©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Ca Regulates the Interaction between Synaptotagmin and Syntaxin 1 (*)

(Received for publication, May 22, 1995; and in revised form, June 29, 1995)

Edwin R. Chapman (§) Phyllis I. Hanson (¶) Seong An Reinhard Jahn

From the Howard Hughes Medical Institute and Departments of Pharmacology and Cell Biology, Boyer Center for Molecular Medicine, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

While there is compelling evidence that the synaptic vesicle protein synaptotagmin serves as the major Ca sensor for regulated exocytosis, it is not known how Ca binding initiates membrane fusion. Here we report that Ca increases the affinity, by approximately 2 orders of magnitude, between synaptotagmin and syntaxin 1, a component of the synaptic fusion apparatus. This effect is specific for divalent cations which can stimulate exocytosis of synaptic vesicles (Ca > Ba, Sr Mg). The Ca-dependence of the interaction was composed of two components with EC values of 0.7 and 180 µM Ca. The interaction is mediated by the carboxyl-terminal region of syntaxin 1 (residues 194-288) and is regulated by a novel Ca-binding site(s) which does not require phospholipids and is not disrupted by mutations that abolish Ca-dependent phospholipid binding to synaptotagmin. We propose that this interaction constitutes an essential step in excitation-secretion coupling.


INTRODUCTION

Exocytosis of synaptic vesicles is strictly controlled by Ca ions (Katz, 1969; Augustine et al., 1987). Presumably, Ca ions initiate conformational changes in proteins which ultimately catalyze membrane fusion. The Ca binding properties of the synaptic vesicle protein synaptotagmin are consistent with the requirements for the exocytotic Ca receptor (Brose et al., 1992; reviewed by DeBello et al.(1993), Popov and Poo(1993), Chapman and Jahn (1994b)). Indeed, gene disruption (Geppert et al., 1994; Broadie et al., 1994; Littleton et al., 1994; Nonet et al., 1994) and microinjection experiments (Elferink et al., 1993; Bommert et al., 1993) have provided strong evidence that synaptotagmin functions as the major Ca sensor in regulated exocytosis. However, the mechanism by which Ca binding to synaptotagmin triggers membrane fusion has yet to be elucidated. To address this issue, efforts have been directed at identifying downstream effectors of Ca-synaptotagmin action. Synaptotagmin was shown to form Ca-independent complexes with neurexins (Petrenko et al., 1991), a family of neuronal cell surface proteins, and the adaptor protein AP-2 (Zhang et al., 1994). Synaptotagmin also interacts with syntaxin 1, an abundant neuronal plasma membrane protein associated with N-type Ca channels (Bennett et al., 1992; Yoshida et al., 1992; Sheng et al., 1994). This property suggests that syntaxin may physically link the calcium receptor to the site of Ca influx.

Syntaxin has been shown recently to form a complex with the synaptic vesicle protein synaptobrevin (VAMP) and the synaptic plasma membrane protein SNAP-25. This complex serves as the membrane receptor for the soluble proteins NSF (^1)(N-ethylmaleimide-sensitive fusion protein) and SNAPs (soluble NSF attachment proteins), factors required for intracellular membrane fusion. Therefore, syntaxin, SNAP-25, and synaptobrevin have been designated as SNAREs (SNAP receptors (Söllner et al., 1993a)). In addition, each of the SNAREs is selectively proteolyzed by clostridial neurotoxins, potent inhibitors of exocytosis (Schiavo et al., 1992; Link et al., 1992; Blasi et al., 1993a, 1993b). Furthermore, disruption of the syntaxin 1 gene in Drosophila abolishes evoked neurotransmission (Schulze et al., 1995). Thus, the complex containing syntaxin, SNAP-25, and synaptobrevin is thought to comprise the core of the exocytotic fusion machine. Consequently, the interaction between syntaxin 1 and synaptotagmin is of particular interest, since it could provide a direct link between the Ca-sensor and the fusion apparatus. In the present study we have characterized the interaction between synaptotagmin and syntaxin 1 and report that it is regulated by Ca ions.


