SNAP-25 is required for a late postdocking step in Ca2+-dependent exocytosis.

The Ca2+-activated fusion of large dense core vesicles (LDCVs) with the plasma membrane is reconstituted in mechanically permeabilized PC12 cells by provision of millimolar MgATP and cytosolic proteins. Ca2+-activated LDCV exocytosis was inhibited completely by the type E but not the type A botulinum neurotoxin (BoNT) even though both BoNTs were equally effective in proteolytically cleaving the synaptosome-associated protein of 25 kDa (SNAP-25). The greater inhibition of exocytosis by BoNT E correlated with a greater destabilization of detergent-extracted complexes consisting of SNAP-25, synaptobrevin, and syntaxin. LDCVs in permeable PC12 cells can be poised at a late postdocking, prefusion state by MgATP-dependent priming processes catalyzed by N-ethylmaleimide sensitive factor and priming in exocytosis proteins. BoNT E completely blocked Ca2+-activated LDCV exocytosis in ATP-primed cells, whereas BoNT A was only slightly inhibitory, implying that the C-terminal region of SNAP-25 (Ile181-Gln197) between the cleavage sites for BoNT E and BoNT A is essential for late postdocking steps. A required role for SNAP-25 at this stage was also indicated by inhibition of Ca2+-activated LDCV fusion in ATP-primed cells by a C-terminal peptide antibody. We conclude that plasma membrane SNAP-25, particularly residues 181-197, is required for Ca2+-regulated membrane fusion at a step beyond LDCV docking and ATP utilization.

Neurotransmitter and peptide hormone secretion require fusion between secretory vesicles and the plasma membrane, an exocytotic process activated by cytoplasmic Ca 2ϩ elevation. Understanding regulated secretion requires identification of molecular components that mediate docking and fusion reactions and delineation of rate-limiting steps that are Ca 2ϩregulated (1). The synaptic vesicle protein synaptobrevin and the presynaptic membrane proteins syntaxin and SNAP- 25 1 are required components of the exocytotic apparatus as indicated by the inhibitory action of BoNTs and Tetx, which involves the specific endoproteolytic cleavage of these protein substrates (2,3). The three toxin substrates were independently identified as receptors for SNAPs, proteins required for the membrane binding of NSF, a late acting component in constitutive membrane fusion reactions (4). A characterized ternary complex containing synaptobrevin, syntaxin, and SNAP-25 was suggested to represent a docking complex that mediates the targeting, docking, or fusion of secretory vesicles (5,6). Identification of an ATP-dependent catalytic activity of NSF/SNAP that promotes the disassembly of ternary complexes in vitro led to the suggestion that a similar reaction in vivo was responsible for late steps in membrane fusion (7).
Ca 2ϩ -triggered secretion in permeable neuroendocrine cells requires ATP; however, the requirement for ATP precedes that for Ca 2ϩ (8,9). ATP hydrolysis is required for prefusion events that prime the exocytotic apparatus, whereas Ca 2ϩ -activated fusion proceeds in the absence of ATP (8 -11). 2 In priming, ATP serves as a substrate for polyphosphoinositide synthesis (10,11) and as a substrate for the SNAP-dependent ATPase activity of NSF that catalyzes rearrangement of docking protein complexes. 2 These studies experimentally identify a late step in the exocytotic pathway beyond LDCV docking and ATP utilization that is proximal to Ca 2ϩ -dependent fusion reactions. To characterize events that lead to or are directly involved in membrane fusion, it is important to identify molecular components that act at this late Ca 2ϩ -dependent step. The studies presented here describe a requirement for SNAP-25 at this and specify a role for the C-terminal region of SNAP-25 (Ile 181 -Gln 197 ) between the cleavage sites for BoNT E and BoNT A.  (12). Secretion assays were conducted either as single stage or as two-stage assays (9,14). For the former, permeable cells were incubated for 15 min at 30°C in KGlu-BSA buffer supplemented with CaCl 2 (to achieve 10 M Ca 2ϩ f ), 0.002 M MgATP, and 0.5 mg/ml rat brain cytosol. Two-stage assays were conducted as separate priming incubations (30 min at 30°C in KGlu-BSA buffer supplemented with 0.002 M MgATP plus 1 mg/ml rat brain cytosol) followed by triggering incubations (1-3 min at 30°C in KGlu-BSA buffer supplemented with 10 M Ca 2ϩ plus 0.5 mg/ml rat brain cytosol) with extensive washing between incubations. [ 3 H]NE release was determined by centrifugation of permeable cells at 800 ϫ g for 30 min and scintillation counting of 3 H in supernatants and in cell pellets to express NE release as percent of the total 3 H. The SNAP-25 antibody used to inhibit secretion is a rabbit polyclonal antibody raised against the C-terminal peptide ANQRATKMLGSG generously provided by M. Takahashi (Tokyo). IgGs were purified by chromatography on protein A-Sepharose.

