©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Synaptic Core Complex of Synaptobrevin, Syntaxin, and SNAP25 Forms High Affinity -SNAP Binding Site (*)

(Received for publication, October 5, 1994; and in revised form, November 12, 1994)

Harvey T. McMahon Thomas C. Südhof (§)

From the Department of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

SNAPs (soluble NSF attachment proteins) are cytoplasmic proteins that bind to specific membrane receptors and mediate the membrane binding of NSF (N-ethylmaleimide-sensitive factor), a protein that is required for membrane fusion reactions. Three synaptic proteins in brain (SNAP25 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), synaptobrevin/VAMP, and syntaxin) were identified as SNAP receptors by affinity chromatography on immobilized alpha-SNAP complexed to NSF (Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. and Rothman, J. E. (1993) Nature 362, 318-324). However, the nature of the alpha-SNAP binding site is unclear. We now show that alpha-SNAP binds tightly to the complex of syntaxin with synaptobrevin. SNAP25 is not required for tight binding of alpha-SNAP to this complex but stabilizes the syntaxin-synaptobrevin complex by forming a trimeric core complex with it. alpha-SNAP does not bind to synaptobrevin individually and binds only weakly to syntaxin and SNAP25 in the absence of synaptobrevin. These data suggest that the complex of the vesicular protein synaptobrevin with the plasma membrane protein syntaxin is required for physiological alpha-SNAP binding. Thus, alpha-SNAP probably functions in a late step of the membrane fusion reaction after the formation of the synaptobrevin-syntaxin-SNAP25 core complex.


INTRODUCTION

In pioneering studies on vesicular membrane transport between Golgi stacks in vitro, Rothman's laboratory demonstrated that a soluble ATPase called NSF (^1)is essential for membrane transport (Wilson et al., 1989). Further studies revealed that NSF acts by binding to the interacting membranes in a reaction mediated by a second class of cytoplasmic proteins called SNAPs (Clary et al., 1990). SNAPs in turn bind to membranes via specific receptors that are thought to be present on vesicular and target membranes and are referred to as v- and t-SNAP receptors, respectively (``SNAREs''). In vitro, NSF and SNAPs are also essential for early endosome fusion (Diaz et al., 1989) and for membrane traffic between the endoplasmic reticulum and the Golgi complex (Beckers et al., 1989), suggesting that these proteins perform a universal function in membrane traffic. Furthermore, mutations in the yeast homologs for NSF and SNAPs result in a secretory defect, demonstrating that these proteins are functionally conserved in evolution (reviewed by Pryer et al.(1992)). Although NSF and SNAPs are required for membrane traffic, their mechanisms of action are unclear.

In the nerve terminal, synaptic vesicles execute a complex life cycle that involves docking, membrane fusion, and budding steps at the plasma membrane and at the endosome. Proteins that participate in these steps are now being characterized. Particular progress has been made in the elucidation of the proteins functioning in the docking and fusion of synaptic vesicles at the plasma membrane. Two independent approaches identified the same three proteins, synaptobrevin/VAMP, syntaxin, and SNAP25 (synaptosomal-associated protein of 25 kDa; no relation to the SNAPs for NSF), as likely core components of the membrane fusion machinery (reviewed by Südhof et al.(1993), Bennett and Scheller(1994), and Ferro-Novick and Jahn(1994)). First, studies on clostridial neurotoxins that block synaptic vesicle membrane fusion revealed that these constitute a family of metalloproteases which specifically cleave either synaptobrevin, syntaxin, or SNAP25 (Link et al., 1992; Schiavo et al., 1992; Blasi et al., 1993a, 1993b). Second, affinity chromatography on immobilized alpha-SNAP complexed to NSF resulted in the purification of the same three proteins, indicating that syntaxin, synaptobrevin, and SNAP25 are SNAP receptors (Söllner et al., 1993a). These studies suggest a central role for syntaxin, synaptobrevin, and SNAP25 in synaptic vesicle exocytosis.