EXPERIMENTAL PROCEDURES

Immunoprecipitation

All manipulations were carried out on ice. Synaptosomes were prepared by homogenizing one to two rat brains in 30 ml of 320 mM sucrose with 10 strokes at 900 rpm using a Teflon glass homogenizer. The homogenate was centrifuged at 5000 rpm for 2 min in an SS34 rotor, and the crude synaptosomes were collected by centrifugation of the supernatant at 11,000 rpm for 12 min in an SS34 rotor. Synaptosomes were solubilized for 45 min at a detergent to protein ratio of 10:1 (w:w) with 1% Triton X-100 in 50 mM HEPES, pH 7.2, 100 mM NaCl, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 20 µg/ml aprotinin). Insoluble material was removed by centrifugation at 50,000 rpm in a TLA 100.3 rotor for 15 min, and samples were then supplemented with 2 mM EGTA or 0.5 mM CaCl(2). Immunoprecipitations were carried out by incubating aliquots of the detergent extract (1 mg of protein) with 15 µl of ascites containing monoclonal antibodies directed against synaptophysin (Cl 7.3) or syntaxin 1 (HPC-1 or Cl 78.2) for 2 h followed by mixing with 30 µl of protein G-Sepharose fast flow (Pharmacia Biotech Inc.) for 1 h. HPC-1 recognizes both syntaxin 1A and 1B and has been described previously (Barnstable et al., 1985). Cl 78.2 is a newly generated monoclonal antibody raised against recombinant full-length syntaxin 1A that also recognizes syntaxin 1A and 1B and will be described in detail elsewhere. Immunoprecipitates were washed four times with the immunoprecipitation buffer and subjected to SDS-PAGE and immunoblot analysis as described (Chapman and Jahn, 1994a). Immunoreactive bands were visualized with I-protein A.

Binding of recombinant proteins was also carried out by immunoprecipitation of syntaxin. Recombinant full-length syntaxin 1A and C2AB were incubated at the indicated concentrations in 20 mM Tris, pH 7.4, 150 mM NaCl, 0.5% Triton X-100 supplemented with EGTA or divalent cations for 4 h. Syntaxin 1 was immunoprecipitated by incubating the samples with affinity purified HPC-1 IgG (7 µg) for 2 h and 12 µl of protein G-Sepharose fast flow (Pharmacia) for 1 h. The immunoprecipitates were washed three times and analyzed by SDS-PAGE and immunoblotting as described above.

Recombinant Proteins

A soluble form of full-length rat syntaxin 1A was prepared as described (Chapman et al., 1994) which contains a His(6)-tag at the amino terminus. Immobilized full-length and truncated syntaxins were prepared by amplifying the indicated regions of syntaxin 1A, using the polymerase chain reaction, and subcloning the products into pGEX-2T (Pharmacia). The resulting GST fusion proteins were expressed in Escherichia coli, purified, and immobilized using glutathione-Sepharose as described (Chapman and Jahn, 1994a). The cytoplasmic domain of rat synaptotagmin I, designated C2AB (residues 97-421; Chapman and Jahn, 1994a) was also prepared as a GST fusion protein by subcloning into pGEX-2T. A motif crucial for Ca-dependent phospholipid binding to the first C2-domain (SDPYVK-L, residues 177-185 (Chapman and Jahn, 1994a; Davletov and Südhof, 1993)) was deleted from C2AB and designated C2ADeltaB. For comparison, the corresponding motif (amino acids 308-316) was also deleted in the second C2-domain of C2AB (referred to as C2ABDelta). Mutagenesis was performed as described (Chapman and Jahn, 1994a) and confirmed by DNA sequencing. These deletion mutants were prepared as GST fusion proteins by subcloning into pGEX-2T as described above. Soluble C2AB, C2ADeltaB, and C2ABDelta were generated by thrombin cleavage as described (Chapman et al., 1994).

Miscellaneous

Phospholipid binding assays were carried out as described previously (Chapman and Jahn, 1994a) by immobilizing GST-C2AB, GST-C2ADeltaB, and GST-C2ABDelta on glutathione-Sepharose. The total radiolabeled phospholipid binding data are plotted in Fig. 6B. Quantitation of the immunoblots was carried out using a Molecular Dynamics PhosphorImager and ImageQuant software. For the Ca dose-response analysis, samples were buffered with 1 mM EGTA and the total Ca added to yield the indicated free Ca concentrations was determined as described previously (Chapman and Jahn, 1994a).


