©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The N-Ethylmaleimide-sensitive Fusion Protein and -SNAP Induce a Conformational Change in Syntaxin (*)

Phyllis I. Hanson (§) , Henning Otto (¶) , Nikki Barton , Reinhard Jahn

From the (1)Department of Pharmacology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06510

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The N-ethylmaleimide-sensitive fusion protein (NSF) plays an essential role in intracellular membrane fusion events and has been implicated in the exocytosis of synaptic vesicles. NSF binds through soluble NSF attachment proteins (SNAPs) to a complex of neuronal membrane proteins comprised of synaptobrevin, syntaxin, and SNAP-25. Disassembly of this complex by NSF is thought to be a critical step in the molecular events which lead to vesicle fusion with the plasma membrane. Here we have studied the interaction of -SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/-SNAP. We find that -SNAP binds directly to syntaxin 1A as well as weakly to SNAP-25, while it does not bind to synaptobrevin II. NSF binds to syntaxin through -SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and -SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. -SNAP binds to a carboxyl-terminal syntaxin fragment (residues 194-288) that also binds synaptobrevin and SNAP-25. However, NSF action on this syntaxin fragment has no effect on the binding of -SNAP or synaptobrevin. This suggests that the conformational change normally induced by NSF in syntaxin depends on an interaction between carboxyl- and amino-terminal domains of syntaxin.


INTRODUCTION

Stimulated release of neurotransmitter from synaptic vesicles is the fundamental process responsible for intercellular communication within the nervous system. In the last few years, major advances have been made in our understanding of synaptic vesicle docking and exocytosis. There is now compelling evidence that neuronal exocytosis operates by the same general mechanism as other intracellular membrane fusion events, with the addition of a specialized regulatory mechanism that enables Ca to rapidly trigger secretion(1, 2, 3) . Understanding which proteins are involved in neuronal exocytosis and how they work together to bring about membrane fusion is now the focus of considerable effort.

Studies of vesicular transport between purified Golgi stacks by Rothman and colleagues (3) led to the isolation and identification of soluble proteins necessary for intracellular membrane fusion. These include the N-ethylmaleimide-sensitive fusion protein (NSF)()and the soluble NSF attachment proteins (SNAPs) which comprise a small family of homologous isoforms, designated as -, -, and -SNAP(4, 5, 6) . NSF and the SNAPs have been shown to participate in most intracellular fusion reactions that are amenable to in vitro analysis, and their homologs in yeast are indispensable for vesicular transport through the secretory pathway(2, 3, 7) . NSF is an ATPase which in the presence of a SNAP promotes membrane fusion between uncoated transport vesicles and their target compartment(8, 9, 10) . NSF does not bind directly to membranes but instead binds to /- and -SNAP which attach to specific membrane receptors (SNAP receptors or SNAREs)(11, 12, 13) .

A set of neuronal SNAP receptors was first identified by Rothman and colleagues(14) . These receptors form a complex that contains the synaptic proteins synaptobrevin (also known as VAMP), syntaxin, and SNAP-25 (synaptosomal associated protein of 25 kDa)(14) . Synaptobrevin is a small integral membrane protein with a single carboxyl-terminal transmembrane domain found at high levels on synaptic vesicles(15, 16) . Syntaxin is likewise an abundant small integral membrane protein with a carboxyl-terminal transmembrane domain but is localized primarily on the plasma membrane(17, 18, 19) . SNAP-25 is palmitoylated on a number of cysteine residues and is also found predominantly on the plasma membrane(19, 20) . The observation that selective cleavage of each of these three proteins by clostridial neurotoxins leads to a complete blockade in neurotransmission provides strong evidence that they are indeed essential in neuronal exocytosis(21, 22, 23, 54) . There are families of proteins related to synaptobrevin and syntaxin expressed outside of the nervous system with varying subcellular distribution; these related proteins are likely to serve as SNAP receptors for other vesicular fusion events(24, 25) . Further evidence for involvement of these protein families in membrane trafficking has been provided by studies in yeast, where relatives of synaptobrevin, syntaxin, and SNAP-25 have been shown to be essential at several stages in the secretory pathway(2) .

Little is known about how NSF, SNAPs, and their membrane receptors actually promote membrane fusion. Some clues, however, have been provided by characterization of the protein-protein interactions between them. In detergent extracts of brain membranes, synaptobrevin, syntaxin, and SNAP-25 bind to each other, forming the 7 S SNAP receptor complex(26) . This SNARE complex binds -SNAP and NSF to form a 20 S particle which is stable in the presence of a non-hydrolyzable ATP analog(12, 26) , and is thought to comprise the core of a molecular membrane fusion machine. Since the SNARE complex contains membrane proteins from both the vesicle (v-SNAREs) and the plasma membrane (t-SNAREs), it has been proposed that selectivity in pairing between v- and t-SNAREs may enable a transport vesicle to choose the appropriate target site at which to dock and fuse(26, 27) . The finding that certain isoforms of syntaxin (syntaxin 1A and 4, but not syntaxin 2 or 3) preferentially interact with synaptobrevin II (27) and that SNAP-25 selectively potentiates these interactions (28) suggests that there is selectivity in the pairing of v- and t-SNAREs as they assemble into a SNARE complex.

NSF catalyzes dissociation of the 20 S SNARE protein complex in the presence of ATP. In this reaction it releases itself as well as synaptobrevin, SNAP-25 and some -SNAP from syntaxin(12, 14, 26) . This disassembly precedes membrane fusion, since assembled SNARE complexes accumulate in yeast secretory mutants deficient in sec18 (NSF) activity(29) . The ATPase activity of NSF is essential for its role in promoting membrane fusion, since site-directed mutants with impaired ATPase activity do not support Golgi transport in vitro(10, 30) . Whether disassembly of the SNARE complex by NSF leads directly to membrane fusion, or serves as a priming step for a subsequent, perhaps Ca stimulated reaction that causes membrane fusion is unknown. Furthermore, exactly how the SNAPs and NSF interact with the individual components of the SNARE complex remains an open question.

We have now studied the interactions between -SNAP, NSF, and purified individual synaptic SNARE proteins (syntaxin, SNAP-25, and synaptobrevin). We find that syntaxin binds with moderate affinity to -SNAP and that the syntaxin--SNAP complex is a target for NSF action. NSF uses the energy derived from ATP hydrolysis to induce a conformational change through -SNAP in syntaxin. This reaction leads to the previously described disassembly of the synaptic fusion complex.


