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Previous Article | Next Article
Volume 17, Number 5, Issue of March 1, 1997 pp. 1596-1603
Copyright ©1997 Society for NeuroscienceEffect of Mutations in Vesicle-Associated Membrane Protein (VAMP) on the Assembly of Multimeric Protein Complexes
Joe C. Hao1, a, Natalie Salem2, a, Xiao-Rong Peng1, Regis B. Kelly2, and Mark K. Bennett1 1 Department of Molecular and Cell Biology, University of California, Berkeley, California 94720, and 2 Department of Biochemistry and Biophysics, Hormone Research Institute, University of California, San Francisco, California 94143
ABSTRACT
The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.
Key words: VAMP; syntaxin; SNAP-25; SNARE complex; synaptic vesicle; exocytosis
INTRODUCTION
Synaptic transmission, the primary means by which neurons communicate with their target cells, is initiated by the regulated secretion of neurotransmitter from the presynaptic nerve terminal. The precise interaction of neurotransmitter-filled synaptic vesicles with the presynaptic plasma membrane and the rapid fusion of these membranes after an action potential-induced influx of calcium constitute the primary steps in neurotransmitter secretion. Recent biochemical and genetic studies have begun to elucidate the molecular mechanisms responsible for neurotransmitter secretion and reveal the fundamental similarity of these mechanisms to those underlying the targeting and fusion of other intracellular transport vesicle intermediates (Bennett and Scheller, 1993
, 1994
Vesicle-associated membrane protein 2 (VAMP-2, also known as synaptobrevin), together with the presynaptic plasma membrane proteins syntaxin 1 and synaptosome-associated protein of 25 kDa (SNAP-25), has been proposed to participate in synaptic vesicle targeting or fusion. Several lines of evidence support such a role, including the observation that a class of potent presynaptic neurotoxins (including tetanus toxin and six types of botulinum toxin) selectively target syntaxin 1, SNAP-25, or VAMP-2 for proteolysis (Niemann et al., 1994
Recent biochemical studies have demonstrated that direct interactions among VAMP-2, syntaxin 1A, and SNAP-25 contribute to the formation of the synaptic SNARE complex (Calakos et al., 1994
MATERIALS AND METHODS
Materials. Rabbit polyclonal antibodies against rat VAMP-2 either were provided by William Trimble [University of Toronto (Gaisano et al., 1994
Preparation of fusion proteins. Constructs for the bacterial expression of glutathione S-transferase (GST) fusion proteins incorporating the full cytoplasmic domain of rat syntaxin 1A (amino acids 4-267) and mouse SNAP-25 (amino acids 1-206) were prepared in the pGEX-KG vector (Guan and Dixon, 1991 C)] was prepared by digesting a vector consisting of full-length SNAP-25 in pGEX-KG with XbaI (which cuts both in the SNAP-25 insert and at the 3
end of the pGEX-KG polylinker), followed by religation. Constructs for the expression GST-VAMP-2 fusion proteins were prepared by PCR amplification of the DNA fragment encoding the cytoplasmic domain of rat VAMP-2 (amino acids 1-94) and various deletion and point mutant forms of VAMP-2 (Grote et al., 1995
Immobilized GST fusion proteins (for use in affinity chromatography experiments) were prepared from bacterial lysates, as previously described (Pevsner et al., 1994 -mercaptoethanol as described (Calakos et al., 1994
Affinity chromatography assay. For affinity chromatography binding studies, GST fusion proteins (either syntaxin 1A or SNAP-25) bound to glutathione-agarose beads were incubated with wild-type or mutant VAMP-2 either in the absence or presence of SNAP-25 (as indicated in the figure legends) in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2.5 mM CaCl2, 0.1% Triton X-100, 0.1% gelatin, and 0.1% bovine serum albumin. After a 2 hr incubation at 4°C, the beads were washed three times with 500 µl of 10 mM HEPES-KOH, pH 7.5, 140 mM potassium acetate, 1 mM MgCl2, 0.1 mM EGTA, and 0.1% Triton X-100 (buffer A) containing 0.1% gelatin and once with buffer A without gelatin. Proteins on the beads were recovered by boiling in SDS-PAGE sample buffer, resolved by SDS-PAGE, and analyzed by Western blotting. The blots were probed with affinity-purified rabbit anti-VAMP-2 antibodies. After incubation with 125I-labeled goat anti-rabbit secondary antibody (ICN Biochemicals, Costa Mesa, CA), VAMP-2 immunoreactivity was visualized by autoradiography and quantitated by phosphorimaging (Molecular Dynamics, Sunnyvale, CA). Known amounts of bacterially expressed VAMP-2 proteins were analyzed simultaneously to normalize for potential differences in immunoreactivity. For titration experiments (see Figs. 2, 4), the 50% effective concentration (EC50), defined as the concentration of VAMP-2 at which half-maximal binding occurs, was estimated from plots of phosphorimaging pixel intensity versus input VAMP-2 protein concentration. 