Figure 6: Distinct structural determinants of synaptotagmin underlie Ca-dependent syntaxin 1 and phospholipid binding. A, Coomassie Blue-stained gel of purified C2AB, C2ADeltaB, and C2ABDelta fused to GST. A motif crucial for Ca-dependent phospholipid binding to the first C2-domain (SDPYVK-L, residues 177-185 (Chapman and Jahn, 1994a) was deleted from C2AB and designated C2ADeltaB. For comparison, the corresponding motif (amino acids 308-316) was also deleted in the second C2-domain of C2AB and is designated C2ABDelta. Recombinant proteins were prepared as described under ``Experimental Procedures,'' immobilized using glutathione-Sepharose, and subjected to SDS-PAGE on 10% gels. The GST-synaptotagmin fusion proteins migrate at approximately 67 kDa; the lower molecular mass bands are proteolytic fragments. Note: the prominent 40-kDa band in the GST-C2ADeltaB lane reflects the increased sensitivity of this deletion mutant to bacterial proteases. B, disruption of Ca-dependent phospholipid binding to synaptotagmin. GST-C2AB, GST-C2ADeltaB, GST-C2ABDelta, and GST alone were immobilized using glutathione-Sepharose (80 pmol/data point) and assayed for Ca-dependent phospholipid binding as described (Chapman and Jahn, 1994a), using liposomes composed of a mixture of 75% phosphatidylcholine and 25% phosphatidylserine and labeled with [^3H]phosphatidylcholine. Phospholipid binding was measured in 50 mM Tris, pH 7.2, 100 mM NaCl with 2 mM EGTA (open bars) or 0.5 mM Ca (solid bars). The figure shows total phospholipid binding to the immobilized proteins (mean values from triplicate determinations). C, disruption of Ca-dependent phospholipid binding does not inhibit Ca-dependent syntaxin 1 binding to synaptotagmin. Ca-dependent binding of syntaxin 1 to wild type (C2AB) and mutant synaptotagmins (C2ADeltaB, C2ABDelta) was assayed as described in Fig. 2.




Figure 2: Phospholipid and divalent cation dependence for the association of synaptotagmin with syntaxin 1. Left panel, Ca-dependent binding of synaptotagmin to syntaxin 1 does not require acidic phospholipids. Recombinant synaptotagmin I (residues 97-421; designated C2AB) and syntaxin 1A (full length) were prepared as described under ``Experimental Procedures.'' C2AB (0.8 µM) was incubated in 20 mM Tris, pH 7.2, 150 mM NaCl, 0.5% Triton X-100, supplemented with EGTA (2 mM), Ca (0.5 mM), or Ca (0.5 mM) plus phospholipids (+PL; 3.7 mM phosphatidylcholine, 1.25 mM phosphatidylserine) in the presence (+) or absence(-) of recombinant syntaxin 1 (0.8 µM). Binding was monitored by immunoprecipitation using a purified anti-syntaxin 1 monoclonal antibody (HPC-1) as described in Experimental Procedures. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting. Immunoreactive bands were visualized using I-protein A and autoradiography. C2AB was precipitated only in the presence of syntaxin 1. Coprecipitation was strongly enhanced by Ca and was independent of negatively charged phospholipids. Right panel, divalent cation specificity for promoting binding of synaptotagmin to syntaxin 1. Binding of synaptotagmin to syntaxin 1 was assayed by coimmunoprecipitation as described in legend for the left panel in the presence of EGTA (2 mM) or the following divalent cation concentrations: Mg alone, 2.5 mM; Ca, Ba, Sr, 0.5 mM each; Mg (5 mM) in the presence of Ca (0.5 mM). Note that equal amounts of syntaxin 1 were immunoprecipitated under all conditions. In the absence of syntaxin 1, C2AB was not detected in the immunoprecipitates (not shown).




RESULTS

To determine the effect of Ca ions on the interaction between syntaxin and synaptotagmin, we immunoprecipitated syntaxin 1 from synaptosomal detergent extracts, using two distinct monoclonal antibodies directed against syntaxin 1, in the presence of Ca or excess Ca chelator and examined the precipitates for the presence of synaptotagmin. As shown in Fig. 1(top panel), Ca dramatically increased the level of synaptotagmin associated with syntaxin 1. We did not observe the calcium-dependent recruitment of additional proteins to the syntaxin immunoprecipitates by either protein staining or immunoblot analysis using antibodies directed against other synaptic proteins (data not shown). In addition, the amount of synaptobrevin and SNAP-25, components of the SNARE complex, bound to syntaxin was not affected by Ca ( Fig. 1and data not shown). Neither synaptotagmin nor syntaxin 1 was detected in control immunoprecipitations using anti-synaptophysin antibodies (Fig. 1, left lanes).