Syntaxin and SNAP25 are plasma membrane proteins that are present as a tight complex (Hayashi et al., 1994; Pevsner et al., 1994). Synaptobrevin is a synaptic vesicle protein that binds only weakly to syntaxin or SNAP25 separately but very tightly to the complex of syntaxin and SNAP25, resulting in the formation of a stable SDS-resistant complex (Hayashi et al., 1994). These findings have led to the concept of a core complex consisting of at least three components: synaptobrevin, syntaxin, and SNAP25. The core complex bridges the synaptic vesicle and plasma membranes and may function in the docking and/or fusion of synaptic vesicles.

The stage at which alpha-SNAP and NSF bind to components of the core complex is not known. It is possible that alpha-SNAP binds to one or more of the components separately, implying a function for SNAPs and NSF in the docking of synaptic vesicles with the plasma membrane. Alternatively, alpha-SNAP and NSF may not bind until the core complex has formed. The later result would imply that the function of alpha-SNAP and NSF is exerted only after docking. In the current experiments, we show that alpha-SNAP binds only weakly to isolated syntaxin or SNAP25 or the syntaxin-SNAP25 complex, and not at all to synaptobrevin. Binding is markedly enhanced after synaptobrevin complexes with syntaxin or the syntaxin-SNAP25 complex. These results imply a post-docking role for alpha-SNAP and NSF.


MATERIALS AND METHODS

Antibodies

Polyclonal antibodies to syntaxin IA (I378), SNAP25B (I733), and alpha-SNAP (J373) were raised against GST-syntaxin (Hata et al., 1993), GST-SNAP25 (Hayashi et al., 1994), and His-tagged alpha-SNAP (Söllner et al., 1993a) expressed in bacteria. Monoclonal antibodies to synaptobrevin (Cl69.1) were a gift of Dr. R. Jahn, and monoclonal antibodies to SNAP25 were obtained from Sternberger Monoclonals Inc.

Plasmid Construction and Protein Expression in Bacteria and COS Cells

The expression plasmid encoding His-tagged alpha-SNAP was a gift of Dr. J. Rothman (Sloan-Kettering Cancer Research Institute, New York, NY). GST-syntaxin IA (residues 1-265; pGEX-SyntIA), GST-syntaxinDeltaA (residues 1-8 and 76-265; pGEX-SyntDelta1), GST-syntaxinDeltaB (residues 1-180; pGEX-Synt771-1030), GST-syntaxin II (residues 1-266; pGEX-SyntII), GST-syntaxin III (residues 1-263; pGEX-SyntIII), GST-syntaxin IV (residues 1-263; pGEX-SyntIV), GST-SNAP25B (residues 1-206; pGEX-SNAP25B), GST-cellubrevin (residues 1-103; pGEX-Ceb), GST-synaptobrevin (residues 1-96; pGEX-18-1), and GST-alpha-SNAP (1-295; pGEX-alpha-SNAP) were constructed with pGexKG (Guan and Dixon, 1991) using standard procedures (Sambrook et al., 1988). Recombinant proteins were purified from expressing bacteria harboring the respective plasmids using affinity purification on glutathione-agarose or nickel columns and stored in phosphate-buffered saline (137 mM NaCl, 10 mM phosphate buffer pH 7.4, 2.7 mM KCl) containing protease inhibitors (10 mg/liter leupeptin, 0.1 g/liter PMSF, 10 mg/liter aprotonin, and 1 mg/liter pepstatin A). cDNAs for SNAP25B, syntaxin Ia, synaptobrevin 2, and alpha-SNAP were subcloned into pCMV vectors (Anderson et al., 1989) and transfected into COS cells using DEAE-dextran (Gorman, 1985). Transfected cells were harvested in phosphate-buffered saline containing protease inhibitors and 1% Nonidet P-40, and total cell extracts were used for binding assays after insoluble material had been removed by centrifugation.