PC12
Immunoprecipitation and Immunoblotting-Permeable PC12 cells were recovered by centrifugation (800 ϫ g for 15 min) and resuspended in lysis buffer (0.02 M HEPES, 0.1 M KCl, 0.002 M EDTA, 0.0005 M ATP, 0.001 M dithiothreitol, 1% Triton X-100, and 0.4 mM phenylmethylsulfonyl fluoride, pH 7.3). Docking complexes isolated from dilute detergent extracts were not formed following detergent extraction since their * This work was supported by United States Public Health Service Grants DK25861, DK40428 (to T. F. J. M.), and NS17742 (to B. R. D.) and an award from the Northwestern Mutual Life Foundation, Wisconsin (to A. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ To whom correspondence should be addressed: Dept. of Biochemistry, University of Wisconsin, 420 Henry Mall, Madison, WI 53706. Tel.: 608-263-2427; Fax: 608-262-3453; E-mail: tfmartin@macc.wisc.edu. 1 The abbreviations used are: SNAP-25, synaptosome-associated protein of 25 kDa; NE, norepinephrine; BoNT, botulinum neurotoxin; Tetx, tetanus toxin; LDCV, large dense core vesicle; NSF, N-ethylmaleimidesensitive factor; SNAP, soluble NSF attachment protein; KGlu, potas-composition was unaffected by extraction volume over a 10-fold range; however, their composition was affected by cell pretreatment conditions that were terminated by washing prior to detergent extraction. Moreover, similar complexes were detected by direct SDS extraction. 2 Detergent extracts, clarified by centrifugation at 100,000 ϫ g for 90 min, were used for immunoprecipitations employing syntaxin (HPC-1, Sigma) or SNAP-25 (Sternberger Monoclonals Inc., Baltimore, MD) monoclonal antibodies conjugated to Protein G-agarose beads by dimethylpimelimidate (Pierce). Immune complexes were recovered by centrifugation at 12,000 ϫ g for 15 min and washed 5 times in lysis buffer, which removed all nonspecifically bound proteins. Recovery of SNAP-25 and syntaxin in immunoprecipitates exceeded 60%. For quantitation of coimmunoprecipitated proteins, all results were normalized to the recovery of the immunoprecipitated syntaxin or SNAP-25. Immunoprecipitates were eluted by boiling in SDS sample buffer (0.0625 M Tris, pH 6.8, 2% sodium dodecyl sulfate, 10% glycerol, 0.0025% bromphenol blue, 0.002% pyronin Y, 0.1 M dithiothreitol), electrophoresed on 10% SDS-polyacrylamide gels, transferred to nitrocellulose membranes, immunoblotted with primary antibodies, and probed with 125 I-protein A (DuPont NEN) or 125 I-goat anti-mouse IgG (DuPont NEN) followed by autoradiography at Ϫ70°C with an intensifying screen. Densitometry of autoradiograms was performed on a Molecular Dynamics personal densitometer SI using ImageQuaNT software. Antibodies used for immunoblotting were: rabbit synaptobrevin-2 antibodies to an N-terminal peptide SATAATVPPAAPAGEGGPPC generously provided by M. Takahashi (Tokyo), rabbit synaptotagmin I antibodies to a cytoplasmic domain glutathione S-transferase fusion protein generously provided by R. H. Scheller (Stanford), and rabbit SNAP-25 antibodies to a C-terminal dodecapeptide generously provided by M. C. Wilson (La Jolla). Syntaxin and SNAP-25 monoclonal antibodies were also used for immunoblotting. BoNT A, BoNT B, and BoNT E were isolated from cultures of Clostridium botulinum and purified as described previously (15)(16)(17). BoNT C and Tetx were generously provided by S. Kozaki (Osaka) and U. Weller (Mainz), respectively. BoNT B and E were nicked to the dichain form with trypsin (17). Immediately before use, BoNTs and Tetx were incubated with 5 mM dithiothreitol for 1 h at 23°C to reduce interchain disulfide bonds.