Fig. 2. Titration of the in vitro binding of wild-type, M46A, and N49A mutant VAMP-2 to GST-syntaxin 1A and GST-SNAP-25. Shown are in vitro binding of wild-type and mutant VAMP-2 at the indicated concentrations to (A) immobilized GST-syntaxin 1A (0.3 µM) and (B) immobilized GST-SNAP-25 (0.3 µM). Bound VAMP-2 was recovered and visualized by Western blotting, as described in Materials and Methods.
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Fig. 4. Potentiation of wild-type and
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Yeast two-hybrid analysis. To allow in-frame insertion into the two-hybrid expression plasmids, we introduced convenient restriction endonuclease sites at each end of the desired cDNA fragment. The coding sequence of the cytoplasmic domain of rat VAMP-2 (amino acids 1-94) and a series of VAMP-2 deletion and point mutants (Grote et al., 1995
Yeast strain SFY526 (MATa, ura3-52, his 3-200, ade 2-101, lys 2-801, trp 1-901, leu 2-3, 112, canr, gal4-542, gal80-538, URA::GAL1-lacZ) (Clontech) was grown in YPD medium (yeast extract, peptone, and dextrose) or on synthetic defined dropout yeast medium lacking the appropriate amino acids (BIO 101, La Jolla, CA). SFY526 was transformed simultaneously with two of the hybrid plasmids by the improved lithium acetate method of Gietz et al. (1992)
Ligand overlay blotting. Wild-type and mutant VAMPs (100 ng each) were separated by SDS-PAGE and transferred to nitrocellulose. Four identical nitrocellulose blots containing the immobilized VAMP-2 proteins were blocked first for 30 min at room temperature in 20 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.05% Tween-20, and 5% nonfat dry milk (blocking buffer). Then the blots were incubated in blocking buffer containing 2 µg/ml of either syntaxin 1A or SNAP-25 alone or a mixture of both proteins. After a 1 hr incubation, the blots were washed in blocking buffer for 5 min and then probed with a monoclonal antibody against syntaxin 1A (HPC-1) or an affinity-purified anti-SNAP-25 antibody. After incubation with 125I-labeled goat anti-mouse or goat anti-rabbit antibodies, the bound proteins were visualized by autoradiography and quantitated by phosphorimaging.
Analysis of SDS-resistant SNARE complexes. Combinations of soluble wild-type or mutant VAMP-2, syntaxin 1A, and SNAP-25 (2 µM each) were incubated overnight (16 hr) at 4°C in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 2.5 mM CaCl2. After addition of 6× SDS-PAGE sample buffer, samples were divided into two aliquots and either boiled for 3 min or incubated at 37°C for 3 min. Samples then were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with an affinity-purified monoclonal antibody against syntaxin 1A [HPC-1 (Barnstable et al., 1985
RESULTS
Deletions within the conserved domain of VAMP-2 eliminate binary interactions with syntaxin 1A and SNAP-25
To identify the domains of VAMP-2 involved in interactions with syntaxin 1A and SNAP-25, we analyzed the binding properties of a series of VAMP-2 mutants (Fig. 1A) both in vitro with an affinity chromatography assay and in vivo with the yeast two-hybrid system. For the affinity chromatography assay, the interaction of soluble recombinant VAMP-2 (either wild-type or mutant) with immobilized GST-syntaxin 1A or GST-SNAP-25 was examined. As shown in Figure 1B,C, the VAMP-2 deletion mutants fell into three categories: (1) those able to interact with either syntaxin 1A or SNAP-25 (
Fig. 1. Affinity chromatography survey of binary and ternary interactions among wild-type and mutant VAMP-2, GST-syntaxin 1A, and GST-SNAP-25. A, Schematic diagram of wild-type VAMP-2 and the VAMP-2 mutants used in the present study. The constructs encode the cytoplasmic domain of wild-type or mutant VAMP-2 fused to the C terminus of either GST or the GAL4 DNA binding domain. TM, Transmembrane domain. B-D, In vitro binding of wild-type and mutant VAMP-2 (10 µM) to (B) immobilized GST-SNAP-25 (0.5 µM), (C) immobilized GST-syntaxin 1A (0.5 µM), and (D) immobilized GST-syntaxin 1A (0.5 µM) in the presence of soluble SNAP-25 (1 µM). Bound VAMP-2 was recovered and quantitated by Western blotting, as described in Materials and Methods. The relative strengths of the interactions are expressed as the percentage of VAMP-2 input recovered on the immobilized GST fusion proteins.
