not To confirm this interaction, we performed (results shown). protein In the we examined how cross-linking experiments using intact synaptosomes. following experiments the This was interaction between and approach prompted by the previous observation that synaptobrevin synaptophysin is synaptophysin related to the of in purified synaptic vesicles can be cross- binding synaptobrevin to the SNARE linked by bifunctional For this reagents to a low complex. purpose, all were protein immunoprecipitates M, and for (Johnston Sudhof, 1990). Cross-linking of proteins in analyzed the presence of syntaxin I. In addition, intact synaptosomes with I was disuccinimyl suberate syntaxin directly immunoprecipitated from the same (DSS) resulted in the formation of an adduct with an synaptosomal extract using a syntaxin-specific monoclonal apparent Mr of -55 000 that reacted with antibodies against both antibody (HPC-1; Barnstable et al., to for 1985) analyze synaptophysin and synaptobrevin the (Figure 3). With the presence of synaptophysin. As shown in Figure 2B, exception of bands corresponding I approximately to the syntaxin was absent when a synaptophysin-specific Mr of synaptobrevin and synaptophysin dimers was and trimers, antibody used for immunoprecipitation. Likewise, respectively, no additional adducts were observed (Figure synaptophysin was absent when a syntaxin I-specific 3). Furthermore, the Mr 55 000 adduct containing was both antibody used. Only the immunoprecipitates obtained proteins was immunoprecipitated with both synaptobrevin- with the synaptobrevin II-specific antibody contained both and synaptophysin-specific antibodies (results not shown). synaptophysin and syntaxin I. To control for specificity, Therefore, it is unlikely that the adduct is due to we also blotted cross- for additional membrane proteins of linking of another protein of similar size. These findings synaptic vesicles containing multiple transmembrane demonstrate that synaptophysin and synaptobrevin are domains (SV2: Bajjalieh et al., 1992; Feany et al., 1992; bound to each other in intact membranes and suggest that and p29: Baumert et al., 1990) and for the NMDA-RI the binding observed in detergent extracts reflects an receptor (Sucher et al., 1993). No co-sedimentation of interaction that occurs in vivo. any of these proteins was observed (Figure 2B). Together, these data demonstrate that the synapse contains at least two distinguishable pools of synaptobrevin: one com- Specificity of the synaptobrevin-synaptophysin plexed to synaptophysin and another complexed to syn- interaction taxin, i.e. the SNARE complex, with no overlap All experiments described so far were performed using between them. antibodies directed against synaptobrevin II, the pre- 226 Synaptobrevin binding to synaptophysin im munostai ning for sybil syp crosslinker + (DSS) I- I+T 66- 456 - _ _ synaptophysin 36 - o 29 > 20 - _ < synaptobrevin 1 4 - dye front Fig. 3. Cross-linking of synaptobrevin and synaptophysin in intact synaptosomes using the bifunctional reagent DSS. After cross-linking, the samples were separated by SDS-PAGE on a 15% gel and analyzed by immunoblotting for bands immunoreactive with synaptobrevin- and synaptophysin- specific monoclonal antibodies. Bands were visualized using the Amersham enhanced chemiluminescence (ECL) kit. The long arrow indicates the position of an adduct of -55 kDa that reacts with antibodies for both proteins. The additional immunoreactive bands of high molecular weight that are caused by DSS correspond approximately to the molecular masses expected for homodimers and homotrimers. dominant synaptobrevin isoform of the central nervous immob. GST-fusion (Elferink et al., 1989). We also examined whether system is able to interact with synaptobrevin I, a synaptophysin protein second neuronal isoform (Elferink et al., 1989), and with the non-neuronal homolog cellubrevin (McMahon et al., 1993). Synaptobrevins I and II and cellubrevin were + synaptosomal ++- - - + extract II''-II II expressed in Escherichia coli as GST fusion proteins and immobilized on glutathione-Sepharose beads. The purity synaptophysin of the fusion proteins was checked by SDS-PAGE and immunoblotting (results not shown). Aliquots of the beads were then incubated with a detergent extract of synapto- am syntaxin a/b somes and tested for binding of synaptophysin, syntaxin I and SNAP-25. As shown in Figure 4, synaptophysin EE - SNAP-25 bound to all three synaptobrevin homologs. However, the interaction of synaptophysin with cellubrevin was much weaker than that with synaptobrevins I and II (Figure 4). Rab 3 SNAP-25 and syntaxin I bound to all three synapto- brevin isoforms (Figure 4). Interestingly, cellubrevin bound relatively more of these two proteins than the brain- synaptotagmin specific isoforms. It is possible that the high degree of I and II interferes synaptophysin binding to synaptobrevins 4. Binding of synaptic proteins to immobilized fusion proteins of I and SNAP-25 (see below Fig. with the binding of syntaxin II (syb II). cellubrevin (ceb), synaptobrevin I (syb I) and synaptobrevin The binding of rab3 and synaptotagmin and Discussion). of each were immobilized on equal amounts protein Approximately levels, demonstrating the specificity was below detectable incubated with Triton X-100 extracts of and glutathione-Sepharose see also Figure 5). Further- of the interactions (Figure 4; the beads were for bound After washing, analyzed synaptosomes. Note that none of the antibodies reacted of the proteins bound to the beads proteins by immunoblotting. more, none analyzed fusion lanes). No binding was observed with any of the proteins (- when the carrier protein glutathione S-transferase only the carrier GST was immobilized (data not shown; when only protein was immobilized (results not shown; see also Figure 5; also 6 and et - and + refer to the see Figure Chapman al., 1994). et al., 1994). Chapman of extracts in the absence or synaptosome presence, respectively, we whether the same investigated assay. Using approach, binding similar to were homologous or synaptophysin proteins to 5 shows that with These homologous synaptophysin. Figure of synaptobrevin. proteins capable interacting similar to that of bound with an a isoform efficiency include synaptoporin, synaptophysin (Knaus synaptoporin the neuronal synapto- and the similar membrane again preferring et 1990), structurally synaptophysin, al., In no of over cellubrevin. p29 et and SCAMP brevins contrast, binding (Baumert al., 1990) (Brand proteins p29 data not was detectable shown). that are less or not or SCAMP (Figure 4; and 1993) (p29) (SCAMP) Castle, 227 et aL L.Edelmann immunoprecipitate was not maintained but rather only increased after pretreatment with SDS. The latter observa- tion is consistent with our suggestion that synaptophysin binding prevents the binding of synaptobrevin to the SNARE complex. Furthermore, it demonstrates that the nature of synaptophysin binding to synaptobrevin is different from that of syntaxin I binding to synaptobrevin. synaptophysin 4~ EP 411 Discussion In this synaptopodn study we have demonstrated that the two synaptic _I *iA vesicle proteins synaptobrevin and synaptophysin form a complex, and that the ._ complexed synaptobrevin appears p 29 not to be available for interaction with syntaxin or SNAP-25. we have shown Furthermore, that the protein- but not binds to immobilized Fig. 5. Synaptoporin p29 synaptobrevins. protein interactions for the responsible synaptophysin- as in followed Binding was performed Figure 4, by immunoblot for and The asterisk synaptobrevin are different from those of analysis synaptophysin, synaptoporin p29. complex the a of the cellubrevin fusion that indicates reactive component is protein SNARE sensitive to complex, increasing ionic strength to not identical p29. and mild SDS treatment. Several previous studies have characterized proteins which bind to and synaptophysin synaptobrevin, for antibody respectively. Bennett et al. used a (1992b) variety of immunoprecipitation detergents, Triton CHAPS and including X-100, octyl- synaptophysin synaptobrevin I1 glucoside, to solubilize proteins of purified synaptic 450 300 vesicles