RESULTS
Release of the neurotransmitter NE from LDCVs in permeable PC12 cells is triggered by 10 M calcium in the presence of ATP and cytosolic proteins (14). Ca 2ϩ -activated release was inhibited by BoNT E and BoNT A (Fig. 1A), confirming previ-ous results (18). The inhibition of Ca 2ϩ -dependent NE secretion by BoNT E was complete whereas that by BoNT A was incomplete even at high concentrations. Since SNAP-25 is a specific substrate for endoproteolysis by both BoNT A and BoNT E (19 -22), we determined the extent of SNAP-25 proteolysis and found that both neurotoxins were equally effective in cleaving Ͼ75% of the SNAP-25 in permeable PC12 cells (Fig. 1B). Since the sites of proteolysis for each toxin (Arg 180 -Ile 181 and Gln 197 -Arg 198 for BoNT E and BoNT A, respectively (21,22)) are near the C terminus of SNAP-25 but 17 residues apart, the preferential inhibition by BoNT E implies that the Ile 181 -Gln 197 domain of SNAP-25 is particularly important for regulated LDCV exocytosis in PC12 cells.
SNAP-25 forms binary complexes with the cytoplasmic domains of both recombinant synaptobrevin and syntaxin (23,24) and participates in stable ternary complex formation with these proteins (23)(24)(25). The C terminus of SNAP-25 appears to be required for interactions with synaptobrevin but not with syntaxin and not for ternary complex formation as determined with BoNT A-treated SNAP-25 (23,24). However, the effect of BoNT-catalyzed SNAP-25 cleavage on in situ docking complexes in cells has not previously been determined. We isolated complexes containing SNAP-25, syntaxin, and synaptobrevin from detergent extracts of permeable PC12 cells by co-immunoprecipitation with SNAP-25 or syntaxin antibodies ( Fig. 2A). Treatment of permeable cells with BoNT E and A resulted in extensive cleavage of SNAP-25 with retention of SNAP-25 fragments in the immune complexes. BoNT E treatment reduced the recovery of synaptobrevin in syntaxin and SNAP-25 immunoprecipitates whereas BoNT A treatment had little effect (Fig.  2, A and B). BoNT E treatment also reduced the recovery of syntaxin in SNAP-25 immunoprecipitates and the recovery of SNAP-25 in syntaxin immunoprecipitates, whereas BoNT A treatment did not. The results indicate that BoNT E treatment markedly destabilized docking complexes whereas BoNT A treatment had little effect. Hence, the degree of destabilization correlated with the severity of inhibition of regulated secretion (see Fig. 1A).
Since the cleavage sites for BoNTs and Tetx in syntaxin, synaptobrevin, and SNAP-25 reside in domains predicted to form coiled-coils that are potentially involved in protein-protein interactions (2), it has been suggested that uncomplexed proteins are the preferred substrates for neurotoxin proteases (2,24). Ternary complexes formed from recombinant protein cytoplasmic domains were found to be resistant to neurotoxin proteolysis (24,26). However, it is unclear that proteolysis in secretory cells is restricted to a point prior to vesicle docking and docking complex formation, since treatment of susceptible neural cells with BoNTs does not alter the number of vesicles docked in an active zone (27). In recent studies, it was shown that LDCVs in permeable PC12 cells are docked but undergo a prefusion, ATP-dependent priming step during which there is NSF-catalyzed disassembly of docking complexes. 2 Hence, priming might allow a postdocking exposure of proteins to neurotoxin cleavage. As shown in Fig. 3, BoNT E treatment following the ATP-dependent priming reaction (P) exerted a greater destabilizing effect on protein complexes compared with treatment without priming (D). These results confirmed the partial disassembly of docking complexes during ATP-dependent priming prior to fusion 2 and imply that BoNT E can act at a late postdocking, prefusion step.