MATERIALS AND METHODS

Plasmid Construction

Plasmids encoding His--SNAP, untagged -SNAP(6) , and His-NSF (31) were kindly provided by S. W. Whiteheart and J. E. Rothman (New York). cDNAs encoding rat syntaxin 1A(17) , synaptobrevin II(15) , and SNAP-25B (22, 32) were generously provided by R. H. Scheller (Stanford University) and T. C. Südhof (Dallas), respectively. Full-length and truncated coding sequences were amplified using the polymerase chain reaction and oligonucleotides containing appropriate restriction sites for subcloning into the indicated plasmid(33) .

Two vector systems were used to generate fusion proteins in Escherichia coli. Expression in the vector pGEX-2T (Pharmacia LKB Biotechnol.) generates fusion proteins with glutathione S-transferase (GST). Expression in the pTrcHis vector (InVitrogen, Portland, OR) generates fusion proteins in which a 6-histidine tag and a 32-45-amino-acid linker sequence are fused to the amino terminus. Full-length - and -SNAP and two fragments of -SNAP (residues 1-154 and 114-295) were amplified using polymerase chain reaction and were subcloned into pGEX-2T. Primers used for amplification included: -SNAP full-length: (sense, EcoRI) 5`-TATAGGATCCATGGACAACTCCGGGAAG-3` (antisense, EcoRI) 5`-ATAGAATTCTTAGCGCAGGTCTTCCTC-3`; -SNAP(1-154): (sense, EcoRI) 5`-TATAGGATCCATGGACAACTCCGGGAAG-3` (antisense, EcoRI) 5`-GCAGAATTCGCCTTTGTAGTAGTCTGC-3`; -SNAP(114-295): (sense, EcoRI) 5`-CGTGAATTCGTATGGGCCGCTTCACCATC-3` (antisense, EcoRI) 5`-ATAGAATTCTTAGCGCAGGTCTTCCTC-3`; -SNAP: (sense, BamHI) 5`-GCGGATCCATGGACAACGCG-3` (antisense, BamHI) 5`-GCGGATCCTCACTTGAGGTCTCCGTC-3`.

Full-length syntaxin 1A in pGEX-2T and pTrcHis has been previously described(34) .

Syntaxin 1A fragments (residues 1-193 and 194-288) were introduced into pGEX-2T after amplification with the following primers: syntaxin(1-193): (sense, EcoRI) 5`-TATGAATTCTTATGAAGGACCGAACCCAG-3` (antisense, EcoRI) 5`-GCGAATTCCTAACTGAGGGCCTGCTTCGA-3`; syntaxin(194-288): (sense, EcoRI) 5`-CGTGAATTCGGATGGAGATCGAGACCAGGCAC-3` (antisense, EcoRI) 5`-ATA-GAATTCCTATCCAAAGATGCCCCCG-3`.

SNAP-25B in pTrcHisA has been previously described (35) as has synaptobrevin II in pGEX-2T(36) . Synaptobrevin II was also introduced into pTrcHisA as a BamHI-EcoRI fragment.

Purification of Fusion Proteins

GST fusion proteins were purified essentially as described (34, 36) except that HEPES-buffered saline (HBS: 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol) was used as the primary buffer. In the purification of proteins retaining their transmembrane domains (full-length syntaxin, synaptobrevin; syntaxin(194-288)) 0.5% Triton X-100 was added to all washing buffers. Synaptobrevin II was separated from GST by treating the glutathione-Sepharose-immobilized fusion protein with 1 unit of thrombin (Sigma) per 500 µl of beads in HBS containing 0.5% Triton X-100 at room temperature for 1 h(37) . Thrombin was then inhibited by addition of 1 mM phenylmethylsulfonyl fluoride, and the supernatant containing released synaptobrevin was collected.

Fusion proteins expressed in pTrcHis were also purified essentially as described previously(34) . Proteins were eluted in steps from Ni-Sepharose (ProBond, InVitrogen) with buffer containing 80 mM, 160 mM, or 400 mM imidazole, pH 7.6. In a typical purification from 1 liter of bacteria, 0.8 ml of resin was used and eluted with two 1-ml fractions at each imidazole concentration. Fractions were analyzed for purity by SDS-PAGE and staining with Coomassie Blue and then dialyzed against HBS and, when appropriate, 0.5% Triton X-100. Syntaxin 1A(1-288) was most concentrated in fractions eluted with 400 mM imidazole, and a typical preparation from 1 liter of bacteria produced 0.5 mg of essentially pure syntaxin. SNAP-25 was most concentrated in fractions eluted with 160 mM imidazole, and a typical preparation from 1 liter of bacteria produced 100 µg of SNAP-25 which was 50-75% pure. Synaptobrevin II was also most concentrated in fractions eluted with 160 mM imidazole, and a typical preparation from 1 liter of bacteria produced 200 µg of synaptobrevin which was 50-75% pure.

His-NSF and His--SNAP (expressed in the pQE9 vector (Qiagen)) were purified essentially according to published procedures(6, 38) . His-NSF was eluted from Ni-Sepharose with a continuous gradient of imidazole (0-500 mM in 20 mM HEPES-KOH, pH 7.8, 200 mM KCl, 2 mM MgCl, 10% glycerol, 1 mM ATP, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride). Aliquots were snap frozen and stored at -70 °C. His--SNAP was eluted from Ni-Sepharose with steps of 80 mM, 160 mM, and 400 mM imidazole as above. A typical preparation of -SNAP from 1 liter of bacteria produced >10 mg of essentially pure protein.

Production of Antibodies

New monoclonal antibodies recognizing - and -SNAP were generated (Fig. 1). Purified -SNAP (cleaved with thrombin from a GST--SNAP fusion protein) was used to immunize BALB/c mice. Two hybridoma cell lines (clones Cl 77.1 and 77.2) were established using standard procedures for fusion, propagation, and screening(39, 40) . A polyclonal rabbit serum recognizing NSF (R32) was generated using purified His-NSF by standard procedures(41) .


Figure 1: Characterization of monoclonal antibodies specific for - and -SNAP. A, immunoblots of total rat brain homogenate were probed with two monoclonal antibodies raised against recombinant -SNAP. Each lane is an immunoblot of S1 homogenate (15 µg) separated on a 12% SDS-polyacrylamide gel. Ascites fluid containing antibody from clone Cl 77.1 or Cl 77.2 (1:5000 dilution) was used to immunodecorate the blots. Proteins recognized were visualized using alkaline phosphatase coupled secondary antibody reagents. B, -SNAP and -SNAP-GST fusion proteins were expressed in bacteria and immobilized on glutathione-Sepharose beads. An immunoblot of 2 µg of each fusion protein is shown. GST alone is included as a control.