to study protein-protein interactions 150 450 NaCI among [mM] synaptic vesicle membrane proteins. An interaction E_4UI@ . _ synaptophysin between synaptophysin and synaptobrevin was observed in which is in with our Triton extracts agreement findings. jpp syn v. _ 11 of interaction However, the specificity this remained synaptobrevin doubtful since other interactions were seen when different were in contrast to our observations. detergents used, The interaction of and is sensitive Fig. 6. synaptobrevin synaptophysin the association of and Furthermore, synaptobrevin synapto- to were as in high ionic strength. Immunoprecipitations performed physin with syntaxin and SNAP-25 was not addressed. In but instead of 140 mM the indicated concentrations of Figure 2, KCI, an earlier Thomas and Betz study, (1990) characterized NaCl were used for and The extraction, precipitation washing. figure the of a membrane named binding protein, physophilin, shows an immunoblot of the analysis immunoprecipitates obtained with anti-synaptophysin and to immobilized vesicles. was (left) anti-synaptobrevin (right) synaptic Binding competed monoclonal antibodies. by synaptophysin. A direct interaction between synapto- physin and physophilin, however, could not be demon- of the strated. Nature Finally, we earlier noticed an interaction between synaptobrevin-synaptophysin interaction synaptobrevin and which at that time was synaptophysin Several were used to approaches gain an into the thought to be non-specific due to its sensitivity to insight high nature of the concentrations of synaptobrevin-synaptophysin interaction. chaotropic agents (Siidhof et al., 1989). we whether In our the First, investigated the interaction is sensitive present study, association between synapto- to ionic Both and physin and increasing strength. synaptobrevin synaptobrevin has been demonstrated using synaptophysin were from extracts three immunoprecipitated independent experimental approaches: co-immuno- containing increasing concentrations of NaCl. As shown precipitation (with identical in results three different in Figure 6, coprecipitation was reduced at 300 mM NaCl detergents), of to binding synaptophysin immobilized and virtually abolished at 450 mM NaCl, suggesting that recombinant synaptobrevin and cross-linking. Although ionic interactions a play major role. Second, we examined we cannot exclude that synaptophysin undergoes additional whether this interaction is sensitive to a brief exposure protein-protein interactions, we did not see any other to SDS. This experiment was prompted proteins in the by the recent synaptophysin-synaptobrevin complex observation that the SNARE complex is resistant to mild (see e.g. the stains of protein the immunoprecipitates in SDS treatment et (Hayashi al., 1994), probably due to the Figure 2A and the control blots in Figure 2B). This stability of coiled-coil interactions. Immunoprecipitation interaction thus appears to be specific and selective. The was performed from an extract that was first solubilized stoichiometry and prevalence of the complex remain to with 1% SDS and then diluted with Triton X-100 to yield be determined. In our experiments we found that at least final concentrations of 0.05% SDS and 1% Triton X- half of the entire synaptophysin pool precipitates with 100. As shown in Figure 7, the pretreatment with SDS synaptobrevin and vice versa (unpublished observations). completely abolished the interaction between synapto- This appears to be far more than complexed to syntaxin physin and synaptobrevin. This was demonstrated by both I and SNAP-25. A detailed quantitation of these inter- silver of staining the electrophoretically separated proteins actions is currently under way. of the immunoprecipitates and immunoblotting. In con- The domains of synaptobrevin that are responsible for trast, the amount of syntaxin I in the synaptobrevin the interaction with synaptophysin remain to be deter- 228 Synaptobrevin binding to synaptophysin total extract extract after IP * . immunoprecipitate synaptobrevin I, - _ h.c. ,-synaptophysin *4*N-syntaxin alb synaptophysin E. _ * I. c syntaxin a/b imio synaptobrevin immunoblot silver stain Fig. 7. SDS pretreatment blocks the synaptobrevin-synaptophysin interaction but increases the binding of syntaxin. Synaptosomal extracts were pretreated with SDS and then incubated with excess Triton X-100 (see Materials and methods), before immunoprecipitation was performed using the synaptobrevin H-specific monoclonal antibody Cl 69.1. All immunoprecipitates were analyzed by SDS-PAGE and immunoblotting for synaptobrevin, synaptophysin and syntaxin I. Left, immunoblots; right, silver stain of the precipitates obtained before and after SDS pretreatment. See the legend to Figure 2 for details. h.c, l.c., heavy and light chain of IgG, respectively. The band visible in the precipitate of the synaptophysin blots corresponds to the heavy chain of IgG that reacted with the secondary antibody. mined. The sensitivity to high ionic strength suggests that to synaptobrevin, although it shares only 58% of amino hydrophilic regions play a major role, implicating the acid residues with synaptophysin. cytoplasmic domains of both proteins. However, truncation Our findings shed new light on the functional role of the transmembrane domain of synaptobrevin or replace- of the vesicle protein synaptophysin and its relatives. ment of this domain with the transmembrane domain of Synaptophysin is a membrane glycoprotein with four syntaxin resulted in a loss of synaptophysin binding, while membrane-spanning domains and an extended cytoplasmic binding of the t-SNAREs syntaxin and SNAP-25 was not tail. This tail contains nine unusual repeats and is phos- affected (unpublished observations; see also Calakos et al., phorylated by c-src kinase on tyrosine residues (for a 1994; Hayashi et al., 1994). Furthermore, a syntaxin Ta review see Jahn and Sudhof, 1994). Although this protein mutant containing the transmembrane domain of synapto- was the first synaptic vesicle protein to be characterized brevin did not bind synaptophysin. Although these negative in molecular terms (Leube et al., 1987; Sudhof et al., results may be due to misfolding of the recombinant 1989), its function has remained unclear. An early study mutant proteins, it is possible that the Ca2' transmembrane reporting binding (Rehm et al., 1986) could not be domain of synaptobrevin is involved in the confirmed binding of (Brose et al., 1992). When purified synapto- synaptophysin. Clearly, the interaction of physin is into synaptobrevin incorporated lipid bilayers it forms non- with synaptophysin is different from that with syntaxin, ion selective, voltage-dependent channels (Thomas et al., which is not sensitive to ionic strength and is independent 1988), but the physiological significance of this observa- of the transmembrane domains (Calakos et tion remains to be established. al., 1994). Interestingly, the primary sequence of synaptophysin The presence of two of non-overlapping pools synapto- to be in appears less conserved evolution than that of brevin, one complexed to and the other synaptophysin et Cowan et synaptobrevin (Sudhof al., 1989; al., 1990; complexed to the SNARE a mechanism complex, suggests for a review see Jahn and with the which Sudhof, 1994), by synaptophysin can control the of ability synapto- transmembrane domains more brevin to enter the SNARE for being highly conserved this complex. Support than other of the molecule. view is results which demonstrate parts Likewise, provided by preliminary synaptophysin to be absent from dense that recombinant SNAP-25 with appears peptide-containing large competes synaptophysin core a review see De Camilli and for to granules (for Jahn, binding synaptobrevin (unpublished observations). 