To further address BoNT sensitivity at a late step, the effects of BoNTs on Ca 2ϩ -activated secretion from ATP-primed permeable PC12 cells were examined. LDCVs in ATP-primed cells are poised at a late stage and engage in the rapid release of NE upon addition of Ca 2ϩ and a cytosolic protein in the absence of

SNAP-25 Requirement for Regulated Exocytosis 20228
ATP (9). 2 BoNT E treatment completely inhibited Ca 2ϩ -triggered neurotransmitter release (Fig. 4A) indicating that SNAP-25 is required for regulated fusion at a late, post-ATP utilization step. This conclusion was also indicated by finding that IgGs from a SNAP-25 C-terminal peptide-specific antiserum inhibited Ca 2ϩ -triggered secretion from ATP-primed permeable cells whereas nonimmune IgGs were without effect (Fig. 4C). In contrast, BoNT A exerted only weak partial inhibitory effects (Fig. 4A), implying that the C-terminal region of SNAP-25 between BoNT E and BoNT A cleavage sites, but not the most C-terminal region, was required for Ca 2ϩ -triggered fusion. The uniqueness of the incomplete inhibition by BoNT A was indicated by the finding that Tetx or BoNT B and BoNT C, which extensively proteolyzed synaptobrevin and syntaxin, respectively (not shown), completely inhibited Ca 2ϩ -activated secretion in ATP-primed cells (Fig. 4B).
There is a Ca 2ϩ -independent pathway for stimulating NE secretion from permeable PC12 cells that is activated by nonhydrolyzable GTP analogs (e.g. GMPPNP) in the absence of Ca 2ϩ , ATP, and cytosolic proteins 3 (28). Although the mechanism of stimulation by GMPPNP remains to be elucidated, this pathway appears to converge with the Ca 2ϩ -activated pathway at a point prior to or at SNAP-25 since GMPPNP-stimulated NE secretion was effectively inhibited by BoNT E and to a much lesser extent by BoNT A (Fig. 4D). In addition, the C-terminal SNAP-25 peptide-specific antibody completely inhibited GMPPNP-stimulated secretion (Fig. 4E). Complete inhibition of GMPPNP-stimulated secretion was also observed with Tetx, BoNT B, and BoNT C, indicating requirements for synaptobrevin and syntaxin as well. DISCUSSION An unanticipated role for SNAP-25 in regulated neurotransmitter secretion was suggested by its recovery on NSF/SNAP affinity columns in a search for membrane SNAP receptors (4) and by its identification as a specific substrate for proteolysis by BoNT E and BoNT A (2, 3). Although a role for SNAP-25 in regulated exocytosis is implied by its discovery as a substrate for BoNTs, its precise role remains unknown. As a component of heteroligomeric complexes with syntaxin and synaptobrevin, it has been suggested to be a presynaptic membrane docking component for the specific targeting of synaptic vesicles to the active zone (7). While our studies did not address the role of SNAP-25 in docking, they did provide evidence for a postdocking role for SNAP-25 in regulated exocytosis.
The majority of the LDCVs in PC12 cells are anchored close to or docked at plasma membrane sites. 2 Ca 2ϩ elevations prompt a single rapid phase of NE release involving most of the LDCVs (9,14). In permeable PC12 cells, putative docking interactions between secretory vesicles and plasma membrane can be detected as detergent-soluble complexes containing synaptobrevin, SNAP-25, and syntaxin. 2 While the majority of LDCVs are docked, Ca 2ϩ -dependent NE secretion still requires ATP-dependent reactions that consist of prefusion activation processes catalyzed by NSF/SNAP 2 and priming in exocytosis proteins (9 -11). ATP-dependent priming activates LDCVs and poises them for Ca 2ϩ -triggered fusion at a step beyond docking and ATP utilization, and Ca 2ϩ -triggered fusion proceeds in the absence of ATP utilization (9). 2 The inhibition of Ca 2ϩ -triggered fusion of LDCVs in ATP-primed cells by BoNT E-catalyzed proteolysis of SNAP-25 and by SNAP-25 antibody implies a postdocking, post-ATP utilization role for SNAP-25 in steps that immediately precede or directly cause membrane fusion.