Immunoprecipitations

Purified proteins were incubated together at the concentrations indicated in a final volume of 100 µl at 4 °C with gentle agitation. Standard binding assay buffer included 20 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM dithiothreitol, and 0.5% Triton X-100. After 4 h, samples were briefly spun to remove any aggregated material (5 min, 12,000 revolutions/min in 4 °C microfuge). After transfer to a fresh tube, 5 µl of ascites fluid (containing 5-15 µg of specific IgG) was added, and incubations were continued for 1 h. 25 µl of a 70% protein G-Sepharose suspension was then added, and samples were incubated for an additional 1 h. The beads were collected by brief centrifugation (30 s, 3000 revolutions/min in microfuge), and then washed three times with 1 ml of buffer. Proteins bound to the beads were finally solubilized with 40 µl of 1.5 SDS sample buffer (42) and heated for 5 min. The monoclonal antibodies used for immunoprecipitation included HPC-1 for syntaxin(43) , Cl 69.1 for synaptobrevin(36) , and Cl 71.2 for SNAP-25 (35).

Binding to Glutathione-Sepharose Immobilized Proteins

Soluble proteins were incubated together with the indicated amount of GST fusion protein immobilized on glutathione-Sepharose beads in standard binding buffer. Incubations were for 4 h, after which the beads were washed and processed as described for protein G-Sepharose beads above.

Disassembly Reactions

Syntaxin was incubated together with -SNAP and, where indicated, synaptobrevin for 2 h in a prebinding reaction. The buffer used was the same as in the binding reactions above, but also contained 2 mM MgCl. Disassembly reactions were initiated by addition of NSF (2-5 µg/100 µl reaction) together with ATP or ATPS (1 mM each), and allowed to proceed for 1 h. When immobilized GST-syntaxin was used in the disassembly reaction, the beads were washed directly after the reaction, and bound proteins were solublized in 1.5 SDS sample buffer as above. When His-syntaxin was used in the disassembly reaction, it was collected by immunoprecipitation with HPC-1 and protein G-Sepharose beads, as described above.

Other Procedures

Samples were separated by SDS-PAGE and transferred to nitrocellulose as described(34) . Blots were incubated with monoclonal antibodies (ascites fluid) or polyclonal sera diluted 1:1000-1:2000 in Blotto (20 mM Tris, pH 7.4, 150 mM NaCl, 5% powdered non-fat milk, and 0.1% Tween 20) overnight at 4 °C. Blots to be probed with I-protein A were washed and incubated with secondary rabbit anti-mouse IgG antibodies (Cappel), washed again and then incubated with 0.1 µCi/ml I-protein A (DuPont NEN) for 1-2 h, and finally washed five to six times over >2 h. After drying, membranes were exposed to x-ray film. For quantitation, radioactive membranes were exposed to Phosphor-screens which were analyzed on a PhosphorImager (Molecular Dynamics) using the ImageQuant software. When alkaline phosphatase-coupled secondary goat anti-mouse or anti-rabbit antibodies were used, immunoreactive bands were visualized by addition of nitro blue tetrazolium (0.33 mg/ml) and 5-bromo-4-chloro-3-indolyl phosphate (0.17 mg/ml). Densitometry (used for relative quantitation of Coomassie-stained bands) was performed using a Visage 2000 Scanner (BioImage Products, MilliGen/Biosearch Division of Millipore, Ann Arbor, MI). Protein concentrations were determined by the method of Bradford using bovine serum albumin as a standard (44) or estimated from Coomassie Blue-stained gels of recombinant proteins.


RESULTS

Production and Characterization of Antibodies Recognizing -SNAP

To study the interaction of -SNAP with its receptors, we raised monoclonal antibodies directed against recombinant -SNAP. Two hybridoma cell lines were developed and are designated Cl 77.1 and Cl 77.2. As shown in Fig. 1A, each recognized two proteins in a rat brain homogenate with apparent molecular masses of 33 and 34 kDa. Based on the high similarity between -SNAP and -SNAP (83% identity(6) ), it is likely that this doublet consists of - and -SNAP. Using recombinant GST fusion proteins encoding -SNAP and -SNAP, we confirmed this specificity (Fig. 1B). Both antibodies recognize - and -SNAP, and neither antibody recognizes GST alone.

-SNAP Binds to Syntaxin and SNAP-25 but Not to Synaptobrevin

In the first series of experiments, we asked whether -SNAP can bind to the individual SNARE proteins syntaxin, SNAP-25, and synaptobrevin. For this purpose we expressed full-length syntaxin 1A (referred to henceforth as syntaxin), synaptobrevin II (referred to as synaptobrevin), SNAP-25, and -SNAP each in bacteria fused to either a 6-histidine (His) or GST tag. The proteins were purified by affinity chromatography (using Ni- or glutathione-Sepharose) and when indicated separated from the GST moiety by thrombin cleavage. To assay for binding, each SNARE protein was incubated separately with increasing concentrations of -SNAP. The SNARE proteins were then collected by immunoprecipitation using specific monoclonal antibodies and analyzed for bound -SNAP by immunoblotting.

When syntaxin was used in the assay, -SNAP bound efficiently (Fig. 2A), with a 50% effective concentration (EC) of between 500 nM and 1 µM. At saturation, about 1 mol of -SNAP was bound for every 3-4 mol of syntaxin collected, as judged by densitometry of Coomassie Blue-stained samples (see, for example, Fig. 3A). Immunoprecipitation of -SNAP by the syntaxin-specific antibody HPC-1 was dependent on the presence of syntaxin, as essentially no -SNAP was detected in control precipitations lacking syntaxin (not shown, but see Fig. 3A). This demonstrates that under these conditions -SNAP did not adsorb significantly to the plastic surface of the test tubes, as has been described when higher concentrations of -SNAP are used (5, 13, 45). We also tested the effect of increasing concentrations of sodium chloride on the binding of -SNAP to syntaxin (Fig. 2B). The interaction between these proteins was unaffected by 500 mM sodium chloride and was reduced by higher sodium chloride concentrations. -SNAP binding to syntaxin is thus relatively salt resistant, as is the binding of synaptobrevin and SNAP-25 to syntaxin(46) .