1990), whereas synaptobrevin is Synaptophysin may function as a molecular switch that present (unpublished the existence of additional allows the association of with the t-SNAREs observations). However, synaptobrevin on these cannot after it dissociates from Such unknown isoforms only a synaptophysin granules synaptophysin. It is that the structural determinants would ensure that binds at be ruled out. regulation synaptobrevin possible only for to the correct time and location to the for instance of synaptophysin t-SNAREs, required binding synaptobrevin is activated. This interaction reside in its thus for when the secondary structure, allowing greater synapse may for control of v-SNAREs in the amino acid This is confirmed reflect a mechanism in variability sequence. general binds well fusion events. It will be to find our observation that membrane out by synaptoporin interesting equally 229 L.Edelmann et aL to recombinant and Binding proteins cross-linking of whether other v-SNAREs such as cellubrevin or the yeast synaptosomes homologs Sncl/Snc2 form similar complexes with partner Recombinant proteins were immobilized on glutathione-Sepharose proteins and what the structural features of such hypo- beads were solubilized in Triton X- (-1-5 beads). Synaptosomes gg/gl thetical partner proteins may be. Furthermore, it remains 100 containing extraction buffer (see above). 10 p1 of beads containing recombinant protein were added to 1 ml extracts. Incubation and washing to established are be how these interactions regulated were performed as for immunoprecipitation (see above). intracellular membrane traffic. during For cross-linking, synaptosomes were resuspended at a protein con- centration of 1.5 mg/ml in Krebs-Ringer buffer (140 mM NaCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM Na2HPO4, 10 mM glucose, 20 mM HEPES, pH 7.4) and pre-warmed for 10 min at room temperature. Materials and methods DSS, dissolved in dimethyl sulfoxide, was then added to yield final concentrations of 5 mM DSS and 5% (v/v) of the solvent. After Antibodies incubation under rotation for 45 min at room temperature, 100 mM For the study of synaptobrevin, we generated a new monoclonal antibody (final concentration) of Tris-Cl (pH 7.4) were added as quencher, since the antibodies described previously (Baumert et al., 1989) were followed by a further incubation for 30 min. The membranes were not suitable for immunoprecipitation experiments (unpublished observa- pelleted in a microfuge (5000 r.p.m. for 3 min) and then resuspended in tions). synthetic peptide (CSATAATVPPAAPAGEG) corresponding 1.5 ml of ice-cold 150 mM NaCl, 20 mM Tris-Cl (pH 7.4) containing to the N-terminus of rat II isoform in synaptobrevin (the predominant 1% (v/v) Triton X-100. After incubation at 4°C for 1 h, insoluble the obtained from Keck CNS), Biopolymer Facility (Yale University, at 40 for 20 material was removed by centrifugation 000 r.p.m. min in was immobilized to New Haven, CT) keyhole limpet hemocyanine a Beckman T1 100 rotor. Fifteen pl aliquots of the supematants were and used for immunization (Schneider et al., 1983) the of Balb/c mice. analyzed by SDS-PAGE and immunoblotting. and were to standard Fusion, propagation screening performed according et A procedures (Kohler and Milstein, 1975; Jahn al., 1985). single Other methods hybridoma line (clone Cl 69.1) was established. was to Laemmli the monoclonal SDS-PAGE performed according (1970) using The antibodies have been described following previously: Bio-Rad Protean II and minigel apparatus. Immunoblotting visualization antibodies directed against synaptophysin (clone C 7.3; Jahn et al., with A was as described et [1251]protein performed (Jahn al., 1985) Brose et rab3 1985), synaptotagmin (clone 41.1; al., 1992), (clone 42.1, unless indicated otherwise Silver of electro- (Figure 3). staining for B and Matteoli et specific rab3A, C; al., 1991), synaptobrevin (clone was carried out to phoretically separated proteins according Morrissey Baumert et NMDA RI et 10.1; al., 1989), receptor (Sucher al., 1993) Protein was determined the bichinoninic acid (1981). using protein assay and rabbit serum directed et Monoclonal against p29 (Baumert al., 1990). the manufacturer's reagent (BCA assay, Pierce, Rockford, IL) following antibodies Ia/b and SV2 were against syntaxin (HPC-1) kindly given by instructions. Dr C.Barnstable Barnstable et and Dr (New Haven, CT; al., 1985) and Monoclonal K.Buckley (Boston, MA; Buckley Kelly, 1985). antibody for was raised recombinant SNAP-25 Cl 71.1, specific SNAP-25, using Acknowledgements and will be described in detail elsewhere and (D.Bruns R.Jahn, manuscript in A rabbit serum directed preparation). against synaptoporin (Fykse The authors wish to thank Nikki Barton and Silke Engers for their skilful a monoclonal directed SCAMP et al., 1993) and antibody against (Brand assistance in recombinant and in the preparing proteins establishing Dr and Castle, 1993) were kindly provided by T.C.Sudhof (Dallas, TX) hybridoma line Cl 69.1. We are grateful to Drs C.Barnstable (New Dr and J.D.Castle (Charlottesville, VA), respectively. Haven, CT), S.Brand and D.Castle (Charlottesville), and K.Buckley for their of antibodies. we (Boston, MA) generous supply Furthermore, thank Drs C.Solimena and P.De Camilli for critical Immunoprecipitation (New Haven, CT) Isolated nerve terminals were from rat cerebral advice and discussions. (synaptosomes) prepared stimulating cortex to standard von Mollard et according procedures (Fischer al., McMahon et Ficoll 1991; al., 1992) using density gradient centrifugation as the last For 1 of purification step. solubilization, mg synaptosomal References was and dissolved in 1 ml of ice-cold extraction buffer protein pelleted mM 20 mM HEPES-KOH 2 mM unless Peterson,K., Shinghal,R. and Scheller,R.H. (1992) (140 KCI, pH 7.3, EDTA, Bajjalieh,S.M., indicated 1% Triton X-100. Ten of ascites Science, 257, 1271-1273. otherwise) containing (v/v) ,ul fluid to -10-30 were added and the Barnstable,C.J., Hofstein,R. and Akagawa,K. (1985) Dev. Brain Res., (corresponding ,ug specific IgG) incubation was carried out for 6-8 h at One hundred p1 of 20, 286-290. 4°C. protein De were Baumert,M., Maycox,P.R., Navone,F., Camilli,P. and Jahn,R. (1989) G-Sepharose suspension (Pharmacia, Piscataway, NJ) added, EMBO 379-384. followed incubation for 1 h. The beads were collected J., 8, by by centrifugation Fischer von and washed three times in extraction buffer Triton X-100. Baumert,M., Takei,K., Hartinger,J., Burger,P.M., Mollard,G., containing De and J. Cell For SDS 1 of was first solubilized in Maycox,P.R., Camilli,P. Jahn,R. (1990) Biol., 110, pretreatment, mg synaptosomes 1285-1294. 50 of extraction buffer 2% ,l containing (w/v) SDS, followed by the Bennett,M.K. and Scheller,R.H. (1993) Proc. Nati Acad. Sci. USA, 90, addition of 950 of extraction buffer containing 1% Triton X-100 to g1 2559-2563. the quench SDS. Immunoprecipitation was then carried out exactly as Bennett,M.K., Calakos,N. and Scheller,R.H. (1992a) Science, 257, described above. 255-259. Bennett,M.K., Calakos,N., Kreiner,I. and Scheller,R.H. (1992b) J. Cell Generation and purification of recombinant proteins Biol., 116, 761-775. cDNAs encoding rat synaptobrevins (VAMP) I and II and syntaxins Ia and Brand,S.H. Castle,J.D. (1993) EMBO J., 12, 3753-3761. and Ib were kindly provided by Dr R.Scheller (Stanford; Elferink et al., Sudhof,T.C. and Brose,N., Petrenko,A.G., Jahn,R. (1992) Science, 256, 1989; Bennett et al., 1992a). cDNA for cellubrevin was a gift from Drs 1021-1025. McMahon and Stidhof (Dallas, TX; McMahon et al., 1993). Full- Buckley,K. and Kelly,R.B. (1985) J. Cell Biol., 100, 1284-1294. or truncated length coding regions were amplified using the PCR with Calakos,N., Bennett,M.K., Peterson,K.E. and Scheller,R.H. (1994) oligonucleotides which contained BamHI and EcoRI restriction sites. Science, 263, 1146-1149. The PCR products were subcloned into the BamHI-EcoRI sites of and Chapman,E.R. Jahn,R. (1994) J. Bio. 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(1990) Biol., 111, Thomas,L., Hartung,K., Langosch,D., Rehm,H., Bamber,E., Franke,W.W. and 1050-1053. Betz,H. (1988) Science, 242, and Scheller,R.H. Proc. Natl Acad. Trimble,W.S., Cowan,D.M. (1988) Sci. 4538-4542. USA, 85, Received on revised on October 1994 August 18, 1994; 26, Note added in proof interaction between After submission of this an paper, synaptobrevin N.Calakos and R.H.Scheller was and synaptophysin reported by (1994) J. Biol. 24534-24537. Chem., 269, http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine.