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To investigate the in vivo interactions between wild-type or deletion mutants of VAMP-2 and either syntaxin or SNAP-25, the yeast two-hybrid system was used (Fields and Song, 1989
Qualitative results from the two-hybrid analysis were obtained by blue/white screening with X-gal indicator. Control experiments in which the yeast were transformed with one or both vectors lacking the insert or with vectors containing a cDNA not related to the SNARE complexes (SV40 large T-antigen) failed to produce detectable
Table 1. Analysis of in vivo interactions between wild-type or mutant VAMP-2 and syntaxin 1A or SNAP-25 with the yeast two-hybrid system
VAMP-2 "bait" Syntaxin 1A "prey" SNAP-25 "prey" Wild-type + ++ + + M46A + + N49A ++ +++ Two-hybrid constructs were prepared and cotransfected into the reporter strain SFY526, as described in Materials and Methods. (white colonies) signal. The number of +'s reflects the intensity of the blue color.
Point mutations in VAMP-2 (M46A and N49A) have opposing effects on binary interactions with syntaxin 1A and SNAP-25
The region of VAMP-2 removed by the
To better characterize the effect of the VAMP-2 point mutations on the in vitro interactions with syntaxin 1A and SNAP-25, we performed affinity chromatography assays with increasing concentrations of soluble VAMP-2. As shown in Figure 2A, the N49A mutant bound to GST-syntaxin 1A to a greater extent and with a higher apparent affinity than wild-type VAMP-2. In comparison with the binding of wild-type VAMP-2 (which was not saturable at concentrations up to 20 µM), the binding of the N49A mutant was strongly potentiated and saturable, with an EC50 of ~4 µM. Similarly, as shown in Figure 2B, the N49A mutant bound to GST-SNAP-25 to a greater extent and with higher apparent affinity (EC50 of 2.5 µM) than wild-type VAMP-2 (EC50 of 6 µM). In contrast to the N49A mutant, the binding of the M46A mutant to both GST-syntaxin 1A (Fig. 2A) and GST-SNAP-25 (Fig. 2B) was significantly weaker than wild-type VAMP-2, failing to reach saturation at even the highest concentration tested (20 µM).
To determine whether the point mutations in VAMP-2 influence the formation of protein complexes in vivo, we used the yeast two-hybrid assay. A quantitative enzyme assay was used to assess the level of 
Fig. 3. Quantitative analysis of the in vivo binding of wild-type, M46A, and N49A mutant VAMP-2 to syntaxin 1A and SNAP-25 with the yeast two-hybrid system. Two-hybrid "bait" (wild-type, M46A, and N49A mutant VAMP-2) and "prey" (syntaxin 1A and SNAP-25) plasmids were prepared as described in Materials and Methods. The reporter strain SFY526 was cotransformed with the indicated plasmid pairs, and the level of
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Mutations within the conserved domain of VAMP-2 do not eliminate the formation of ternary SNARE complexes
Another characteristic of VAMP-2 is its ability to form ternary complexes with the combination of syntaxin 1A and SNAP-25. The formation of the ternary complexes can be monitored by the potentiation of VAMP-2 binding to GST-syntaxin 1A in the presence of soluble SNAP-25 (Pevsner et al., 1994
Previous studies have demonstrated that the N-terminal 80 amino acids of SNAP-25 are sufficient for syntaxin 1A binding, whereas the C-terminal 25 amino acids are necessary for VAMP-2 binding (Chapman et al., 1994 Ternary, but not binary, SNARE complex formation can be detected by ligand overlay blotting