Figure 2: -SNAP binding to syntaxin 1A, SNAP-25, and synaptobrevin II. A, -SNAP binding to syntaxin. His-syntaxin 1A (300 nM) was incubated with increasing concentrations of His--SNAP (0-5 µM), and then collected by immunoprecipitation using a syntaxin-specific monoclonal antibody (HPC-1) and protein G-Sepharose. Precipitated proteins were visualized by SDS-PAGE and immunoblotting for -SNAP (Cl 77.2) or syntaxin (HPC-1). Bound antibody was visualized using I-protein A and autoradiography. Note that equal amounts of syntaxin were recovered in each precipitation. B, effect of NaCl on -SNAP binding to syntaxin. His-syntaxin (300 nM) was incubated with 5 µM His--SNAP in the presence of increasing concentrations of NaCl as indicated. Immunoprecipitation and immunoblotting was carried out as in A. C, -SNAP binding to SNAP-25. His-SNAP-25 (300 nM) was incubated with increasing concentrations of His--SNAP (0-5 µM), and collected by immunoprecipitation using the SNAP-25-specific monoclonal antibody Cl 71.2. Proteins were visualized by immunoblotting as above using specific antibodies (polyclonal rabbit SNAP-25 antibody, Cl 77.2 for -SNAP). D, effect of NaCl on -SNAP binding to SNAP25. His-SNAP-25 (300 nM) was incubated with 5 µM His--SNAP in the presence of increasing concentrations of NaCl as indicated. E, -SNAP binding to synaptobrevin. Synaptobrevin II (300 nM, prepared by thrombin cleavage from GST-synaptobrevin fusion protein) was incubated with -SNAP (0.7-5 µM) and immunoprecipitated using the synaptobrevin II-specific antibody (Cl 69.1) (36). Immunoprecipitates were subjected to immunoblotting in parallel with the samples shown in A using antibodies recognizing -SNAP or synaptobrevin. The exposure shown is the same as shown in A; even after a four times longer exposure no -SNAP signal could be detected (not shown).




Figure 3: Binding of NSF to the -SNAP-syntaxin complex and dissociation of the complex by ATP. A, effect of NSF on the binary syntaxin--SNAP complex. Syntaxin (0.9 µM) was incubated with -SNAP (5 µM) in a buffer containing Mg for 2 h prior to addition of NSF (0.3 µM) together with ATP or ATPS (1 mM) as indicated. After an additional 30 min, the syntaxin was collected by immunoprecipitation as in Fig. 2. The precipitated proteins were separated by SDS-PAGE and visualized by protein staining with Coomassie Blue. Note that -SNAP was recovered in the immunoprecipitate only when syntaxin was included (compare lanes 1 and 3). NSF only bound to syntaxin in the presence of -SNAP (compare lanes 5 and 7) and a non-hydrolyzable ATP analog (compare lanes 7 and 8). B, effect of NSF on synaptobrevin binding to syntaxin. A similar experiment to that shown in A, except that each reaction contained synaptobrevin, and the proteins recovered in the immunoprecipitates were detected by immunoblotting. Syntaxin (0.5 µM), synaptobrevin (0.5 µM), and -SNAP (5 µM) were preincubated and then treated with NSF as above. Proteins collected in the washed immunoprecipitate pellets were subjected to SDS-PAGE and immunoblotting. Bound proteins were visualized using specific antibodies (see ``Materials and Methods'') and I-protein A followed by autoradiography, except for synaptobrevin for which alkaline phosphatase-coupled secondary antibody and color substrates were used.



SNAP-25 also bound -SNAP, but with lower affinity since the binding was not saturated at the highest concentration of -SNAP tested (10 µM) (Fig. 2C and not shown). Furthermore, the binding of -SNAP to SNAP-25 was reduced by increasing the sodium chloride concentration to only 300 mM (Fig. 2D).

In contrast to its binding to syntaxin and SNAP-25, -SNAP did not appreciably bind to synaptobrevin (Fig. 2E). No -SNAP was immunoprecipitated together with synaptobrevin even when the -SNAP concentration was as high as 10 µM (Fig. 2E and not shown). To be sure that the antibody used for immunoprecipitation (Cl 69.1(36) ) did not itself interfere with an interaction between -SNAP and synaptobrevin, we repeated the experiment using a GST--SNAP fusion protein immobilized on glutathione-Sepharose. We incubated immobilized GST--SNAP with up to 10 µM synaptobrevin, and after washing did not detect any specific binding between synaptobrevin and -SNAP (data not shown).

Interaction of NSF with the -SNAP-Syntaxin Complex

As outlined in the Introduction, Rothman and collaborators (26) have demonstrated that -SNAP must bind to its receptors on the membrane before NSF can bind and act. Furthermore, ATP hydrolysis by NSF leads to a dissociation of the SNAP receptor (SNARE) complex, with a release of synaptobrevin II, SNAP-25, -SNAP and NSF from syntaxin(26) . Given our finding that -SNAP can bind to syntaxin in the absence of other SNAREs, we tested whether the purified syntaxin--SNAP complex alone could bind NSF, and whether ATP hydrolysis can dissociate this complex.

Syntaxin was incubated with -SNAP, NSF, or both in the presence of either MgATP or non-hydrolyzable MgATPS. Syntaxin was then immunoprecipitated, and the collected material was visualized by protein staining. As shown in Fig. 3A, -SNAP binding to syntaxin was not affected by Mg or nucleotide (Fig. 3A, lanes 3 and 4). NSF bound to the -SNAP-syntaxin complex in the presence of non-hydrolyzable ATPS demonstrating that syntaxin alone is a bona fide SNAP receptor which can promote NSF binding to -SNAP (compare Fig. 3A, lane 7 to lanes 5 and 6). However, when ATP was present, neither -SNAP nor NSF coprecipitated with syntaxin. The lack of -SNAP binding to syntaxin must be due to a significant change in their affinity for each other because the -SNAP concentration in this experiment (5 µM) was sufficient to saturate binding to syntaxin prior to the addition of NSF (see Fig. 2). The effect of ATP was abolished when the samples were incubated in the presence of 1 mM NEM (data not shown), a condition known to inactivate NSF(45, 47) . We conclude that ATP hydrolysis by NSF caused dissociation of -SNAP and NSF from syntaxin in the absence of any other proteins. We refer to this new, non-binding, conformation of syntaxin as syntaxin*.

Virtually identical results were obtained when the vesicle protein synaptobrevin was added to the experiment. In this case, syntaxin was prebound to -SNAP and synaptobrevin before NSF and ATP or ATPS were added (Fig. 3B). Synaptobrevin coprecipitated with syntaxin (Fig. 3B, lane 1), although the binding was substoichiometric at the concentrations used (0.5 µM synaptobrevin, 0.5 µM syntaxin), and bound synaptobrevin was not readily seen by protein staining with Coomassie Blue. Immunoprecipitated proteins were therefore visualized by immunoblotting. The syntaxin-synaptobrevin interaction was unaffected by binding of -SNAP to syntaxin (Fig. 3B, compare lanes 1 and 3). However, when NSF dissociated the -SNAP-syntaxin complex in the presence of ATP, synaptobrevin no longer coprecipitated with syntaxin (Fig. 3B, compare lanes 7 and 8). Syntaxin* thus no longer binds well to synaptobrevin. Parallel experiments using recombinant SNAP-25 suggested that its binding to syntaxin* was also decreased by NSF action (data not shown).()These findings suggest that the conformational change induced by NSF and -SNAP in purified recombinant syntaxin is the same change previously shown to disrupt v-SNARE-t-SNARE pairing in the complete SNARE complex isolated from brain(26) . Furthermore, tests of our NSF and -SNAP proteins on SNARE complexes from brain detergent extracts demonstrated that similar concentrations of each were required for effective disassembly of the complex (data not shown).