Edelmann, L.; Hanson, P.I.; Chapman, E.R.; Jahn, R.
The EMBO Journal , Volume 14 (2) – Jan 1, 1995
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Springer Journals
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Copyright © European Molecular Biology Organization 1995
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10.1002/j.1460-2075.1995.tb06995.x
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Abstract

The EMBO Journal vol.14 no.2 pp.224-231, 1995 Synaptobrevin binding to synaptophysin: a potential mechanism for controlling the exocytotic fusion machine Rothman and to the current Lambert Edelmann, Phyllis lHanson, Warren, 1994). According conserved membrane form model, a set of highly proteins Reinhard Jahn' Edwin R.Chapman and fusion machine. In neuronal the core of an exocytotic Howard Hughes Medical Institute and Departments of Pharmacology these include the vesicle exocytosis proteins synaptic and Cell Biology, Yale University School of Medicine, New Haven, Trimble et 1988; protein synaptobrevin (VAMP; al., CT 06510, USA et and the membrane Baumert al., 1989) synaptic proteins ICorresponding author et al., synaptosome-associated protein (SNAP)-25 (Oyler Communicated P.De Camilli 1989) and syntaxin I (Bennett et al., 1992a; Inoue and by lines of evidence Akagawa, 1992). Several independent The synaptic vesicle protein synaptobrevin (VAMP) have been support this model. First, all three proteins has recently been implicated as one of the key proteins identified recently as sole targets of clostridial neurotoxins. involved in exocytotic membrane fusion. It interacts a of that These neurotoxins include group proteins with the synaptic membrane proteins syntaxin I and irreversibly block exocytosis in neurons and which are synaptosome-associated protein (SNAP)-25 to form a responsible for tetanus and botulism. The light chains of complex which precedes exocytosis [Sollner et al. these toxins are metallo-endoproteases with high (1993b) Cell, 75, 409-418]. Here we demonstrate that selectivity for their respective substrates (reviewed by to vesicle the majority of synaptobrevin is bound the Niemann et al., 1994). Second, all three proteins are in No protein synaptophysin detergent extracts. syn- significantly homologous to proteins identified in yeast taxin I was found in this complex when synaptophysin- by genetic approaches. These proteins are essential for specific antibodies were used for immunoprecipitation. intracellular membrane traffic including exocytosis with the Conversely, no synaptophysin was associated (reviewed by Bennett and Scheller, 1993; Ferro-Novick I when synaptobrevin-syntaxin complex syntaxin- and I and Jahn, 1994). Third, synaptobrevin, syntaxin specific antibodies were used for immunoprecipitation. have been shown to function as mem- SNAP-25 recently bound to Thus, the synaptobrevin pool synaptophysin for a set of soluble that are brane receptors proteins I and is not available for binding to syntaxin SNAP-25, membrane fusion in cell-free fusion required for assays and vice versa. Synaptobrevin-synaptophysin binding These include N- (Sollner et al., 1993a). proteins chemical in was also demonstrated by cross-linking sensitive factor a-SNAP and ethylmaleimide (NSF), y- recombinant isolated nerve terminals. Furthermore, SNAP here the for soluble NSF (SNAP being acronym II bound and synaptobrevin efficiently synaptophysin Both NSF and a-SNAP are attachment proteins). highly its isoform but not the more synaptoporin, distantly conserved in evolution and correspond to the yeast proteins related synaptic vesicle protein p29. Recombinant in Sudhof et Seci 8 and Sec 17, respectively (reviewed al., synaptobrevin I bound with similar efficiency, whereas and 1993; Ferro-Novick and Jahn, 1994; Rothman the non-neuronal isoform cellubrevin a lower displayed Warren, 1994). affinity towards synaptophysin. Treatment with high The interactions between these soluble factors and in NaCl concentrations resulted a dissociation of the in membrane proteins have been studied detergent extracts, synaptobrevin-synaptophysin complex. In addition, resulting in a model describing the sequence of events in the interaction of synaptobrevin with synaptophysin membrane fusion (Sollner et al., 1993b). According to was abolished low amounts of irreversibly by SDS, this study, the vesicle protein synaptobrevin binds to while the interaction with syntaxin was enhanced. I and SNAP-25 in the membrane, syntaxin synaptic plasma interacts We conclude that synaptophysin selectively resulting in the docking of synaptic vesicles at the site of in which excludes the with synaptobrevin a complex release. This complex also contains small amounts of t-SNAP receptors syntaxin I and SNAP-25, suggesting synaptotagmin, a synaptic vesicle membrane protein a role for synaptophysin in the control of exocytosis. to mediate Ca21 and thought signaling (Popov Poo, Key words: exocytosis/SNAREs/synaptobrevin-VAMP/ 1993). Once this complex is formed, a-SNAP binds and synaptophysin/syntaxin synaptotagmin is displaced, followed by the binding of NSF. When ATP is hydrolyzed by NSF, the complex This disassembles. event may reflect the changes in protein-protein interactions that underlie membrane Introduction fusion. Synaptobrevin, SNAP-25 and syntaxin I have Neurotransmitters are released from neurons by Ca2+- therefore also been named SNAP receptors (SNAREs), dependent exocytosis of synaptic vesicles. In recent years, with the vesicle protein synaptobrevin designated v- major progress has been made in our understanding of the SNARE, and syntaxin I and SNAP-25 which reside on molecular mechanisms underlying exocytotic membrane the target membrane designated t-SNAREs (Sollner et al., fusion (reviewed by Ferro-Novick and Jahn, 1994; 1993b). Each intracellular fusion event appears to have 224 to synaptophysin Synaptobrevin binding its specific set of v-SNAREs and t-SNAREs, while NSF, synaptobrevin recombinant function as general fusion factors a-SNAP and y-SNAP isoform in all of these events (Rothman and Warren, 1994). 11 11 I 11 suggests that the membrane proteins Recent evidence syntaxin I, SNAP-25 and synaptobrevin have the ability to bind to each other without any additional factors or 48 - Using recombinant proteins, it has been shown energy. CO) can interact directly with syntaxin and o that synaptobrevin 33 .. that SNAP-25 enhances this binding (Calakos et al., 1994; 29 - Pevsner et al., 1994b). The binding is mediated by distinct domains, involving the cytoplasmic portion of synaptobrevin (residues 1-94) and a confined domain of which is adjacent to the transmembrane domain syntaxin (residues 194-267; Calakos et al., 1994). Furthermore, we have recently demonstrated that the N-terminal portion of SNAP-25 binds with high affinity to the same region of syntaxin I. In addition, synaptobrevin binds directly to SNAP-25 involving both C- and N-terminal portions of SNAP-25 (Chapman et al., 1994; Pevsner et al., 1994b). Fig. 1. Isoform specificity of synaptobrevin antibodies. Recombinant Structural analysis has shown that the domains of all three synaptobrevins I and II were expressed as fusion proteins linked to proteins participating in these interactions have a high by glutathione-Sepharose affinity chromatography and