Figure 1: Coprecipitation of synaptotagmin with syntaxin 1 from rat brain detergent extracts is stimulated by Ca. Syntaxin 1 (syx) was immunoprecipitated from rat brain detergent extracts as described under ``Experimental Procedures'' using monoclonal antibodies HPC-1 and 78.2 in the presence of EGTA (2 mM, -Ca) or Ca (0.5 mM, +Ca). As a control, synaptophysin (syp) was immunoprecipitated in parallel with monoclonal antibody Cl 7.3. The immunoprecipitates were separated by SDS-PAGE and analyzed by immunoblotting for synaptotagmin, syntaxin 1, synaptophysin, and synaptobrevin. Immunoreactive bands were visualized using I-protein A and autoradiography. Note that equal levels of syntaxin 1 were precipitated by the antibodies under all conditions. As expected, synaptophysin antibodies did not precipitate syntaxin 1 or synaptotagmin but efficiently coprecipitated synaptobrevin with synaptophysin, in agreement with our earlier observations (Edelmann et al., 1995).



To determine whether the Ca-dependent coprecipitation is due to a direct interaction between syntaxin 1 and synaptotagmin, we repeated the syntaxin immunoprecipitations using purified recombinant proteins (Fig. 2, left panel). Full-length syntaxin 1A was mixed with the cytoplasmic portion of synaptotagmin I (amino acids 97-421). This domain contains two repeats of a region homologous to the C2-domains found in Ca-dependent isoforms of protein kinase C (Perin et al., 1990) and is designated C2AB. As shown in Fig. 2(left panel), addition of Ca results in a 6-fold increase (determined by phosphorimage analysis) in the level of C2AB which coprecipitates with syntaxin 1. Surprisingly, this increase did not require, and was not enhanced by, negatively charged phospholipids (Fig. 2, left panel). Since Ca binding to isolated synaptotagmin requires the presence of negatively charged phospholipids (Brose et al., 1992), these findings suggest that a novel Ca binding site regulates the synaptotagmin bullet syntaxin 1 interaction (discussed in more detail below).

We next compared the effects of different divalent cations on the syntaxin 1-synaptotagmin interaction. As shown in Fig. 2(right panel), Ba and Sr ions promoted the association of syntaxin 1 and synaptotagmin, albeit less potently than Ca. In contrast, Mg was virtually inactive and, in addition, failed to antagonize the effects of Ca, even at high concentrations (5 mM; Fig. 2, right panel). These findings agree with physiological studies demonstrating that Ba and Sr, but not Mg, ions can partially substitute for Ca in regulated exocytosis (Augustine et al., 1987).

The Ca dependence of the interaction was determined by incubating rat brain detergent extracts, containing native synaptotagmin, with immobilized syntaxin under varying Ca concentrations. Full-length syntaxin 1A was expressed as a GST fusion protein and immobilized using glutathione-Sepharose. After extensive washing, binding of synaptotagmin was determined by immunoblot analysis. As shown in Fig. 3, the Ca dependence of the syntaxin-synaptotagmin interaction appears to be composed of two distinct components. By curve fitting these individual components (see legend, Fig. 3) we estimate that the EC values for the high and low affinity binding sites are 0.7 and 180 µM Ca, respectively. There is evidence that at least four calcium ions bind to the calcium sensor in a cooperative manner to activate synaptic vesicle fusion (for example, see Heidelberger et al.(1994)). Determining whether there is a cooperative relationship between the Ca concentration and the interaction between syntaxin and synaptotagmin will require the dissection of the high and low Ca affinity components.


Figure 3: Ca dependence of synaptotagmin-syntaxin binding. Full-length syntaxin 1 was prepared as a GST-fusion protein and immobilized using glutathione-Sepharose. The immobilized protein (0.5 nmol) was incubated with 1 mg of the Triton X-100 rat brain synaptosomal extract (1 mg/ml) in the presence of 2 mM EGTA or 1 mM EGTA and sufficient Ca to yield the indicated free Ca concentration, for 4 h at 4 °C. In parallel, GST alone was incubated with the detergent extract as a control. Beads were washed three times and the association of native synaptotagmin determined by SDS-PAGE and immunoblot analysis using anti-synaptotagmin 1 monoclonal antibody Cl 41.1. Immunoreactive bands were visualized with I-protein A (upper panel). These data were quantified by phosphorimaging and are plotted in the lower panel. Error bars represent the standard deviation from the mean of three independent experiments. Two distinct inflection points in the binding curve corresponding to high and low affinity Ca binding sites were observed. Curves were fit to each of these two components using a logistic equation (De Lean et al., 1978). From this analysis, EC values of 0.7 and 180 µM Ca were calculated and are denoted by arrows (lower panel). E corresponds to assays carried out in 2 mM EGTA.