How stable is the conformational change in syntaxin induced by NSF? Non-binding syntaxin* was prepared using -SNAP, NSF, and ATP as described above. After a 1-h incubation, the ATPase activity of NSF was inhibited by chelating Mg with EDTA (Fig. 4, lanes 3 and 4), and incubations were continued for an additional 1 or 2 h. A control reaction in which ATPS was present throughout was included to measure maximal levels of -SNAP and NSF binding to syntaxin (lane 1), as was a reaction in which Mg was not chelated so that NSF remained active throughout the experiment (lane 2). Syntaxin was then collected from all samples by immunoprecipitation. As shown in Fig. 4, -SNAP and NSF gradually rebound to syntaxin when the ATPase activity of NSF was inhibited. This suggests that the syntaxin conformation induced by NSF is only transiently stable, and in the absence of other factors or ongoing NSF activity ``syntaxin*'' will relax back to its original ``syntaxin'' conformation.


Figure 4: Rebinding of NSF and -SNAP to syntaxin after quenching of NSF activity. Syntaxin (500 nM) was incubated with -SNAP (1 µM), and NSF (0.3 µM) in the presence of either MgATPS (lane 1) or MgATP (lanes 2-4). After the first hour, EDTA was added (5 mM) to the samples shown in lanes 3 and 4. Samples in lanes 1-3 were incubated for 1 h, and the syntaxin was then immunoprecipitated. The sample in lane 4 was incubated for 2 h prior to starting the immunoprecipitation. Immunoprecipitated proteins were visualized by SDS-PAGE and immunoblotting, using I-protein A for detection.



-SNAP Binds to a Carboxyl-terminal Fragment of Syntaxin

The cytoplasmic portion of syntaxin 1A contains a carboxyl-terminal domain (amino acid residues 194-265) which is sufficient to allow synaptobrevin(27) , SNAP-25(34) , the 1 subunit of the class B N-type Ca channel(49) , and synaptotagmin()to bind. An amino-terminal fragment of syntaxin (residues 1-193) also binds directly to the carboxyl-terminal domain (residues 194-265), suggesting an intramolecular association(27) . To determine which part of syntaxin is responsible for the binding of -SNAP, we generated deletion mutants of syntaxin fused to GST. Full-length, amino-terminal (residues 1-193), and carboxyl-terminal (residues 194-288) syntaxin-GST fusion proteins were immobilized on glutathione-Sepharose and then incubated with -SNAP. After extensive washing, bound -SNAP was detected by immunoblotting. -SNAP bound to full-length GST-syntaxin but not to control beads containing the GST protein alone (Fig. 5A). -SNAP also bound efficiently to the carboxyl-terminal syntaxin fragment and did not bind to the amino-terminal fragment (Fig. 5A). -SNAP therefore binds to the same domain on syntaxin (residues 194-288) which has previously been shown to interact with the SNARE proteins. Since most work characterizing the interactions of purified recombinant syntaxin with other proteins has been done using the cytoplasmic domain of syntaxin (residues 1-265), we also tested the effect of deleting the trans-membrane domain from syntaxin on -SNAP binding. As shown in Fig. 5A, removal of this short sequence reduced the binding of -SNAP to syntaxin. This is reminiscent of the effects of this same deletion on the cleavage of syntaxin by the neurotoxin BoNT/C1 (23) and suggests that the transmembrane domain is important for maintaining syntaxin in a conformation permissive for binding some of its partners.


Figure 5: Domain mapping for -SNAP-syntaxin binding and test of NSF activity on the -SNAP-syntaxin(194-288) complex. A, binding of -SNAP to GST fusion proteins encoding full-length syntaxin, an amino-terminal fragment (syntaxin residues 1-193), a carboxyl-terminal fragment (syntaxin residues 194-288), a cytoplasmic domain fragment (syntaxin residues 1-265) and, as a control, GST alone. GST fusion proteins (10 µg) were immobilized on glutathione-Sepharose and incubated with -SNAP (5 µM). After extensive washing the material bound to the beads was subject to electrophoresis and immunoblotting for -SNAP. The bound -SNAP was detected using the 77.2 antibody and I-protein A. B, binding of His-syntaxin to GST fusion proteins encoding full-length -SNAP, an amino-terminal fragment (-SNAP residues 1-154), and a carboxyl-terminal fragment (-SNAP residues 114-295). GST fusion proteins (20 µg) immobilized on glutathione Sepharose were incubated with His-syntaxin (2.5 µM). Bound material was collected and visualized as in A using the syntaxin-specific monoclonal antibody HPC-1. C, effect of NSF activity on -SNAP and synaptobrevin binding to syntaxin GST fusion proteins. Full-length (1-288) syntaxin or its carboxyl-terminal fragment (194-288) immobilized on glutathione-Sepharose was incubated with synaptobrevin and -SNAP. After a pre-binding period (2 h, 4 °C) NSF and either Mg-ATPS or Mg-ATP was added as shown. After 30 min, the beads were washed extensively and proteins remaining bound visualized by electrophoresis and immunoblotting.



In parallel experiments, we tested two fragments of -SNAP for their ability to bind syntaxin (Fig. 5B). Neither an amino-terminal (residues 1-154) nor a carboxyl-terminal (residues 114-295) -SNAP fragment fused to GST was able to bind syntaxin. Syntaxin did bind tightly to full-length -SNAP-GST. An intact -SNAP appears therefore to be important for the binding of syntaxin, and we cannot define a subdomain responsible for syntaxin binding.

NSF Fails to Disassemble -SNAP and Synaptobrevin from the Syntaxin Carboxyl-terminal Fragment

We next investigated whether the carboxyl-terminal domain of syntaxin bound to -SNAP will allow NSF to bind and disassemble the complex (Fig. 5C). For this experiment, equal amounts of full-length syntaxin or the carboxyl-terminal fragment (syntaxin residues 199-288), expressed as GST-fusion proteins, were immobilized on glutathione-Sepharose. The beads were preincubated with synaptobrevin and -SNAP, and NSF was then added together with either ATPS or ATP. After a 1-h reaction, the beads were washed, and bound material was analyzed by immunoblotting. When full-length GST-syntaxin was used, NSF was able to dissociate -SNAP and synaptobrevin, in agreement with results described above. When the carboxyl-terminal fragment of syntaxin was used, synaptobrevin, -SNAP, and NSF all bound. However, no dissociation of -SNAP or synaptobrevin was observed in the presence of ATP. The binding of NSF itself to -SNAP and syntaxin was reduced in the presence of ATP, suggesting that it induced a change in -SNAP which had no effect on the syntaxin fragment. This suggests that while the carboxyl-terminal fragment contains the sequences necessary for binding -SNAP, as well as synaptobrevin and SNAP-25, it is not able to assume the syntaxin* conformation induced by NSF.