GST, purified propensity to form coiled-coils, suggesting that the cleaved by thrombin prior to SDS-PAGE and immunoblotting (-5 ,ug/ SNARE-protein complexes are primarily stabilized by lane). The following antibodies were examined: a polyclonal rabbit serum, raised against a peptide (CSAPAQPPAEGTEGA) such interactions (Chapman et al., 1994). corresponding to the N-terminal portion of synaptobrevin I; Based on these findings, one would expect that mem- monoclonal antibody Cl 69.1, raised against an N-terminal peptide branes bind to each other and fuse whenever the correct corresponding to synaptobrevin II; and monoclonal antibody Cl 10.1, t-SNAREs and v-SNAREs are paired. However, it is raised against gel-purified synaptobrevin (Baumert et aL, 1989). The bands with an Mr of -30 000 correspond to the positions of the difficult to imagine that these membrane 'glues' are uncleaved GST fusion proteins. constitutively active during membrane recycling or bio- synthesis and degradation. In fact, recent observations show that in neurons the t-SNAREs are widely distributed Results over the axonal plasmalemma despite the fact that vesicle docking and release only occurs at specialized sites with Synaptophysin copurifies with synaptobrevin upon (E.P.Garcia, P.S.McPherson, high topological precision immunoprecipitation P.DeCamilli, manuscript submit- interact with synaptobrevin, K.Takei, T.Chilcote and To identify proteins which P.De communication). Therefore it (clone Cl 69.1) ted; Camilli, personal we generated a new monoclonal antibody SNAREs are controlled by other proteins to the N-terminal domain is likely that the using a peptide corresponding ability to enter the SNARE complex. II To test its specificity, which regulate their of synaptobrevin as the antigen. a candidate for such a regulatory protein has I and II were separated Recently, recombinant rat synaptobrevin been characterized. This was named rb-Secl, m- and transferred to nitrocellulose. The protein by SDS-PAGE 1 is for II, with no cross- Sec or munc- 18 due to its homology to the corresponding antibody specific synaptobrevin in elegans towards synaptobrevin I (Figure 1), cellubrevin proteins yeast and the nematode Caenorhabditis reactivity et al., 1994; Pevsner et al., other in brain extracts (results not shown). (Hata et al., 1993; Garcia or any protein in or unc-18 gene result in monoclonal synaptobrevin II was 1994a). Deficiencies the SEC] Using this antibody, that the from Triton X-100 extracts of a loss of exocytosis, demonstrating corresponding immunoprecipitated for membrane fusion (Novick The immunoprecipitate was separated by gene products are essential synaptosomes. silver et Munc-18 interacts SDS-PAGE and the were visualized by et al., 1980; Hosono al., 1992). proteins revealed that with that this protein is An of the protein pattern directly syntaxin I, suggesting staining. analysis an control element regulating the function of in addition to antibody-derived bands, single major important an of 38 000 co- I et Garcia et al., 1994; Pevsner protein band with approximate syntaxin (Hata al., 1993; Mr with 2A). We tested et the function of munc-18 precipitated synaptobrevin (Figure al., 1994a). Although precise to synaptophysin, a is not it is possible that this protein whether this protein corresponded yet established, controls the membrane of Mr 38 000 present in functions as a molecular switch which ability major integral protein understood vesicles whose function is still of I to enter the SNARE complex. synaptic poorly syntaxin a review see Jahn and Immunoblotting For the vesicle protein synaptobrevin, corresponding (for Sudhof, 1994). revealed that a of We have now for major proportion have not been identified. analyzed synaptophysin regulators with II inter- coprecipitated synaptobrevin (Figure whether synaptobrevin undergoes protein-protein synaptophysin we immuno- To confirm this other than the of the 2B, left lanes). interaction, actions with proteins components a monoclonal Here we that binds precipitated synaptophysin directly using SNARE report synaptobrevin complex. Jahn et and the with C analyzed antibody (clone 7.3; al., 1985) to the vesicle synaptophysin high specificity. protein for the of in nerve terminal extracts synaptobrevin. immunoprecipitate presence This interaction predominates that a at the of to the 2A shows major protein migrating to the Figure and is alternative binding synaptobrevin with of synapto- SNARE position synaptobrevin coprecipitated complex. 225 L.Edelmann et aL for antibody mmuJnoprecipitation/ _. }- _om. synaptotagmin _W _ _ T + synaptosomal h.c. +l+ -] extra ct __ _ _ synaptophysin 66- _* . piII_ _I synaptobrevin h. c. r_ _ w~~~- synapto- 29 - physin CO I a/b __g~dEI~4I4II) syntaxin 0 _~uI SV2 2 0- synapto- brevin - p29 14 - -W qP0 42MVW NM DA-receptor Fig. 2. of from Triton extracts of Immunoprecipitation synaptobrevin-containing X-100 Protein protein complexes synaptosomes. (A) pattern obtained after of the electrophoretic separation precipitates 15% obtained with H and (SDS-PAGE, gel) synaptobrevin (syb)- synaptophysin antibodies h.c. and indicate the of the (syp)-specific (silver staining). l.c. and Immunoblot positions IgG heavy light chain, respectively. (B) analysis of the extract used as the synaptosomal detergent (synapt. extr.) and the starting material, immunoprecipitate (IP) remaining supernatant (S) for synaptotagmin II and chain of the antibodies I, synaptophysin, synaptobrevin syntaxin. that reacted with h.c., IgG heavy immunoprecipitating the detection Note that no detectable of the vesicle I was observed. system. coprecipitation protein synaptotagmin syx, syntaxin Ia/b. The of this with physin. identity band synaptobrevin was Cross-linking of synaptophysin and synaptobrevin The confirmed by middle results described so far indicate that in the immunoblotting (Figure 2B, lanes). synapse Virtually identical results were obtained when major proportion of synaptobrevin is octylgluco- complexed to side or CHAPS instead of Triton X-100 were used for synaptophysin, probably on the surface of synaptic vesicles. membrane solubilization not To confirm this interaction, we performed (results shown). protein In the we examined how cross-linking experiments using intact synaptosomes. following experiments the This was interaction between and approach prompted by the previous observation that synaptobrevin synaptophysin is synaptophysin related to the of in purified synaptic vesicles can be cross- binding synaptobrevin to the SNARE linked by bifunctional For this reagents to a low complex. purpose, all were protein immunoprecipitates M, and for (Johnston Sudhof, 1990). Cross-linking of proteins in analyzed the presence of syntaxin I. In addition, intact synaptosomes with I was disuccinimyl suberate syntaxin directly immunoprecipitated from the same (DSS) resulted in the formation of an adduct with an synaptosomal extract using a syntaxin-specific monoclonal apparent Mr of -55 000 that reacted with antibodies against both antibody (HPC-1; Barnstable et al., to for 1985) analyze synaptophysin and synaptobrevin the (Figure 3). With the presence of synaptophysin. As shown in Figure 2B, exception of bands corresponding I approximately to the syntaxin was absent when a synaptophysin-specific Mr