The affinity of the synaptotagmin-syntaxin interaction was determined by titrating syntaxin 1 with increasing concentrations of C2AB in the presence of Ca or EGTA. Again, binding was assayed by coprecipitation and immunoblot analysis (Fig. 4, left panels). In the presence of Ca, syntaxin 1 was saturated at low micromolar C2AB concentrations (EC 0.5 µM). At saturation, the binding stoichiometry approached 1:1 (Fig. 4, right panel). In the presence of EGTA, only weak binding was observed, even at the highest concentration of synaptotagmin tested (Fig. 4). Assuming that the binding stoichiometry at saturation is not affected by Ca, we estimate that Ca shifts the affinity of the interaction by approximately two orders of magnitude (Fig. 4, lower panel).


Figure 4: Concentration dependence of synaptotagmin binding to syntaxin 1 in the presence and absence of Ca. Left panel, a fixed amount of syntaxin 1 (0.5 µM) was incubated with increasing amounts of C2AB in 2 mM EGTA or 0.5 mM Ca. Binding was assayed by immunoprecipitation and immunoblot analysis as described under ``Experimental Procedures.'' The bands immunoreactive for synaptotagmin were quantified by phosphorimaging. These data are plotted in the lower panel (closed circles, binding measured in Ca; open circles, binding measured in EGTA). For the estimation of the EC, (0.5 µM in Ca), curves were fit to the data using a logistic equation (De Lean et al., 1978). Right panel, Coomassie Blue staining of the anti-syntaxin 1 immunoprecipitates obtained after incubating 10 µM C2AB with or without 0.5 µM syntaxin 1 in 2 mM EGTA or 0.5 mM Ca. Note that the staining of C2AB and syntaxin 1 is of approximately equal intensity in the presence of Ca. H and L denote the heavy and light chains, respectively, of the HPC-1 IgG used for immunoprecipitation.



To understand in more detail the interaction between syntaxin 1 and synaptotagmin, we investigated the structural determinants which mediate Ca-dependent binding. As in the Ca titration experiments, full length and truncated forms of syntaxin 1A were expressed as GST fusion proteins (Fig. 5A). These fusion proteins were immobilized using glutathione-Sepharose and incubated with synaptosomal detergent extracts in the presence of Ca or EGTA. After washing, binding of synaptotagmin, and, as controls, synaptobrevin and Rab 3A, were determined by immunoblot analysis (Fig. 5B). As expected, immobilized full-length syntaxin 1 bound to synaptotagmin in a Ca-dependent manner. This effect was specific for synaptotagmin, since Ca did not modulate the amount of synaptobrevin bound to immobilized syntaxin 1 and did not cause other synaptic proteins (e.g. Rab 3A, Fig. 5B) to become associated with the immobilized syntaxin. Synaptotagmin did not bind to residues 1-193 of syntaxin but bound efficiently, in a Ca-dependent manner, to residues 194-288 (Fig. 5B). Interestingly, the same region of syntaxin 1 also mediates its interaction with SNAP-25 (Chapman et al., 1994), synaptobrevin (Calakos et al., 1994; Fig. 5B), alpha-SNAP (Hanson et al., 1995; Kee et al., 1995) and N-type Ca-channels (Sheng et al., 1994). This region of syntaxin is composed of heptad repeats with a high probability of forming coiled-coils (Chapman et al., 1994; Kee et al., 1995), suggesting that at least some of these interactions may be mediated by intermolecular coiled coils.