DISCUSSION

NSF is an essential protein in numerous intracellular membrane fusion reactions(3) . Using detergent extracts from brain, Rothman and colleagues (14, 26) have demonstrated that -SNAP and NSF bind to an assembled complex of membrane proteins consisting of synaptobrevin, syntaxin, and SNAP-25. NSF drives an ATP-dependent disassembly of this complex, and this event is thought to be intimately associated with membrane fusion(26) . We have shown here that NSF and -SNAP interact with purified syntaxin and that NSF retains its ability to induce a conformational change in syntaxin in this simplified system. This conformational change leads to formation of what we refer to as syntaxin*, which no longer binds well to -SNAP or the other SNARE complex proteins. Formation of syntaxin* and the resulting disassembly of the SNARE complex may lead directly to membrane fusion as previously proposed(26) , or may instead serve as an essential priming step which prepares syntaxin and the other SNARE proteins for a downstream, possibly Ca triggered, interaction leading to fusion. Essential features of a model based on our results are depicted schematically in Fig. 6.


Figure 6: Model showing the effect of NSF and -SNAP on syntaxin. Syntaxin is shown on the presynaptic plasma membrane before and after the conformational change catalyzed by NSF. Filled stretches of syntaxin (two near the amino terminus, and one toward the carboxyl terminus) are regions predicted to participate in -helical coiled-coil interactions. Syntaxin can bind SNAP-25 on the presynaptic plasma membrane and synaptobrevin on an incoming synaptic vesicle to form a fully assembled SNAP receptor complex. Binding of SNAP-25 and synaptobrevin is mediated by syntaxin sequences located between amino acid residues 194 and 265, and is predicted to depend on -helical coiled coil interactions (34). -SNAP likewise binds to this carboxyl-terminal domain of syntaxin and can bind in either the absence or presence of SNAP-25 and synaptobrevin. NSF binds to the -SNAP-syntaxin complex, again in either the absence or presence of SNAP-25 and synaptobrevin. The NSF--SNAP-syntaxin complex remains stable in the absence of hydrolyzable ATP (i.e. with ATPS or EDTA). Addition of ATP and its hydrolysis by NSF leads via -SNAP to a conformational change in syntaxin. Syntaxin after NSF action, designated syntaxin*, no longer binds -SNAP, synaptobrevin, or SNAP-25. As shown, this is likely a result of binding of the amino-terminal coiled-coil domains of syntaxin to its own carboxyl-terminal domain. These intramolecular interactions preclude binding of other proteins to the carboxyl-terminal domain. Induction of syntaxin* and the resulting dissociation of synaptobrevin and SNAP-25 may lead directly to membrane fusion, or may be a priming step which prepares the molecules for a subsequent fusion triggering reaction.



The t-SNAREs syntaxin 1A and SNAP-25 each bind specifically to -SNAP and are therefore bona fide SNAP receptors, while the v-SNARE synaptobrevin II does not directly bind -SNAP (Fig. 2). Syntaxin binds -SNAP with significantly higher affinity than SNAP-25 does, suggesting that syntaxin is likely to be the predominant -SNAP receptor. -SNAP may in fact bind simultaneously to syntaxin and SNAP-25 in the neuronal plasma membrane, since these two proteins are largely co-localized and are complexed to each other(19) . This is particularly likely since -SNAP binds to the same domain of syntaxin which binds SNAP-25 (amino acids 194-288; Fig. 5), and also binds to the same domain of SNAP-25 which binds syntaxin (amino acids 1-100).()

Current models suggest that vesicles need to dock on their target membrane, resulting in the assembly of the SNARE complex, before SNAPs can bind and NSF can act. However, our data demonstrate that purified syntaxin is sufficient to serve as an effective binding partner for -SNAP. Furthermore, NSF binds specifically to the -SNAP-syntaxin complex, and ATP hydrolysis leads to the disassembly of this ternary complex in the absence of other proteins (Fig. 3A). We believe that disassembly of the NSF--SNAP-syntaxin complex is primarily due to a major conformational change in syntaxin, which is induced by NSF and transmitted through -SNAP, for the following reasons. First, syntaxin also loses affinity for synaptobrevin (Fig. 3B). Since synaptobrevin does not interact with -SNAP directly, this suggests that a conformational change has been induced in syntaxin which interferes with its ability to bind synaptobrevin. Second, no disassembly of -SNAP or synaptobrevin could be induced from the carboxyl-terminal fragment of syntaxin, despite efficient binding of -SNAP and NSF and clear evidence of NSF activity (Fig. 5C). This is difficult to explain if NSF only affects -SNAP or synaptobrevin. Finally, one would expect that all free -SNAP (which does not interact with NSF unless it is bound to its membrane receptor) would have to be shifted to a non-binding conformation if a change in -SNAP would be solely responsible for the observed disassembly. This is difficult to envision given that -SNAP was present in significant excess over its membrane receptors in our experiments. The effects of NSF on fully assembled SNARE complexes containing syntaxin, synaptobrevin, and SNAP-25 are likely to be mediated by the same conformational change in syntaxin, since syntaxin* also shows reduced ability to bind synaptobrevin and SNAP-25 (Fig. 3B).

The carboxyl-terminal domain of syntaxin responsible for -SNAP binding (Fig. 5) has been shown previously to interact with synaptobrevin, SNAP-25, the 1 subunit of the class B N-type Ca channel, and synaptotagmin(27, 34, 49) . This domain contains a region of six heptad repeats which have a high propensity to participate in -helical coiled-coil protein-protein interactions (see Ref. 34 for discussion). Such heptad repeats are also found in the syntaxin-binding domains of SNAP-25 and synaptobrevin, and it has been proposed that the protein-protein interactions between members of the SNARE complex are mediated by assembly of coiled-coil structures(27, 34, 46) . -SNAP also contains several regions with propensity to form coiled-coils (amino acids 5-28, 127-150, and 166-188(6) ), and there are hydrophobic forces involved in the binding between -SNAP and syntaxin as shown by its relative insensitivity to high salt concentrations (Fig. 2B). We speculate that -SNAP also binds to the carboxyl-terminal domain of syntaxin via a coiled-coil interaction. Synthetic peptides corresponding to two of these predicted coiled coil regions have recently been reported to interfere with -SNAP function in exocytosis, implicating these domains in functionally important protein-protein interactions(51) .