Protein Binding

GST fusion proteins (0.1-0.3 nmol) bound to glutathione-agarose were incubated at 4 °C under gentle agitation in the presence or absence of purified His-tagged alpha-SNAP in the indicated amounts and/or cell extracts from COS cells transfected with the appropriate vectors (generally between 1 and 100 pmol of expressed protein, depending on the efficiency of transfection) in buffer A (0.15 M NaCl, 10 mM HEPES-NaOH pH 7.4, 1 mM EGTA, and protease inhibitors (10 mg/liter leupeptin, 0.1 g/liter PMSF, 10 mg/liter aprotonin, and 1 mg/liter pepstatin A)) containing 1% Nonidet P-40. Beads were washed three times (Fig. 1Fig. 2Fig. 3Fig. 4) or four times (Fig. 5) in a 100-fold excess of buffer A with 1% Nonidet P-40 at 4 °C. The number and temperature of the washes is critical because of the low affinity of the interaction of syntaxin with alpha-SNAP or with synaptobrevin, and the conditions for all experiments were kept constant. Quantification of binding was carried out by immunoblotting using I-labeled secondary antibodies and a Fuji Bio-Imaging Analyzer.


Figure 1: Weak binding of alpha-SNAP to syntaxin and SNAP25 but not to synaptobrevin. GST-alpha-SNAP, GST-SNAP25, and GSTsyntaxin fusion proteins (0.1 nmol) encoded by the indicated pGEX vectors were bound to glutathione-agarose and incubated with extracts from COS cells transfected with syntaxin IA, SNAP25, and synaptobrevin II expression vectors (pCMV-syntaxin, -SNAP25, and -Syb2, respectively). After washing, proteins bound to the beads were analyzed by SDS-PAGE and immunoblotting with the indicated antibodies. The binding of SNAP25 and syntaxin to each other is stronger than either to alpha-SNAP, while binding of alpha-SNAP to synaptobrevin was not detected.




Figure 2: Binding of alpha-SNAP to different isoforms of syntaxin, SNAP25, and cellubrevin. GST fusion proteins of syntaxins IA-IV (GST-SyntIA, -II, -III, and -IV), SNAP25 (GST-SNAP25), and cellubrevin (GST-Ceb) were bound to glutathione-agarose beads and incubated with purified alpha-SNAP. Beads were washed, and bound proteins were analyzed by SDS-PAGE followed by Coomassie Blue staining (top and middlepanels) and immunoblotting with antibodies to alpha-SNAP (bottompanel). The top and middlepanels depict Coomassie-stained gels of the GST fusion proteins before and after the binding experiments, respectively. Purified alpha-SNAP in amounts corresponding to the quantity added to each experimental condition was loaded in the rightlanes of both gels for comparison. The bottompanel displays an immunoblot for alpha-SNAP of the material shown in the middlepanel. Arrows point to alpha-SNAP, and numbers on the left show positions of molecular weight standards.




Figure 3: Binding of alpha-SNAP to syntaxin IA. GST fusion proteins encoding the complete cytoplasmic domains of syntaxin IA (GST-Synt1A, amino acids 1-265), a mutant syntaxin containing a deletion of most of the N-terminal sequences (GST-SyntDeltaA, amino acids 1-8 and 76-265) and a mutant syntaxin with a deletion of the C-terminal sequences (GST-SyntDeltaB, amino acids 1-180) were bound to glutathione-agarose beads and incubated with purified alpha-SNAP (rightlane). Beads were washed and the pellet fractions were analyzed by SDS-PAGE, followed by Coomassie Blue staining (toppanel) or immunoblotting for alpha-SNAP (bottompanel). Arrows point to the position of alpha-SNAP.




Figure 4: alpha-SNAP-binding to syntaxin: potentiation by synaptobrevin. Extracts from COS cells transfected with cytomegalovirus expression vectors encoding alpha-SNAP, SNAP25, and synaptobrevin 2 (Syb2) (containing approximately 10 pmol of expressed protein) were incubated with 100 pmol of GST-syntaxin overnight. Bound proteins were analyzed by immunoblotting with antibodies to alpha-SNAP (toppanel), SNAP25 (middlepanel), and synaptobrevin 2 (bottompanel) using enhanced chemiluminescence detection. Note that the exposure shown was chosen to demonstrate the potentiation of alpha-SNAP binding to syntaxin by synaptobrevin. Almost no alpha-SNAP binding to syntaxin in the absence of synaptobrevin is apparent in this exposure; it can, however, be detected with longer exposures (data not shown).