Figure 5: Residues 194-288 of syntaxin 1 mediate Ca-dependent synaptotagmin binding. A, Coomassie Blue-stained gel of full-length and truncated forms of syntaxin 1 fused to GST. GST, full-length and truncated forms of syntaxin 1A fused to GST, were purified using glutathione-Sepharose and subjected to SDS-PAGE on 15% gels. Recombinant proteins were visualized by staining with Coomassie Blue. B, identification of the synaptotagmin binding domain of syntaxin. The GST-syntaxin 1 fusion proteins (0.5 nmol) were immobilized using glutathione-Sepharose and incubated with Triton X-100 extract of synaptosomes (0.5 mg of protein) in the presence of 2 mM EGTA(-) or 0.5 mM Ca (+) for 4 h at 4 °C. Beads were extensively washed and subjected to SDS-PAGE and immunoblot analysis to detect synaptotagmin and, as controls, synaptobrevin and Rab 3A. Immunoblots were visualized with I-protein A. Total corresponds to 10 µg of the synaptosomal extract.



It is notable that the removal of the transmembrane domain of syntaxin (residues 266-288) resulted in decreased synaptotagmin binding activity and also diminished the ability of syntaxin to bind synaptobrevin (Fig. 5B) and alpha-SNAP (Hanson et al., 1995). In addition, insertion of the transmembrane region into membranes is required for cleavage of syntaxin by botulinum neurotoxin C1 (Blasi et al., 1993b). While it is unlikely that the transmembrane domain participates in direct contacts with other proteins, these data suggest it may be essential for oligomerization and/or folding of syntaxin into its correct conformation.

The data described above demonstrate that Ca regulates the interaction between syntaxin 1 and synaptotagmin in the absence of phospholipids. As mentioned above, this contrasts with the finding that purified synaptotagmin binds Ca only in the presence of phospholipids (Brose et al., 1992), a property conferred by the first C2-domain (Davletov and Südhof, 1993; Chapman and Jahn, 1994a; Fukuda et al., 1994). Within this domain, a short sequence of highly conserved residues (SDPYVK-L) has been identified that is crucial for Ca binding. Deletion of, or point mutations within, this motif abolish Ca-dependent phospholipid binding to the isolated first C2-domain (Davletov and Südhof, 1993; Chapman and Jahn, 1994a). In contrast, the isolated second C2-domain does not bind to phospholipids in a Ca-dependent manner, even though this domain contains the SDPYVK-L motif (Fukuda et al., 1994). To determine the role of these motifs in Ca-dependent syntaxin 1 binding, we prepared mutant synaptotagmins (Fig. 6A) which contained this deletion in either the first (designated C2ADeltaB) or second C2-domain (designated C2ABDelta). The Ca-dependent phospholipid and syntaxin 1 binding properties of these mutants were then compared.

Deletion of the conserved motif within the first C2-domain abolished Ca-dependent phospholipid binding, whereas deletion of the corresponding motif within the second C2-domain had no effect (Fig. 6B). In contrast, binding of both deletion mutants to syntaxin 1 was stimulated by Ca to the same extent as that of the wild type cytoplasmic domain of synaptotagmin (Fig. 6C). These results clearly demonstrate that Ca-dependent binding of synaptotagmin to syntaxin 1 involves structural features distinct from those required for the Ca-dependent interaction of synaptotagmin with phospholipids. Therefore, Ca regulates the synaptotagmin-syntaxin interaction via a novel Ca-binding site within the complex.


DISCUSSION

Recent studies have provided insights into the sequence of events that may lead to bilayer fusion (reviewed by Rothman and Warren(1994) and Jahn and Ferro-Novick(1994)). An early step in this pathway includes assembly of the SNARE proteins synaptobrevin, syntaxin 1, and SNAP-25, linking the target membrane to the incoming carrier vesicle. The assembled SNARE complex then recruits alpha-SNAP, enabling NSF to bind. NSF is an ATPase and upon hydrolysis of ATP dissociates the SNARE complex, an event proposed to result in membrane fusion (Söllner et al., 1993b).

How can the Ca-stimulated interaction between synaptotagmin and syntaxin 1 be integrated into this model? Söllner et al. (1993b) observed that in neuronal detergent extracts, a small (substoichiometric) amount of synaptotagmin is associated with syntaxin 1 which could be displaced by the addition of exogenous alpha-SNAP (Söllner et al., 1993b). It was therefore suggested that synaptotagmin interacts with the SNARE complex before alpha-SNAP and NSF bind and dissociate the complex. This view, however, is difficult to reconcile with the dramatic increase in affinity of synaptotagmin for syntaxin upon Ca influx. Rather, we believe that in the nerve terminal the association of synaptotagmin with syntaxin 1 occurs after dissociation of the complex by NSF (O'Conner et al., 1994). In this scenario, the NSF-dependent dissociation of the complex can be viewed as an ATP-dependent priming step that is necessary but not sufficient for membrane fusion. For exocytosis to proceed, dissociation needs to be succeeded by the Ca-dependent association of synaptotagmin with syntaxin 1. Such a model would provide an explanation for the apparent lack of ATP dependence in the final step of exocytosis and puts the Ca sensor, synaptotagmin, closer to the fusion event (Hay and Martin, 1992; Bittner and Holz, 1992; Thomas et al., 1993; Neher and Zucker, 1993).