Although the carboxyl-terminal domain of syntaxin contains the binding site for -SNAP, the amino-terminal part of the molecule appears to play an essential role in the conformational change induced by NSF. This is documented by our finding that the carboxyl-terminal fragment of syntaxin will support binding of -SNAP and synaptobrevin but not their dissociation by NSF (Fig. 5C). Apparently, NSF induces an interaction between the carboxyl- and amino-terminal regions of syntaxin which results in loss of affinity for -SNAP and other SNARE proteins. In fact, it has previously been reported that amino- and carboxyl-terminal fragments of syntaxin bind to each other and that this association prevents the binding of synaptobrevin(27) . Since the amino-terminal portion of syntaxin also contains domains with a high propensity for the formation of coiled-coil interactions, it is possible that the non-binding syntaxin* conformation is stabilized by such interactions between amino- and carboxyl-terminal regions of the molecule (see Fig. 6). NSF catalyzes, through -SNAP, the transition between two syntaxin conformations. NSF can therefore be thought of as a member of the growing family of ``molecular chaperone'' type proteins, which are important in regulating protein conformation and the oligomerization of multi-subunit protein complexes(52, 53) .

The finding that syntaxin alone can serve as a receptor for -SNAP and NSF poses the intriguing question of whether in an intact neuron, where high concentrations of -SNAP and NSF are present, they will bind to syntaxin that is not part of an assembled SNARE complex. This is not a trivial consideration since syntaxin is widely distributed in nonsynaptic areas of neuronal plasma membranes with only a small portion likely to be in a SNARE complex at any given time(19, 35) . Previous reports have suggested that -SNAP and NSF are both partially membrane associated, and may therefore be interacting with membrane receptors which are not part of a docking complex(47, 50) . It is possible that the fully assembled SNARE complex has a higher affinity for binding -SNAP than the individual components do(48) . However, our preliminary observations using purified recombinant proteins suggest that affinity of -SNAP for syntaxin is similar to its affinity for the SNARE complex. A further possibility is that NSF effectively induces fusion only when it acts on an appropriately assembled oligomeric SNAP receptor complex. It will be interesting to study the subcellular localization of -SNAP and NSF in neurons more closely to see if they bind to membranes only at sites where vesicles dock, or if they instead bind diffusely to membranes with a distribution similar to that of syntaxin(19) .


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.

§
Supported by the Helen Hay Whitney Foundation. To whom correspondence should be addressed: Howard Hughes Medical Institute, Boyer Center for Molecular Medicine, Rm. 247, Yale University Medical School, New Haven, CT 06510. Tel.: 203-737-4455; Fax: 203-787-5334.

Supported by the Deutsche Forschungsgemeinschaft.

The abbreviations used are: NSF, N-ethylmaleimide sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; v-SNARE, vesicle SNARE; t-SNARE, target SNARE; SNAP-25, synaptosomal associated protein of 25 kDa; GST, glutathione S-transferase; HBS, HEPES-buffered saline; PAGE, polyacrylamide gel electrophoresis; ATPS, adenosine 5`-O-(thiotriphosphate).

Otto, H., Hanson, P. I., Chapman, E. R., Blasi, J., and Jahn, R. (1995) Biochem. Biophys. Res. Commun., in press.

E. R. Chapman, P. I. Hanson, S. An, and R. Jahn (1995), manuscript submitted for publication.

P. Hanson and R. Jahn, unpublished observations.


ACKNOWLEDGEMENTS

We wish to thank S. W. Whiteheart and J. E. Rothman for cDNAs encoding His-NSF, -SNAP, and -SNAP, R. H. Scheller and M. K. Bennett for cDNAs encoding syntaxin and synaptobrevin, and T. C. Südhof for cDNA encoding SNAP-25. We are grateful to Silke Engers for her outstanding work in generating the hybridoma cell lines. We thank E. R. Chapman for helpful discussions and several expression vector constructions, and J. M. Edwardson, D. Bruns, members of the Jahn laboratory, and P. DeCamilli for helpful discussions.