of synaptobrevin and synaptophysin dimers was and trimers, antibody used for immunoprecipitation. Likewise, respectively, no additional adducts were observed (Figure synaptophysin was absent when a syntaxin I-specific 3). Furthermore, the Mr 55 000 adduct containing was both antibody used. Only the immunoprecipitates obtained proteins was immunoprecipitated with both synaptobrevin- with the synaptobrevin II-specific antibody contained both and synaptophysin-specific antibodies (results not shown). synaptophysin and syntaxin I. To control for specificity, Therefore, it is unlikely that the adduct is due to we also blotted cross- for additional membrane proteins of linking of another protein of similar size. These findings synaptic vesicles containing multiple transmembrane demonstrate that synaptophysin and synaptobrevin are domains (SV2: Bajjalieh et al., 1992; Feany et al., 1992; bound to each other in intact membranes and suggest that and p29: Baumert et al., 1990) and for the NMDA-RI the binding observed in detergent extracts reflects an receptor (Sucher et al., 1993). No co-sedimentation of interaction that occurs in vivo. any of these proteins was observed (Figure 2B). Together, these data demonstrate that the synapse contains at least two distinguishable pools of synaptobrevin: one com- Specificity of the synaptobrevin-synaptophysin plexed to synaptophysin and another complexed to syn- interaction taxin, i.e. the SNARE complex, with no overlap All experiments described so far were performed using between them. antibodies directed against synaptobrevin II, the pre- 226 Synaptobrevin binding to synaptophysin im munostai ning for sybil syp crosslinker + (DSS) I- I+T 66- 456 - _ _ synaptophysin 36 - o 29 > 20 - _ < synaptobrevin 1 4 - dye front Fig. 3. Cross-linking of synaptobrevin and synaptophysin in intact synaptosomes using the bifunctional reagent DSS. After cross-linking, the samples were separated by SDS-PAGE on a 15% gel and analyzed by immunoblotting for bands immunoreactive with synaptobrevin- and synaptophysin- specific monoclonal antibodies. Bands were visualized using the Amersham enhanced chemiluminescence (ECL) kit. The long arrow indicates the position of an adduct of -55 kDa that reacts with antibodies for both proteins. The additional immunoreactive bands of high molecular weight that are caused by DSS correspond approximately to the molecular masses expected for homodimers and homotrimers. dominant synaptobrevin isoform of the central nervous immob. GST-fusion (Elferink et al., 1989). We also examined whether system is able to interact with synaptobrevin I, a synaptophysin protein second neuronal isoform (Elferink et al., 1989), and with the non-neuronal homolog cellubrevin (McMahon et al., 1993). Synaptobrevins I and II and cellubrevin were + synaptosomal ++- - - + extract II''-II II expressed in Escherichia coli as GST fusion proteins and immobilized on glutathione-Sepharose beads. The purity synaptophysin of the fusion proteins was checked by SDS-PAGE and immunoblotting (results not shown). Aliquots of the beads were then incubated with a detergent extract of synapto- am syntaxin a/b somes and tested for binding of synaptophysin, syntaxin I and SNAP-25. As shown in Figure 4, synaptophysin EE - SNAP-25 bound to all three synaptobrevin homologs. However, the interaction of synaptophysin with cellubrevin was much weaker than that with synaptobrevins I and II (Figure 4). Rab 3 SNAP-25 and syntaxin I bound to all three synapto- brevin isoforms (Figure 4). Interestingly, cellubrevin bound relatively more of these two proteins than the brain- synaptotagmin specific isoforms. It is possible that the high degree of I and II interferes synaptophysin binding to synaptobrevins 4. Binding of synaptic proteins to immobilized fusion proteins of I and SNAP-25 (see below Fig. with the binding of syntaxin II (syb II). cellubrevin (ceb), synaptobrevin I (syb I) and synaptobrevin The binding of rab3 and synaptotagmin and Discussion). of each were immobilized on equal amounts protein Approximately levels, demonstrating the specificity was below detectable incubated with Triton X-100 extracts of and glutathione-Sepharose see also Figure 5). Further- of the interactions (Figure 4; the beads were for bound After washing, analyzed synaptosomes. Note that none of the antibodies reacted of the proteins bound to the beads proteins by immunoblotting. more, none analyzed fusion lanes). No binding was observed with any of the proteins (- when the carrier protein glutathione S-transferase only the carrier GST was immobilized (data not shown; when only protein was immobilized (results not shown; see also Figure 5; also 6 and et - and + refer to the see Figure Chapman al., 1994). et al., 1994). Chapman of extracts in the absence or synaptosome presence, respectively, we whether the same investigated assay. Using approach, binding similar to were homologous or synaptophysin proteins to 5 shows that with These homologous synaptophysin. Figure of synaptobrevin. proteins capable interacting similar to that of bound with an a isoform efficiency include synaptoporin, synaptophysin (Knaus synaptoporin the neuronal synapto- and the similar membrane again preferring et 1990), structurally synaptophysin, al., In no of over cellubrevin. p29 et and SCAMP brevins contrast, binding (Baumert al., 1990) (Brand proteins p29 data not was detectable shown). that are less or not or SCAMP (Figure 4; and 1993) (p29) (SCAMP) Castle, 227 et aL L.Edelmann immunoprecipitate was not maintained but rather only increased after pretreatment with SDS. The latter observa- tion is consistent with our suggestion that synaptophysin binding prevents the binding of synaptobrevin to the SNARE complex. Furthermore, it demonstrates that the nature of synaptophysin binding to synaptobrevin is different from that of syntaxin I binding to synaptobrevin. synaptophysin 4~ EP 411 Discussion In this synaptopodn study we have demonstrated that the two synaptic _I *iA vesicle proteins synaptobrevin and synaptophysin form a complex, and that the ._ complexed synaptobrevin appears p 29 not to be available for interaction with syntaxin or SNAP-25. we have shown Furthermore, that the protein- but not binds to immobilized Fig. 5. Synaptoporin p29 synaptobrevins. protein interactions for the responsible synaptophysin- as in followed Binding was performed Figure 4, by immunoblot for and The asterisk synaptobrevin are different from those of analysis synaptophysin, synaptoporin p29. complex the a of the cellubrevin fusion that indicates reactive component is protein SNARE sensitive to complex, increasing ionic strength to not identical p29. and mild SDS treatment. Several previous studies have characterized proteins which bind to and synaptophysin synaptobrevin, for antibody respectively. Bennett et al. used a (1992b) variety of immunoprecipitation detergents, Triton CHAPS and including X-100, octyl- synaptophysin synaptobrevin I1 glucoside, to solubilize proteins of purified synaptic 450 300 vesicles to study protein-protein interactions 150 450 NaCI among [mM] synaptic vesicle membrane proteins. An interaction E_4UI@ . _ synaptophysin between synaptophysin and synaptobrevin was observed in which is in with our Triton extracts agreement findings. jpp syn v. _ 11 