To further characterize the interactions of VAMP-2 with syntaxin 1A and SNAP-25, we performed ligand overlay blotting. The VAMP-2 proteins were immobilized by electrophoretic transfer to nitrocellulose membranes after SDS-PAGE. Then the membranes were incubated with either syntaxin 1A, SNAP-25, or a mixture of syntaxin 1A and SNAP-25. Sites of syntaxin 1A or SNAP-25 binding were visualized (Fig. 5A,B) and quantitated (Fig. 5C) by Western blotting with syntaxin 1A and SNAP-25 specific antibodies. Neither syntaxin 1A alone or SNAP-25 alone was capable of interacting with immobilized wild-type or VAMP-2 mutants. However, in the presence of both syntaxin 1A and SNAP-25, ternary complexes were able to form with each of the immobilized VAMP-2 proteins. The level of syntaxin 1A and SNAP-25 binding varied among the different mutants, with the lowest level of binding observed with deletion mutants
Fig. 5. Analysis of binary and ternary SNARE protein interactions via ligand overlay blotting. A, Wild-type and mutant VAMP-2 proteins (100 ng each) were resolved by SDS-PAGE and transferred to nitrocellulose. After an incubation with 2 µg/ml of either syntaxin 1A (upper) or both syntaxin 1A and SNAP-25 (lower), the blots were probed for syntaxin 1A immunoreactivity, as described in Materials and Methods. B, Blots identical to those described in A were incubated with 2 µg/ml of either SNAP-25 (upper) or both SNAP-25 and syntaxin 1A (lower) and probed for SNAP-25 immunoreactivity. C, Relative binding intensity of syntaxin 1A (open bars) and SNAP-25 (solid bars) to immobilized VAMP-2 proteins, as determined by quantitation of the blots shown in the lower panels of A and B. All values were normalized to those for wild-type VAMP-2.
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A subset of VAMP-2 deletion mutants eliminates the formation of SDS-resistant ternary SNARE complexes
The formation of heat-labile, SDS-resistant multimeric complexes composed of VAMP-2, syntaxin 1A, and SNAP-25 initially was described by Hayashi et al. (1994)
Fig. 6. Formation of heat-labile, SDS-resistant ternary complexes with wild-type and mutant forms of VAMP-2. Combinations of soluble wild-type or mutant VAMP-2, syntaxin 1A, and SNAP-25 (2 µM each) were incubated for 16 hr at 4°C. After SDS-PAGE sample buffer was added, samples were divided into two aliquots and either incubated at 37°C (a) or boiled (b). After SDS-PAGE and transfer to nitrocellulose, multimeric complexes were detected by using an antibody against syntaxin 1A and visualized by autoradiography. Binary controls are shown in the three pairs of lanes at the far left. The molecular weight (MW) markers are in kDa.
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Fig. 7. Heat lability of SDS-resistant ternary complexes generated by using wild-type, M46A, and N49A mutant VAMP-2. Ternary SNARE complexes containing wild-type VAMP-2 (A), M46A (B), or N49A (C) were assembled as described in Figure 6 and divided into 14 aliquots. After incubation at the indicated temperatures, the samples were resolved by SDS-PAGE and analyzed by immunoblotting with an affinity-purified anti-VAMP-2 antibody and visualized with a chemiluminescence detection system. The molecular weight (MW) markers are expressed in kDa.