Figure 5: Quantitative analysis of alpha-SNAP binding to syntaxin in the presence and absence of synaptobrevin. His-tagged alpha-SNAP at the indicated concentrations was bound to immobilized GST-syntaxin (300 pmol) in the presence and absence of synaptobrevin 2 (approximately 20 pmol) in a 0.25-ml reaction volume. Samples were analyzed by quantitative immunoblotting using I-labeled secondary antibodies and detected with a Fuji Bio-Imaging Analyzer. Note that under the conditions used in this experiment, it was not possible to saturate alpha-SNAP binding to syntaxin IA in the absence of synaptobrevin.



Miscellaneous Procedures

SDS-PAGE was carried out as described by Laemmli(1970) using 7% or 13% gels as appropriate. Detection of proteins during immunoblotting was performed with enhanced chemiluminescence (Amersham Corp.) or I-labeled secondary antibodies.


RESULTS

Weak Binding of alpha-SNAP to Syntaxin and SNAP25

In order to investigate alpha-SNAP binding to the three proteins present in the synaptic vesicle membrane fusion complex (synaptobrevin, syntaxin, and SNAP25), the three proteins were individually expressed by transfection in COS cells. Extracts from transfected COS cells were incubated with a GST-alpha-SNAP fusion protein attached to glutathione-agarose beads, and bound proteins were analyzed by SDS-PAGE and immunoblotting. Weak binding of GST-alpha-SNAP to syntaxin 1A and to SNAP25 but not to synaptobrevin 2 was observed (Fig. 1), suggesting that the plasma membrane proteins syntaxin and SNAP25 individually are only weak SNAP receptors, and the vesicle protein synaptobrevin is not a SNAP receptor.

As a positive control for the binding reaction, the binding of syntaxin 1A, SNAP25, and synaptobrevin 2 to each other was analyzed (Fig. 1). In agreement with our previous experiments (Hayashi et al., 1994), syntaxin and SNAP25 were found to bind strongly to each other independent of which protein was expressed as a native protein in COS cells or as a GST fusion protein, thereby validating the binding reactions. Furthermore, synaptobrevin binding to syntaxin was almost undetectable in the absence of SNAP25 but greatly potentiated by addition of SNAP25, leading to the formation of the stable synaptobrevin-syntaxin-SNAP25 core complex. In contrast, the addition of SNAP25 had no potentiating effect on synaptobrevin binding to alpha-SNAP (Fig. 1, right lanes).

In a complementary set of experiments, we investigated the binding of purified His-tagged alpha-SNAP to GST fusion proteins with different syntaxins, SNAP25, and cellubrevin (Fig. 2). Cellubrevin is a ubiquitous homolog of synaptobrevin that can replace synaptobrevin in the syntaxin-SNAP25-synaptobrevin complex (McMahon et al., 1993) and was used instead of synaptobrevin because its GST fusion protein is more stable than that of synaptobrevin. Coomassie Blue staining and immunoblotting revealed that most syntaxin isoforms and SNAP25 but not cellubrevin were capable of binding alpha-SNAP. More robust binding signals were observed in these experiments than in those described in Fig. 1because high, saturating concentrations of alpha-SNAP were used (Fig. 2, right lane). This experiment confirms the conclusion of Fig. 1that syntaxin and SNAP25 but not synaptobrevins can serve as SNAP-binding proteins.

A large number of proteins has been shown to bind to syntaxin, including the members of the core complex (synaptobrevin and SNAP25), synaptotagmin, Munc18, and potentially Ca channels. Thus it is important to map the sites of interaction of these proteins on syntaxin, and preliminary studies have indicated that the core complex components interact primarily via the C-terminal sequences of syntaxin, whereas the N terminus of syntaxin is required for Munc18 binding (Hata et al., 1993; Hayashi et al., 1994). We therefore tested the ability of N- and C-terminal deletion mutants to bind alpha-SNAP, revealing that the C terminus of syntaxin is essential for alpha-SNAP binding, whereas the N terminus is not as critical (Fig. 3). These data suggest that the alpha-SNAP-binding site on syntaxin is similar to that for SNAP25 and synaptobrevin (Hayashi et al., 1994).