of interaction However, the specificity this remained synaptobrevin doubtful since other interactions were seen when different were in contrast to our observations. detergents used, The interaction of and is sensitive Fig. 6. synaptobrevin synaptophysin the association of and Furthermore, synaptobrevin synapto- to were as in high ionic strength. Immunoprecipitations performed physin with syntaxin and SNAP-25 was not addressed. In but instead of 140 mM the indicated concentrations of Figure 2, KCI, an earlier Thomas and Betz study, (1990) characterized NaCl were used for and The extraction, precipitation washing. figure the of a membrane named binding protein, physophilin, shows an immunoblot of the analysis immunoprecipitates obtained with anti-synaptophysin and to immobilized vesicles. was (left) anti-synaptobrevin (right) synaptic Binding competed monoclonal antibodies. by synaptophysin. A direct interaction between synapto- physin and physophilin, however, could not be demon- of the strated. Nature Finally, we earlier noticed an interaction between synaptobrevin-synaptophysin interaction synaptobrevin and which at that time was synaptophysin Several were used to approaches gain an into the thought to be non-specific due to its sensitivity to insight high nature of the concentrations of synaptobrevin-synaptophysin interaction. chaotropic agents (Siidhof et al., 1989). we whether In our the First, investigated the interaction is sensitive present study, association between synapto- to ionic Both and physin and increasing strength. synaptobrevin synaptobrevin has been demonstrated using synaptophysin were from extracts three immunoprecipitated independent experimental approaches: co-immuno- containing increasing concentrations of NaCl. As shown precipitation (with identical in results three different in Figure 6, coprecipitation was reduced at 300 mM NaCl detergents), of to binding synaptophysin immobilized and virtually abolished at 450 mM NaCl, suggesting that recombinant synaptobrevin and cross-linking. Although ionic interactions a play major role. Second, we examined we cannot exclude that synaptophysin undergoes additional whether this interaction is sensitive to a brief exposure protein-protein interactions, we did not see any other to SDS. This experiment was prompted proteins in the by the recent synaptophysin-synaptobrevin complex observation that the SNARE complex is resistant to mild (see e.g. the stains of protein the immunoprecipitates in SDS treatment et (Hayashi al., 1994), probably due to the Figure 2A and the control blots in Figure 2B). This stability of coiled-coil interactions. Immunoprecipitation interaction thus appears to be specific and selective. The was performed from an extract that was first solubilized stoichiometry and prevalence of the complex remain to with 1% SDS and then diluted with Triton X-100 to yield be determined. In our experiments we found that at least final concentrations of 0.05% SDS and 1% Triton X- half of the entire synaptophysin pool precipitates with 100. As shown in Figure 7, the pretreatment with SDS synaptobrevin and vice versa (unpublished observations). completely abolished the interaction between synapto- This appears to be far more than complexed to syntaxin physin and synaptobrevin. This was demonstrated by both I and SNAP-25. A detailed quantitation of these inter- silver of staining the electrophoretically separated proteins actions is currently under way. of the immunoprecipitates and immunoblotting. In con- The domains of synaptobrevin that are responsible for trast, the amount of syntaxin I in the synaptobrevin the interaction with synaptophysin remain to be deter- 228 Synaptobrevin binding to synaptophysin total extract extract after IP * . immunoprecipitate synaptobrevin I, - _ h.c. ,-synaptophysin *4*N-syntaxin alb synaptophysin E. _ * I. c syntaxin a/b imio synaptobrevin immunoblot silver stain Fig. 7. SDS pretreatment blocks the synaptobrevin-synaptophysin interaction but increases the binding of syntaxin. Synaptosomal extracts were pretreated with SDS and then incubated with excess Triton X-100 (see Materials and methods), before immunoprecipitation was performed using the synaptobrevin H-specific monoclonal antibody Cl 69.1. All immunoprecipitates were analyzed by SDS-PAGE and immunoblotting for synaptobrevin, synaptophysin and syntaxin I. Left, immunoblots; right, silver stain of the precipitates obtained before and after SDS pretreatment. See the legend to Figure 2 for details. h.c, l.c., heavy and light chain of IgG, respectively. The band visible in the precipitate of the synaptophysin blots corresponds to the heavy chain of IgG that reacted with the secondary antibody. mined. The sensitivity to high ionic strength suggests that to synaptobrevin, although it shares only 58% of amino hydrophilic regions play a major role, implicating the acid residues with synaptophysin. cytoplasmic domains of both proteins. However, truncation Our findings shed new light on the functional role of the transmembrane domain of synaptobrevin or replace- of the vesicle protein synaptophysin and its relatives. ment of this domain with the transmembrane domain of Synaptophysin is a membrane glycoprotein with four syntaxin resulted in a loss of synaptophysin binding, while membrane-spanning domains and an extended cytoplasmic binding of the t-SNAREs syntaxin and SNAP-25 was not tail. This tail contains nine unusual repeats and is phos- affected (unpublished observations; see also Calakos et al., phorylated by c-src kinase on tyrosine residues (for a 1994; Hayashi et al., 1994). Furthermore, a syntaxin Ta review see Jahn and Sudhof, 1994). Although this protein mutant containing the transmembrane domain of synapto- was the first synaptic vesicle protein to be characterized brevin did not bind synaptophysin. Although these negative in molecular terms (Leube et al., 1987; Sudhof et al., results may be due to misfolding of the recombinant 1989), its function has remained unclear. An early study mutant proteins, it is possible that the Ca2' transmembrane reporting binding (Rehm et al., 1986) could not be domain of synaptobrevin is involved in the confirmed binding of (Brose et al., 1992). When purified synapto- synaptophysin. Clearly, the interaction of physin is into synaptobrevin incorporated lipid bilayers it forms non- with synaptophysin is different from that with syntaxin, ion selective, voltage-dependent channels (Thomas et al., which is not sensitive to ionic strength and is independent 1988), but the physiological significance of this observa- of the transmembrane domains (Calakos et tion remains to be established. al., 1994). Interestingly, the primary sequence of synaptophysin The presence of two of non-overlapping pools synapto- to be in appears less conserved evolution than that of brevin, one complexed to and the other synaptophysin et Cowan et synaptobrevin (Sudhof al., 1989; al., 1990; complexed to the SNARE a mechanism complex, suggests for a review see Jahn and with the which Sudhof, 1994), by synaptophysin can control the of ability synapto- transmembrane domains more brevin to enter the SNARE for being highly conserved this complex. Support than other of the molecule. view is results which demonstrate parts Likewise, provided by preliminary synaptophysin to be absent from dense that recombinant SNAP-25 with appears peptide-containing large competes synaptophysin core a review see De Camilli and for to granules (for Jahn, binding synaptobrevin (unpublished observations). 