The effect of Ca on the affinity of the synaptotagmin-syntaxin interaction is likely to reflect a conformational change in either one or both of these proteins. The significance of the high and low affinity Ca-dependent components is currently under investigation. However, the component which displays a low affinity for Ca (EC = 180 µM) is the first calcium-dependent interaction which corresponds to the calcium dependence of neurotransmitter release reported in neurons (EC = 194 µM; Heidelberger et al., 1994). We therefore propose that the putative conformational changes associated with this component functions in the late acting triggering step in exocytosis.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 203-737-4454; Fax: 203-737-1763.

Supported by the Helen Hay Whitney Foundation.

(^1)
The abbreviations used are: NSF, N-ethlymaleimide-sensitive fusion factor; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor.


ACKNOWLEDGEMENTS

We thank M. K. Bennett and R. H. Scheller for providing the rat syntaxin 1A clone, T. C. Südhof for providing the rat synaptotagmin I clone and anti-synaptotagmin rabbit antisera, and C. Barnstable for providing the HPC-1 hybridoma cell line. We also thank N. Barton and S. Engers for excellent technical assistance, Drs. H. Otto and M. Quillan for assistance with the phosphorimaging and curve fitting analyses, respectively, and Drs. D. Bruns, W. Annaert, M. Edwardson, and U. Kistner for critically reading this manuscript. Finally, we thank Drs. P. DeCamilli, T. Galli, and C. Walch-Solimena for helpful discussions.

Note Added in Proof-Similar observations have recently been reported by Li, C., Ullrich, B., Zhang, J. Z., Anderson, R. G. W., Brose, N., and Südhof, T. C.(1995) Nature375, 594-599.