The Syntaxin-Synaptobrevin Complex Forms a High Affinity Receptor for alpha-SNAP

Previous studies on the formation of the synaptobrevin-syntaxin-SNAP25 complex revealed that the binding of syntaxin to SNAP25 creates a high affinity site for synaptobrevin, whereas syntaxin and SNAP25 individually show only weak binding of synaptobrevin (Hayashi et al., 1994; see also Fig. 1). alpha-SNAP binding to either SNAP25 or syntaxin is also rather weak, raising the question if alpha-SNAP binds to these proteins individually or if the true alpha-SNAP binding site is formed by the complex between two or all three of the components of the core complex. To investigate this question, alpha-SNAP, SNAP25, and synaptobrevin 2 were expressed individually or in various combinations by transfection in COS cells. The interactions of these proteins with GST-syntaxin attached to agarose beads were studied using immunoblotting of the bound proteins (Fig. 4). Under the conditions of the experiment shown, only weak binding of syntaxin alone to alpha-SNAP was observed because of the much lower levels of alpha-SNAP in the transfected COS cells than in the reactions using purified bacterial recombinant proteins described in Fig. 1Fig. 2Fig. 3. Addition of synaptobrevin to syntaxin caused a dramatic increase in alpha-SNAP binding, whereas addition of SNAP25 to syntaxin had no effect. Note that in Fig. 1, synaptobrevin binding to syntaxin was also potentiated by SNAP25. Thus, although both syntaxin and SNAP25 individually bind alpha-SNAP weakly, addition of synaptobrevin selectively increases alpha-SNAP binding to syntaxin. Finally, Fig. 4confirms our previous observation that synatobrevin binding to syntaxin is greatly enhanced in the presence of SNAP25. Since SNAP25 forms a tight complex with syntaxin, our data suggest that alpha-SNAP binding occurs to the complex only after synaptobrevin has bound to form the trimeric core complex.

To quantitate the difference in alpha-SNAP binding to syntaxin in the absence or presence of synaptobrevin, we performed binding experiments that were analyzed with I-labeled secondary antibodies. Increasing amounts of recombinant His-tagged alpha-SNAP were added in these experiments to a constant amount of GST-syntaxin bound to glutathione-agarose beads in the presence or absence of synaptobrevin 2. After incubations, bound proteins were recovered from the washed beads and quantitatively analyzed by immunoblotting (Fig. 5). Although syntaxin binds alpha-SNAP in the absence of synaptobrevin, the concentrations of alpha-SNAP required for binding are much higher than in the presence of synaptobrevin. Binding of alpha-SNAP to syntaxin saturates at low alpha-SNAP concentrations (below micromolar) in the presence of synaptobrevin but does not saturate in the absence of synaptobrevin under the conditions of the experiments. Thus, synaptobrevin binding to syntaxin greatly increases alpha-SNAP binding, either by forming a composite receptor surface for alpha-SNAP or by inducing a conformational change in syntaxin that results in high affinity binding.

A difference in affinity for alpha-SNAP between syntaxin and the core complex of syntaxin-synaptobrevin-SNAP25 would only be physiologically relevant if alpha-SNAP were a limiting component in brain and not present in saturating concentrations. To address this question, we performed quantitative immunoblotting experiments on syntaxin IA and alpha-SNAP in total brain homogenate using GST-syntaxin and His-tagged alpha-SNAP as standards for controlling for antibody affinities. Although these experiments did not allow the calculation of a true concentration of these proteins in brain, they did reveal that syntaxin IA is at least 100 times more abundant than alpha-SNAP (data not shown). In spite of its low abundance, alpha-SNAP was found to be highly enriched in the putative synaptic vesicle membrane fusion complex isolated by immunoprecipitation with syntaxin antibodies from brain, with an enrichment that exceeded that of the other components of the complex (synaptobrevin and SNAP25). Thus the affinity for alpha-SNAP is likely to be a governing determinant in the definition of the alpha-SNAP receptor.


DISCUSSION

There is general agreement that the synaptobrevin, syntaxin, and SNAP25 form a synaptic core complex that is important for synaptic vesicle exocytosis. However, the role of the complex in synaptic vesicle docking (e.g. Pevsner et al.(1994)) or membrane fusion (e.g. Hayashi et al.(1994)) is unclear. Furthermore, the function of the interaction of synaptotagmin I, alpha-SNAP, and Munc18-1 with components of the complex has been controversial (Hata et al., 1993; Pevsner et al., 1994; Söllner et al., 1993b), although recent studies on murine synaptotagmin I mutants reveal that synaptotagmin I has an essential function in the late stages of the fusion reaction (Geppert et al., 1994).