1990), whereas synaptobrevin is Synaptophysin may function as a molecular switch that present (unpublished the existence of additional allows the association of with the t-SNAREs observations). However, synaptobrevin on these cannot after it dissociates from Such unknown isoforms only a synaptophysin granules synaptophysin. It is that the structural determinants would ensure that binds at be ruled out. regulation synaptobrevin possible only for to the correct time and location to the for instance of synaptophysin t-SNAREs, required binding synaptobrevin is activated. This interaction reside in its thus for when the secondary structure, allowing greater synapse may for control of v-SNAREs in the amino acid This is confirmed reflect a mechanism in variability sequence. general binds well fusion events. It will be to find our observation that membrane out by synaptoporin interesting equally 229 L.Edelmann et aL to recombinant and Binding proteins cross-linking of whether other v-SNAREs such as cellubrevin or the yeast synaptosomes homologs Sncl/Snc2 form similar complexes with partner Recombinant proteins were immobilized on glutathione-Sepharose proteins and what the structural features of such hypo- beads were solubilized in Triton X- (-1-5 beads). Synaptosomes gg/gl thetical partner proteins may be. Furthermore, it remains 100 containing extraction buffer (see above). 10 p1 of beads containing recombinant protein were added to 1 ml extracts. Incubation and washing to established are be how these interactions regulated were performed as for immunoprecipitation (see above). intracellular membrane traffic. during For cross-linking, synaptosomes were resuspended at a protein con- centration of 1.5 mg/ml in Krebs-Ringer buffer (140 mM NaCl, 5 mM NaHCO3, 1 mM MgCl2, 1.2 mM Na2HPO4, 10 mM glucose, 20 mM HEPES, pH 7.4) and pre-warmed for 10 min at room temperature. Materials and methods DSS, dissolved in dimethyl sulfoxide, was then added to yield final concentrations of 5 mM DSS and 5% (v/v) of the solvent. After Antibodies incubation under rotation for 45 min at room temperature, 100 mM For the study of synaptobrevin, we generated a new monoclonal antibody (final concentration) of Tris-Cl (pH 7.4) were added as quencher, since the antibodies described previously (Baumert et al., 1989) were followed by a further incubation for 30 min. The membranes were not suitable for immunoprecipitation experiments (unpublished observa- pelleted in a microfuge (5000 r.p.m. for 3 min) and then resuspended in tions). synthetic peptide (CSATAATVPPAAPAGEG) corresponding 1.5 ml of ice-cold 150 mM NaCl, 20 mM Tris-Cl (pH 7.4) containing to the N-terminus of rat II isoform in synaptobrevin (the predominant 1% (v/v) Triton X-100. After incubation at 4°C for 1 h, insoluble the obtained from Keck CNS), Biopolymer Facility (Yale University, at 40 for 20 material was removed by centrifugation 000 r.p.m. min in was immobilized to New Haven, CT) keyhole limpet hemocyanine a Beckman T1 100 rotor. Fifteen pl aliquots of the supematants were and used for immunization (Schneider et al., 1983) the of Balb/c mice. analyzed by SDS-PAGE and immunoblotting. and were to standard Fusion, propagation screening performed according et A procedures (Kohler and Milstein, 1975; Jahn al., 1985). single Other methods hybridoma line (clone Cl 69.1) was established. was to Laemmli the monoclonal SDS-PAGE performed according (1970) using The antibodies have been described following previously: Bio-Rad Protean II and minigel apparatus. Immunoblotting visualization antibodies directed against synaptophysin (clone C 7.3; Jahn et al., with A was as described et [1251]protein performed (Jahn al., 1985) Brose et rab3 1985), synaptotagmin (clone 41.1; al., 1992), (clone 42.1, unless indicated otherwise Silver of electro- (Figure 3). staining for B and Matteoli et specific rab3A, C; al., 1991), synaptobrevin (clone was carried out to phoretically separated proteins according Morrissey Baumert et NMDA RI et 10.1; al., 1989), receptor (Sucher al., 1993) Protein was determined the bichinoninic acid (1981). using protein assay and rabbit serum directed et Monoclonal against p29 (Baumert al., 1990). the manufacturer's reagent (BCA assay, Pierce, Rockford, IL) following antibodies Ia/b and SV2 were against syntaxin (HPC-1) kindly given by instructions. Dr C.Barnstable Barnstable et and Dr (New Haven, CT; al., 1985) and Monoclonal K.Buckley (Boston, MA; Buckley Kelly, 1985). antibody for was raised recombinant SNAP-25 Cl 71.1, specific SNAP-25, using Acknowledgements and will be described in detail elsewhere and (D.Bruns R.Jahn, manuscript in A rabbit serum directed preparation). against synaptoporin (Fykse The authors wish to thank Nikki Barton and Silke Engers for their skilful a monoclonal directed SCAMP et al., 1993) and antibody against (Brand assistance in recombinant and in the preparing proteins establishing Dr and Castle, 1993) were kindly provided by T.C.Sudhof (Dallas, TX) hybridoma line Cl 69.1. We are grateful to Drs C.Barnstable (New Dr and J.D.Castle (Charlottesville, VA), respectively. Haven, CT), S.Brand and D.Castle (Charlottesville), and K.Buckley for their of antibodies. we (Boston, MA) generous supply Furthermore, thank Drs C.Solimena and P.De Camilli for critical Immunoprecipitation (New Haven, CT) Isolated nerve terminals were from rat cerebral advice and discussions. (synaptosomes) prepared stimulating cortex to standard von Mollard et according procedures (Fischer al., McMahon et Ficoll 1991; al., 1992) using density gradient centrifugation as the last For 1 of purification step. solubilization, mg synaptosomal References was and dissolved in 1 ml of ice-cold extraction buffer protein pelleted mM 20 mM HEPES-KOH 2 mM unless Peterson,K., Shinghal,R. and Scheller,R.H. (1992) (140 KCI, pH 7.3, EDTA, Bajjalieh,S.M., indicated 1% Triton X-100. Ten of ascites Science, 257, 1271-1273. otherwise) containing (v/v) ,ul fluid to -10-30 were added and the Barnstable,C.J., Hofstein,R. and Akagawa,K. (1985) Dev. Brain Res., (corresponding ,ug specific IgG) incubation was carried out for 6-8 h at One hundred p1 of 20, 286-290. 4°C. protein De were Baumert,M., Maycox,P.R., Navone,F., Camilli,P. and Jahn,R. (1989) G-Sepharose suspension (Pharmacia, Piscataway, NJ) added, EMBO 379-384. followed incubation for 1 h. The beads were collected J., 8, by by centrifugation Fischer von and washed three times in extraction buffer Triton X-100. Baumert,M., Takei,K., Hartinger,J., Burger,P.M., Mollard,G., containing De and J. Cell For SDS 1 of was first solubilized in Maycox,P.R., Camilli,P. Jahn,R. (1990) Biol., 110, pretreatment, mg synaptosomes 1285-1294. 50 of extraction buffer 2% ,l containing (w/v) SDS, followed by the Bennett,M.K. and Scheller,R.H. (1993) Proc. Nati Acad. Sci. USA, 90, addition of 950 of extraction buffer containing 1% Triton X-100 to g1 2559-2563. the quench SDS. Immunoprecipitation was then carried out exactly as Bennett,M.K., Calakos,N. and Scheller,R.H. 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