The paradox that BoNT A and BoNT E are equally effective in proteolyzing SNAP-25 whereas only the latter substantially inhibits Ca 2ϩ -activated exocytosis in permeable PC12 cells can be resolved by the suggestion that the most C-terminal residues Arg 198 3. BoNT E preferentially destabilizes docking protein complexes following ATP-dependent priming. Permeable PC12 cells were preincubated at 30°C for 30 min with 1 mg/ml rat brain cytosol in the absence (D, deprimed) or presence (P, primed) of 2 mM MgATP. An equivalent number of primed permeable cells was washed and reincubated at 30°C for 5 min in the presence of 10 M Ca 2ϩ and 0.5 mg/ml rat brain cytosol to elicit secretion (T, triggered). Permeable cells were subsequently incubated for 5 min at 30°C in the absence (C) or presence (E) of 4 ϫ 10 Ϫ7 M BoNT E. Triton-soluble protein complexes were isolated by immunoprecipitation with SNAP-25 or syntaxin antibodies and analyzed by immunoblotting for synaptobrevin. SNAP-25 and syntaxin recoveries differed by less than 20% in the parallel samples. Results are representative of three similar experiments. essential for late steps of regulated LDCV exocytosis than the region Ile 181 -Gln 197 that is additionally removed by BoNT E cleavage. An alternative possibility that the Ile 181 -Gly 206 SNAP-25 fragment generated by BoNT E cleavage, which is retained in ternary complexes, interferes with fusion events seems unlikely since direct tests of this and related C-terminal peptides failed to reveal inhibition (not shown).
Prior studies of recombinant protein interactions indicated an essential role for C-terminal SNAP-25 residues in synaptobrevin/SNAP-25 but not syntaxin/SNAP-25 interactions (23,24). However, BoNT A-treated SNAP-25 was as impaired in binary interactions with synaptobrevin as was BoNT E-treated SNAP-25 (24), indicating that the role for Ile 181 -Gly 206 in a late postdocking step cannot reside with synaptobrevin interactions alone. BoNT E-treated SNAP-25 was much more strongly impaired than BoNT A-treated SNAP-25 in assembling into a highly stable, SDS-resistant ternary complex (24) whereas BoNT A-cleaved SNAP-25 functioned normally in the formation and NSF-catalyzed disassembly of docking complexes (13,26). We found a correlation between toxin efficacy in inhibiting secretion and the destabilization of heteromeric complexes isolated by co-immunoprecipitation. This correlation between toxin efficacy in inhibition of secretion and docking complex stability is intriguing and likely indicates that Ile 181 -Gln 197 , located in a region of SNAP-25 predicted to form coiled-coils (23), is required for interactions in a multiprotein complex. Since the C terminus of SNAP-25 is not required for interactions with syntaxin (23,24) and since NSF acts to promote the dissociation of SNAP-25 from synaptobrevin at a prefusion step (7), 2 it is possible that the Ile 181 -Gln 197 region of SNAP-25 is required for interactions with proteins other than syntaxin or synaptobrevin to form complexes that are directly involved in catalyzing membrane fusion. FIG. 4. SNAP-25 is required for a stage of regulated secretion following ATP-dependent priming. A, BoNT inhibition of Ca 2ϩactivated secretion in ATP-primed permeable PC12 cells. Permeable PC12 cells were preincubated with 2 mM MgATP plus 1 mg/ml rat brain cytosol for 30 min at 30°C, chilled, washed extensively, and further incubated with indicated concentrations of BoNTs (q, BoNT E; E, BoNT A) for 5 min at 30°C. Subsequently 10 M Ca 2ϩ plus 0.5 mg/ml rat brain cytosol was added for a 3-min secretion incubation at 30°C. B, inhibition of Ca 2ϩ -activated secretion in ATP-primed permeable PC12 cells by Clostridium neurotoxins. Experiments were performed as in panel A with 10 Ϫ6 M Tetx or BoNT C or 3 ϫ 10 Ϫ7 M BoNT B, BoNT E, or BoNT A. C, inhibition of Ca 2ϩ -activated secretion in ATP-primed permeable PC12 cells by SNAP-25 C-terminal peptide antibody. Permeable cells were ATP-primed, washed, and further incubated on ice for 60 min with indicated concentrations of IgG purified from SNAP-25 C-terminal peptide antiserum (q) or nonimmune serum (E) prior to a 3-min secretion incubation with Ca 2ϩ at 30°C. D, inhibition of GMPPNP-stimulated NE secretion by BoNT E and A. Permeable PC12 cells were incubated with the indicated concentrations of BoNTs for 5 min at 30°C prior to conducting a 30-min secretion incubation with 3 M Mg-GMPPNP in the absence of Ca 2ϩ . E, inhibition of GMPPNP-activated secretion in permeable PC12 cells by Clostridium neurotoxins and SNAP-25 peptide antibody. Permeable PC12 cells were incubated with SNAP-25 antibody (called SNAP-25) or control IgG for 60 min on ice or with indicated toxins for 5 min at 30°C prior to a 30-min secretion incubation at 30°C conducted with 3 M Mg-GMPPNP.