In the current study, we confirm previous results (Hayashi et al., 1994) demonstrating that synaptobrevin 2 binds much more tightly to the complex of syntaxin IA and SNAP25 than to either component individually and forms an SDS-resistant stable complex, referred to as core complex. We then show that alpha-SNAP binds only weakly to either syntaxin IA or SNAP25 alone and not to synaptobrevin 2. However, addition of synaptobrevin 2 to syntaxin IA or to the physiologically occurring syntaxin-SNAP25 complex causes a dramatic enhancement in alpha-SNAP binding, probably due to a shift in affinity. Quantitative immunoblotting revealed that alpha-SNAP is present at a concentration more than 100-fold lower in brain than in syntaxin, suggesting that alpha-SNAP is limiting. Together these results demonstrate that the synaptic core complex of synaptobrevin, syntaxin, and SNAP25 and not the individual components serves as an alpha-SNAP receptor. The high affinity SNAP binding site could be formed by a composite receptor surface on syntaxin and synaptobrevin or could be induced in syntaxin allosterically by synaptobrevin binding.

A model based on these findings is depicted in Fig. 6. Syntaxin is thought to be normally present as a heterodimeric complex with either Munc18 or SNAP25 in the plasma membrane. During or after synaptic vesicle docking, the syntaxin-SNAP25 dimer forms a stable trimeric complex with synaptobrevin. This complex, the core complex, becomes SDS-resistant through a conformational change (Hayashi et al., 1994) and serves as the binding site for the consecutive binding of SNAPs and NSF. It is unclear if the formation of the SDS-resistant complex precedes SNAP binding since alpha-SNAP also binds to the less stable synaptobrevin-syntaxin complex that is not SDS-resistant. However, since after complex formation SDS resistance occurs spontaneously, it seems likely that the SDSresistant complex represents the true SNAP receptor. The sequential binding of first SNAP and then NSF to the complex is suggested by the absence of a SNAP-NSF complex in the cytosol. NSF is a homotrimer in which all three subunits have to be functional for NSF to be active in fusion reactions (Whiteheart et al., 1994). At this point it is unknown if the core complex has one or several binding sites for alpha-SNAP, since SNAP25 exhibited weak binding of alpha-SNAP, which could not be significantly enhanced by synaptobrevin (Fig. 1) but could still be utilized after formation of the high affinity complex between syntaxin-synaptobrevin and SNAP25. The preferential binding of alpha-SNAP to the core complex and not to individual components implies a role for SNAPs and NSF late in the fusion reaction, most likely in the step that prepares the vesicle and plasma membranes for the actual fusion reaction. It is tempting to speculate that the function of NSF is to prepare the membranes bridged by the core complex for the actual fusion reaction, possibly by multimerizing several core complexes into a prefusion state.


Figure 6: Model of the assembly sequence of the putative core complex for synaptic vesicle membrane fusion. In the first step, synaptobrevin (Syb) on the vesicle membrane (VM) and syntaxin 1 and SNAP25 on the plasma membrane (PM) are thought to form a complex (7SComplex). This step may be identical with the docking of synaptic vesicles or may immediately follow docking of synaptic vesicles. The synaptobrevin-syntaxin-SNAP25 complex is then converted into a stable, SDS-resistant form (Hayashi et al., 1994), which serves as a receptor for the sequential binding of SNAPs and of NSF (steps 3 and 4).




FOOTNOTES

*
This work was supported by a grant from the Ross Perot Family Foundation. 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: Dept. of Molecular Genetics and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-5022; Fax: 214-648-6426.

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


ACKNOWLEDGEMENTS

We thank Drs. J. Rothman, R. Scheller, Y. Hata, and J. Zhang for plasmids, and Ewa Borowicz and Izabella Leznicki for excellent technical assistance. We thank Drs. M. S. Brown and J. L. Goldstein for invaluable advice.


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