REFERENCES

  1. Augustine, G. J., Charlton, M. P., and Smith, S. J. (1987) Annu. Rev. Neurosci. 10,633-693 [CrossRef][Medline] [Order article via Infotrieve]
  2. Barnstable, C. J., Hofstein, R., and Akagawa, K. (1985) Dev. Brain Res. 20,286-290
  3. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science 257,255-259 [Medline] [Order article via Infotrieve]
  4. Bittner, M. A., and Holz, R. W. (1992) J. Biol. Chem. 267,16219-16225 [Abstract/Free Full Text]
  5. Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., De Camilli, P., Südhof, T. C., Niemann, H., and Jahn, R. (1993a) Nature 365,160-162 [CrossRef][Medline] [Order article via Infotrieve]
  6. Blasi, J., Chapman, E. R., Yamasaki, S., Binz, T., Niemann, H., and Jahn, R. (1993b) EMBO J. 12,4821-4828 [Abstract]
  7. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363,163-165 [CrossRef][Medline] [Order article via Infotrieve]
  8. Broadie, K., Bellen, H. J., DiAntonio, A., Littleton, J. T., and Schwarz, T. L. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10727-10731 [Abstract/Free Full Text]
  9. Brose, N., Petrenko, A. G., Südhof, T. C., and Jahn, R. (1992) Science 256,1021-1025 [Medline] [Order article via Infotrieve]
  10. Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263,1146-1149 [Medline] [Order article via Infotrieve]
  11. Chapman, E. R., and Jahn, R. (1994a) J. Biol. Chem. 269,5735-5741 [Abstract/Free Full Text]
  12. Chapman, E. R., and Jahn, R. (1994b) Semin. Neurosci. 6,159-165 [CrossRef]
  13. Chapman, E. R., An, S., Barton, N., and Jahn, R. (1994) J. Biol. Chem. 269,27427-27432 [Abstract/Free Full Text]
  14. Davletov, B. A., and Südhof, T. C. (1993) J. Biol. Chem. 268,26386-26390 [Abstract/Free Full Text]
  15. De Lean, A., Munson, P. J., and Rodbard, D. (1978) Am. J. Physiol. 235,E97-E102
  16. DeBello, W. M., Betz, H., and Augustine, G. J. (1993) Cell 74,947-950 [Medline] [Order article via Infotrieve]
  17. Edelmann, L., Hanson, P., Chapman, E. R., and Jahn, R. (1995) EMBO J. 14,224-231 [Abstract]
  18. Elferink, L. A., Peterson, M. R., and Scheller, R. H. (1993) Cell 72,153-159 [Medline] [Order article via Infotrieve]
  19. Fukuda, M., Aruga, J., Niinobe, M., Aimoto, S., and Mikoshiba, K. (1994) J. Biol. Chem. 269,29206-29211 [Abstract/Free Full Text]
  20. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C., and Südhof, T. C. (1994) Cell 79,717-727 [Medline] [Order article via Infotrieve]
  21. Hanson, P. I., Otto, H., Barton, N., and Jahn, R. (1995) J. Biol. Chem. 270,16955-16961 [Abstract/Free Full Text]
  22. Hay, J. C., and Martin, T. F. J. (1992) J. Cell Biol. 119,139-151 [Abstract]
  23. Heidelberger, R., Heinemann, C., and Matthews, G. (1994) Nature 371,513-515 [CrossRef][Medline] [Order article via Infotrieve]
  24. Jahn, R., and Ferro-Novick, S. (1994) Nature 370,191-193 [CrossRef][Medline] [Order article via Infotrieve]
  25. Katz, B. (1969) Sherrington Lecture X , Charles C. Thomas, Springfield, IL
  26. Kee, Y., Lin, R. C., Hsu, S., and Scheller, R. H. (1995) Neuron 14,991-998 [Medline] [Order article via Infotrieve]
  27. Link, E., Edelmann, L., Chou, J. H., Binz, T., Yamasaki, S., Eisel, U., Baumert, M., Südhof, T. C., Niemann, H., and Jahn, R. (1992) Biochem. Biophys. Res. Commun. 189,1017-1023 [Medline] [Order article via Infotrieve]
  28. Littleton, T., Stern, M., Perin, M., and Bellen, H. J. (1994) Proc. Natl. Acad. Sci. U. S. A. 91,10888-10892 [Abstract/Free Full Text]
  29. Neher, E., and Zucker, R. S. (1993) Neuron 10,21-30 [Medline] [Order article via Infotrieve]
  30. Nonet, M. L., Grundahl, K., Meyer, B. J., and Rand, J. B. (1993) Cell 73,1291-1305 [Medline] [Order article via Infotrieve]
  31. O'Conner, V., Augustine, G. J., and Betz, H. (1994) Cell 76,785-787 [Medline] [Order article via Infotrieve]
  32. Perin, M., Fried, V. A., Mignery, G. A., Jahn, R., and Südhof, T. C. (1990) Nature 343,260-263
  33. Petrenko, A., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M., and Südhof, T. C. (1991) Nature 353,65-68 [CrossRef][Medline] [Order article via Infotrieve]
  34. Popov, S. V., and Poo, M. (1993) Cell 73,1247-1249 [Medline] [Order article via Infotrieve]
  35. Rothman, J. E., and Warren, G. (1994) Curr. Biol. 4,220-233 [Medline] [Order article via Infotrieve]
  36. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., Polverino de Laureto, P., DasGupta, B. R., and Montecucco, C. (1992) Nature 359,832-835 [CrossRef][Medline] [Order article via Infotrieve]
  37. Schulze, K. L., Broadie, K., Perin, M. S., and Bellen, H. J. (1995) Cell 80,311-320 [Medline] [Order article via Infotrieve]
  38. Sheng, Z. H., Rettig, J., Takahashi, M., and Catterall, W. A. (1994) Neuron 13,1303-1313 [Medline] [Order article via Infotrieve]
  39. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993a) Nature 362,318-324 [CrossRef][Medline] [Order article via Infotrieve]
  40. Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993b) Cell 75,409-418 [Medline] [Order article via Infotrieve]
  41. Thomas, P., Wong, J. G., Lee, A. K., and Almers, W. (1993) Neuron 11,93-104 [Medline] [Order article via Infotrieve]
  42. Yoshida, A., Oho, C., Omori, A., Kuwahara, R., Ito, T., and Takahashi, M. (1992) J. Biol. Chem. 276,24925-24928 [Medline] [Order article via Infotrieve]
  43. Zhang, J. Z., Davletov, B. A., Südhof, T. C., and Anderson, R. G. W. (1994) Cell 78,751-760 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.