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To investigate the possible relationship among the different SDS-resistant complexes and to determine whether the complexes formed with the VAMP-2 point mutants display differential stability, we performed melting curve experiments. Mixtures of syntaxin 1A, SNAP-25, and either wild-type VAMP-2, M46A, or N49A were incubated at increasing temperatures before SDS-PAGE and Western blot analysis with an antibody against VAMP-2. With wild-type VAMP-2 (Fig. 7A), the 50 and 200 kDa complexes began to dissociate at a slightly lower temperature (55-61°C) than the 100 kDa complex (61-67°C), with all three complexes being fully dissociated by 79°C. Because none of the complexes increases in abundance during the dissociation process, it is unlikely that temperature-induced interconversion of the complexes is occurring. The SDS-resistant complexes formed with the M46A point mutant seemed to be less stable than wild-type VAMP-2 complexes (Fig. 7B), whereas the SDS-resistant complexes formed with the N49A point mutant were less stable than both wild-type VAMP-2 and M46A mutant complexes (Fig. 7C). This observation demonstrates that the enhanced ability of the N49A mutant to form binary complexes with both syntaxin 1A and SNAP-25 (Figs. 2, 3) does not result in the formation of SDS-resistant ternary complexes with enhanced thermal stability. Rather, the reduced stability of the M46A and N49A mutant complexes may be a consequence of disruption of the integrity of the central region of VAMP-2, the importance of which in SDS resistance was clearly established by the behavior of the
DISCUSSION
Formation of binary complexes
We have examined the binary interactions between VAMP-2 and either syntaxin 1A or SNAP-25, both in vitro with an affinity chromatography assay and in vivo with the yeast two-hybrid system. The similar results obtained with the two assays provide confidence that the interactions being monitored accurately reflect those responsible for complex assembly at the synapse. Deletion mutagenesis was used to delineate the regions of VAMP-2 required for its binding to syntaxin 1A and SNAP-25. Our results suggest that VAMP-2 contains a SNAP-25 binding site that encompasses at least amino acids 41-60 and a syntaxin 1A binding site that encompasses at least amino acids 31-70. The fact that deletion mutations in either of the two domains in VAMP-2 predicted to form coiled coils (amino acids 30-56 or 57-88) can eliminate syntaxin 1A or SNAP-25 binding is consistent with the prospect that coiled coils play an important role in these interactions. Two amino acid substitutions within the conserved domain of VAMP-2, M46A and N49A, have strong but opposite effects on sorting to synaptic vesicles (Grote et al., 1995
These results clearly demonstrate that point mutations that influence the sorting of VAMP-2 also affect its association with members of the SNARE complex. However, the correlation between binary complex formation and synaptic vesicle targeting does not extend to the deletion mutants of VAMP-2. For example, the
An alternative explanation for the properties of the VAMP-2 point mutants is that they differentially influence the conformational state of VAMP-2. Although a synthetic peptide corresponding to the cytoplasmic domain of VAMP-2 is not highly structured in solution (Cornille et al., 1994 -SNAP/NSF may include the generation of a closed conformation of syntaxin 1A that is unable to interact with either VAMP-2 or SNAP-25 (Pevsner et al., 1994
Assembly of ternary complexes
Several methods have been used to investigate the requirements for assembly of ternary SNARE complexes. Two of these methods, the affinity chromatography assay and the ligand overlay assay, clearly demonstrate that assembly is a cooperative process. Although some (affinity chromatography assay) or all (ligand overlay assay) of the VAMP-2 proteins failed to form binary complexes with either syntaxin 1A or SNAP-25, they all were able to form ternary complexes. In addition, a truncated form of SNAP-25 [SNAP-25 (
One of the striking properties of the ternary complexes that form among syntaxin 1A, SNAP-25, and VAMP-2 is their resistance to SDS denaturation at low temperature. Evidence that these SDS-resistant complexes may be physiologically important is provided by the following observations: (1) they are detectable in SDS extracts of brain tissue (Hayashi et al., 1994
Although the in vitro assembly of SNARE complexes is well established, much less is known about their structure or in vivo functional importance. Our results demonstrate that the domains of VAMP-2 required for the assembly of binary, ternary, and SDS-resistant ternary complexes are distinct. These observations establish a foundation for the more detailed structural studies that will be required to define precisely the protein-protein interactions that lead to the assembly of distinct binary and ternary SNARE complexes. Furthermore, mutant forms of VAMP with well characterized effects on the assembly of SNARE complexes should prove to be useful tools for localization of SNARE complex functions within the ordered set of reactions that lead to neurotransmitter secretion.
FOOTNOTES
Received Oct. 17, 1996; revised Dec. 9, 1996; accepted Dec. 17, 1996.
a These authors contributed equally to this work.
This work was supported by National Institutes of Health Grants NS 09878 and DA 10154 (R.B.K.) and GM 51313 (M.K.B.), the Alfred P. Sloan Foundation (M.K.B.), the McKnight Fund for Neuroscience (M.K.B.), and a Fonds National de la Recherche Suisse fellowship (N.S.). We thank William Trimble and Colin Barnstable for antibodies to VAMP-2 and syntaxin 1, respectively, and Eric Grote for VAMP-2 deletion and point mutant constructs and valuable discussions throughout the course of this work.
Correspondence should be addressed to Dr. Mark K. Bennett, Department of Molecular and Cell Biology, Life Sciences Addition, University of California, Berkeley, CA 94720.
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