REFERENCES
  1. Bennett, M. K., and Scheller, R. H. (1994) Annu. Rev. Biochem.63, 63-100 [CrossRef][Medline] [Order article via Infotrieve]
  2. Ferro-Novick, S., and Jahn, R. (1994) Nature370, 191-193 [CrossRef][Medline] [Order article via Infotrieve]
  3. Rothman, J. E. (1994) Nature372, 55-63 [CrossRef][Medline] [Order article via Infotrieve]
  4. Wilson, D. W., Wilcox, C. A., Flynn, G. C., Chen, E., Kuang, W. J., Henzel, W. J., Block, M. R., Ullrich, A., and Rothman, J. E. (1989) Nature339, 355-359 [CrossRef][Medline] [Order article via Infotrieve]
  5. Clary, D. O., Griff, I. C., and Rothman, J. E. (1990) Cell61, 709-721 [Medline] [Order article via Infotrieve]
  6. Whiteheart, S. W., Griff, I. C., Brunner, M., Clary, D. O., Mayer, T., Buhrow, S. A., and Rothman, J. E. (1993) Nature362, 353-355 [CrossRef][Medline] [Order article via Infotrieve]
  7. Whiteheart, S. W., and Kubalek, E. W. (1995) Trends Cell Biol.5, 64-68 [CrossRef]
  8. Malhotra, V., Orci, L., Glick, B. S., Block, M. R., and Rothman, J. E. (1988) Cell54, 221-227 [Medline] [Order article via Infotrieve]
  9. Orci, L., Malhotra, V., Amherdt, M., Serafini, T., and Rothman, J. E. (1989) Cell56, 357-368 [Medline] [Order article via Infotrieve]
  10. Whiteheart, S. W., Rossnagel, K., Buhrow, S. A., Brunner, M., Jaenicke, R., and Rothman, J. E. (1994) J. Cell Biol.126, 945-954 [Abstract]
  11. Weidman, P. J., Melancon, P., Block, M. R., and Rothman, J. E. (1989) J. Cell Biol.108, 1589-1596 [Abstract]
  12. Wilson, D. W., Whiteheart, S. W., Wiedmann, M., Brunner, M., and Rothman, J. E. (1992) J. Cell Biol.117, 531-538 [Abstract]
  13. Whiteheart, S. W., Brunner, M., Wilson, D. W., Wiedmann, M., and Rothman, J. E. (1992) J. Biol. Chem.267, 12239-12243 [Abstract/Free Full Text]
  14. Söllner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P., and Rothman, J. E. (1993) Nature362, 318-324 [CrossRef][Medline] [Order article via Infotrieve]
  15. Elferink, L. A., Trimble, W. S., and Scheller, R. H. (1989) J. Biol. Chem.264, 11061-11064 [Abstract/Free Full Text]
  16. Baumert, M., Maycox, P. R., Navone, F., DeCamilli, P., and Jahn, R. (1989) EMBO J.8, 379-384 [Abstract]
  17. Bennett, M. K., Calakos, N., and Scheller, R. H. (1992) Science257, 255-259 [Medline] [Order article via Infotrieve]
  18. Inoue, A., Obata, K., and Akagawa, K. (1992) J. Biol. Chem.267, 10613-10619 [Abstract/Free Full Text]
  19. Garcia, E. P., McPherson, P. S., Chilcote, T. J., Takei, K., and DeCamilli, P. (1995) J. Cell Biol.129, 105-120 [Abstract]
  20. Bark, I. C. (1993) J. Mol. Biol.233, 67-76 [CrossRef][Medline] [Order article via Infotrieve]
  21. Schiavo, G., Benfenati, F., Poulain, B., Rossetto, O., DeLaureto, P. P., DasGupta, B. R., and Montecucco, C. (1992) Nature359, 832-835 [CrossRef][Medline] [Order article via Infotrieve]
  22. Blasi, J., Chapman, E. R., Link, E., Binz, T., Yamasaki, S., DeCamilli, P., Südhof, T. C., Niemann, H., and Jahn, R. (1993) Nature365, 160-163 [CrossRef][Medline] [Order article via Infotrieve]
  23. Blasi, J., Chapman, E. R., Yamasaki, S., Binz, T., Niemann, H., and Jahn, R. (1993) EMBO J.12, 4821-4828 [Abstract]
  24. Bennett, M. K., Garcia-Arraras, J. E., Elferink, L. A., Peterson, K., Fleming, A. M., Hazuka, C. D., and Scheller, R. H. (1993) Cell74, 863-873 [Medline] [Order article via Infotrieve]
  25. McMahon, H. T., Ushkaryov, Y. A., Edelmann, L., Link, E., Binz, T., Niemann, H., Jahn, R., and Südhof, T. C. (1993) Nature364, 346-349 [CrossRef][Medline] [Order article via Infotrieve]
  26. Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell75, 409-418 [Medline] [Order article via Infotrieve]
  27. Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science263, 1146-1149 [Medline] [Order article via Infotrieve]
  28. Pevsner, J., Hsu, S.-C., Braun, J. E. A., Calakos, N., Ting, A. E., Bennett, M. K., and Scheller, R. H. (1994) Neuron13, 353-361 [Medline] [Order article via Infotrieve]
  29. Sgaard, M., Tani, K., Ye, R. R., Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J. E., and Söllner, T. (1994) Cell78, 937-948 [Medline] [Order article via Infotrieve]
  30. Sumida, M., Hong, R.-M., and Tagaya, M. (1994) J. Biol. Chem.269, 20636-20641 [Abstract/Free Full Text]
  31. Tagaya, M., Wilson, D. W., Brunner, M., Arango, N., and Rothman, J. E. (1993) J. Biol. Chem.268, 2662-2666 [Abstract/Free Full Text]
  32. Binz, T., Blasi, J., Yamasaki, S., Baumeister, A., Link, E., Südhof, T., Jahn, R., and Niemann, H. (1994) J. Biol. Chem.269, 1617-1620 [Abstract/Free Full Text]
  33. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  34. Chapman, E. R., An, S., Barton, N., and Jahn, R. (1994) J. Biol. Chem.269, 27427-27432 [Abstract/Free Full Text]
  35. Walch-Solimena, C., Blasi, J., Edelmann, L., Chapman, E. R., Fischer von Mollard, G., and Jahn, R. (1995) J. Cell Biol.128, 637-645 [Abstract]
  36. Edelmann, L., Hanson, P. I., Chapman, E. R., and Jahn, R. (1995) EMBO J.14, 224-231 [Abstract]
  37. Guan, K. L., and Dixon, J. E. (1991) Anal. Biochem.192, 262-267 [Medline] [Order article via Infotrieve]
  38. Wilson, D. W., and Rothman, J. E. (1992) Methods Enzymol.219, 309-318 [Medline] [Order article via Infotrieve]
  39. Köhler, G., and Milstein, C. (1975) Nature256, 495-497 [Medline] [Order article via Infotrieve]
  40. Jahn, R., Schiebler, W., Ouimet, C., and Greengard, P. (1985) Proc. Natl. Acad. Sci. U. S. A.82, 4137-4141 [Abstract]
  41. Harlow, E., and Lane, D. (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
  42. Laemmli, U. K. (1970) Nature227, 680-685 [Medline] [Order article via Infotrieve]
  43. Barnstable, C. J., Hofstein, R., and Akagawa, K. (1985) Dev. Brain Res.20, 286-290
  44. Bradford, M. M. (1976) Anal. Biochem.72, 248-254 [CrossRef][Medline] [Order article via Infotrieve]
  45. Morgan, A., Dimaline, R., and Burgoyne, R. D. (1994) J. Biol. Chem.269, 29347-29350 [Abstract/Free Full Text]
  46. Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Südhof, T. C., and Niemann, H. (1994) EMBO J.13, 5051-5061 [Abstract]
  47. Glick, B. S., and Rothman, J. E. (1987) Nature326, 309-312 [CrossRef][Medline] [Order article via Infotrieve]
  48. McMahon, H. T., and Südhof, T. C. (1995) J. Biol. Chem.270, 2213-2217 [Abstract/Free Full Text]
  49. Sheng, Z.-H., Rettig, J., Takahashi, M., and Catterall, W. A. (1994) Neuron13, 1303-1313 [Medline] [Order article via Infotrieve]
  50. Clary, D. O., and Rothman, J. E. (1990) J. Biol. Chem.265, 10109-10117 [Abstract/Free Full Text]
  51. DeBello, W. M., O'Connor, V., Dresbach, T., Whiteheart, S. W., Wang, S. S.-H., Schweizer, F. E., Betz, H., Rothman, J. E., and Augustine, G. J. (1995) Nature373, 626-630 [CrossRef][Medline] [Order article via Infotrieve]
  52. Rothman, J. E. (1989) Cell59, 591-601 [Medline] [Order article via Infotrieve]
  53. Hendrick, J. P., and Hartl, F. U. (1993) Annu. Rev. Biochem.62, 349-384 [CrossRef][Medline] [Order article via Infotrieve]
  54. Link, E., Edelmann, L., Chou, J. H., Binz, T., Eisel, U., Baumert, M., Sudhof, T. C., Niemann, H., and Jahn, R. (1992) Biochem. Biophys. Res. Commun.189, 1017-1023 [Medline] [Order article via Infotrieve]

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