Dirk Fasshauer

SNARE [soluble NSF (N-ethylmaleimide-sensitive fusion protein) attachment protein receptor] proteins are essential for membrane fusion and are conserved from yeast to humans. Sequence alignments of the most conserved regions were mapped onto the recently solved crystal structure of the heterotrimeric synaptic fusion complex. The association of the four alpha-helices in the synaptic fusion complex structure produces highly conserved layers of interacting amino acid side chains in the center of the four-helix bundle. Mutations in these layers reduce complex stability and cause defects in membrane traffic even in distantly related SNAREs. When syntaxin-4 is modeled into the synaptic fusion complex as a replacement of syntaxin-1A, no major steric clashes arise and the most variable amino acids localize to the outer surface of the complex. We conclude that the main structural features of the neuronal complex are highly conserved during evolution. On the basis of these features we have reclassified SNARE proteins into Q-SNAREs and R-SNAREs, and we propose that fusion-competent SNARE complexes generally consist of four-helix bundles composed of three Q-SNAREs and one R-SNARE.

Assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) syntaxin 1, SNAP-25, and synaptobrevin 2 is thought to be the driving force for the exocytosis of synaptic vesicles. However, whereas exocytosis is triggered at a millisecond time scale, the SNARE-mediated fusion of liposomes requires hours for completion, which challenges the idea of a key role for SNAREs in the final steps of exocytosis. We found that liposome fusion was dramatically accelerated when a stabilized syntaxin/SNAP-25 acceptor complex was used. Thus, SNAREs do have the capacity to execute fusion at a speed required for neuronal secretion, demonstrating that the maintenance of acceptor complexes is a critical step in biological fusion reactions.

SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors α-, β-, and γ-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant α-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in α-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion. SNAP-25, syntaxin, and synaptobrevin play a key role in the regulated exocytosis of synaptic vesicles, but their mechanism of action is not understood. In vitro, the proteins spontaneously assemble into a ternary complex that can be dissociated by the ATPase N-ethylmaleimide-sensitive fusion protein and the cofactors α-, β-, and γ-SNAP. Since the structural changes associated with these reactions probably form the basis of membrane fusion, we have embarked on biophysical studies aimed at elucidating such changes in vitro using recombinant proteins. All proteins were purified in a monomeric form. Syntaxin showed significant α-helicity, whereas SNAP-25 and synaptobrevin exhibited characteristics of largely unstructured proteins. Formation of the ternary complex induced dramatic increases in α-helicity and in thermal stability. This suggests that structure is induced in SNAP-25 and synaptobrevin upon complex formation. In addition, the stoichiometry changed from 2:1 in the syntaxin-SNAP-25 complex to 1:1:1 in the ternary complex. We propose that the transition from largely unstructured monomers to a tightly packed, energetically favored ternary complex connecting two membranes is a key step in overcoming energy barriers for membrane fusion. Neurons release their neurotransmitters by the Ca2+-dependent exocytosis of synaptic vesicles. In recent years, several membrane proteins have been identified which are required for exocytotic membrane fusion. These proteins include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) 1The abbreviations used are: VAMP, vesicle-associated membrane protein; NSF,N-ethylmaleimide-sensitive fusion protein; SNAP, soluble NSF attachment protein; SNARE, SNAP receptor; SNAP-25, synaptosomal associated protein of 25 kDa; PAGE, polyacrylamide gel electrophoresis, PCR, polymerase chain reaction; TCEP, Tris(2-carboxyethyl)phosphine hydrochloride); MALLS, multiangle laser light scattering. and the synaptic membrane proteins syntaxin and SNAP-25, collectively referred to as SNAREs. Synaptobrevin and syntaxin both contain a single transmembrane domain at the C terminus (1Bennett M.K. Calakos N. Scheller R.H. Science. 1992; 257: 255-259Crossref PubMed Scopus (1077) Google Scholar, 2Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). SNAP-25 does not contain a transmembrane domain but carries palmitoyl side chains attached to cysteine residues in the middle of the sequence (3Hess D.T. Slater T.M. Wilson M.C. Skene J.H.P. J. Neurosci. 1992; 12: 4634-4641Crossref PubMed Google Scholar, 4Veit M. Söllner T.H. Rothman J.E. FEBS Lett. 1996; 385: 119-123Crossref PubMed Scopus (205) Google Scholar). Homologues of these proteins have been identified in many eukaryotic cells including yeast, suggesting that fusion of trafficking vesicles with their respective target membranes is mediated by a conserved mechanism (2Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar,5Ferro-Novick S. Jahn R. Nature. 1994; 370: 191-193Crossref PubMed Scopus (561) Google Scholar, 6Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (312) Google Scholar, 7Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 8Augustine G.J. Burns M.E. DeBello W.M. Pettit D.L. Schweizer F.E. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 659-701Crossref PubMed Scopus (117) Google Scholar). While the evidence linking synaptobrevin, SNAP-25, and syntaxin to exocytosis is compelling, their precise role is unknown. In detergent extracts of brain membranes, the three proteins form a tight complex (9Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar). A ternary complex with properties similar to the native complex can be formed using recombinant proteins lacking their transmembrane anchors (11Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). Both native and recombinant complexes can be disassembled by the concerted action of the ATPase NSF and the protein α-SNAP (9Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar, 10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar, 12Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar, 13Hanson P.I. Otto H. Barton N. Jahn R. J. Biol. Chem. 1995; 270: 16955-16961Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). The latter two proteins are soluble, abundant, and highly conserved through evolution. They are essential for the fusion of trafficking vesicles with their target membranes (9Söllner T. Bennett M.K. Whiteheart S.W. Scheller R.H. Rothman J.E. Cell. 1993; 75: 409-418Abstract Full Text PDF PubMed Scopus (1586) Google Scholar,10Söllner T. Whiteheart S.W. Brunner M. Erdjument-Bromage H. Geromanos S. Tempst P. Rothman J.E. Nature. 1993; 362: 318-324Crossref PubMed Scopus (2637) Google Scholar). The assembly and disassembly of the SNARE proteins has not yet been integrated into a coherent picture of exocytosis. However, any interference with these reactions seriously inhibits membrane fusion (6Calakos N. Scheller R.H. Physiol. Rev. 1996; 76: 1-29Crossref PubMed Scopus (312) Google Scholar, 7Rothman J.E. Wieland F.T. Science. 1996; 272: 227-234Crossref PubMed Scopus (1026) Google Scholar, 8Augustine G.J. Burns M.E. DeBello W.M. Pettit D.L. Schweizer F.E. Annu. Rev. Pharmacol. Toxicol. 1996; 36: 659-701Crossref PubMed Scopus (117) Google Scholar). To further understand these essential reactions, one needs to learn more about the structural and energetic properties of assembly and disassembly. Therefore, we have begun intensive biophysical and biochemical studies of the SNAREs and of their complexes. Nondenaturing gel electrophoresis, multiangle laser light scattering (MALLS), and CD spectroscopy were used to investigate structural properties of the individual proteins and their complexes. Dramatic changes in structure, oligomerization, and thermal stability upon complex formation were observed. All recombinant proteins were expressed as His6-tagged fusion proteins. Subcloning was performed using standard techniques (14Saiki R.K. Gelfand D.H. Stoffel S. Scharf S.J. Higuchi R. Horn G.T. Mullis K.B. Ehrlich H.A. Science. 1988; 239: 487-491Crossref PubMed Scopus (13517) Google Scholar). For all polymerase chain reactions (PCR)Pfu DNA polymerase was used. For synaptobrevin 2, the coding sequence for the cytoplasmic domain (i.e. residues 1–96) was amplified by PCR using the primers 5′-CCCGGATCCATATGTCGGCTACCGCTGCCACCGTC-3′ and 5′-CGCGGGATCCCTCGAGTTACATCATCTTGAGGTTTTTCCA-3′ and subsequently subcloned into the pET-15b vector (Novagen) using the NdeI and XhoI restriction sites. This resulted in a fusion protein with an N-terminal His6 tag that is cleavable with thrombin. The cDNA encoding for rat synaptobrevin 2 (15Elferink L.A. Trimble W.S. Scheller R.H. J. Biol. Chem. 1989; 264: 11061-11064Abstract Full Text PDF PubMed Google Scholar) was kindly provided by R. H. Scheller (Stanford University). For the expression of SNAP-25 and syntaxin, expression vectors (referred to as pHO vectors) with versatile polylinker and a C-terminal His6 tag were constructed. First, a short linker consisting of the oligonucleotides 5′-AATTGGTCGAGCC-3′ and 5′-AGCTGGCTCGACC-3′ was inserted between the EcoRI and HindIII site of pET-11c and pET-11d (kindly provided by F. W. Studier and A. H. Rosenberg (16Studier F.W. Rosenberg A.H. Dunn J.J. Dubendorff J.W. Methods Enzymol. 1990; 185: 60-89Crossref PubMed Scopus (6006) Google Scholar)), resulting in the deletion of both cleavage sites. The vector pHO2d was constructed by inserting DNA sequence containing a new multiple cloning site followed by bases coding for a His6 tag and a stop codon. This insert was generated by annealing the partially complementary primers p1 (5′-CGCCATATGGCCATGGTACCCGGGTCGACAAGCTTGAATTCGCAC-3′) and p2 (5′-GGCGGATCCTATCAGTGATGGTGGTGATGGTGCGAATTCAAGCTTGT-3′) and filling the missing 3′ ends by Pfu DNA polymerase activity. The product was cut with NcoI and BamHI, gel-purified, and inserted into the corresponding sites of the modified pET-11d vector. In analogy, the vector pHO2c was constructed from the modified pET-11c vector using the primer p3 (5′-GGGATTCCATATGGTACCCGGGTCGACAAGCTTGAATTCGCAC-3′) instead of primer p1 for the construction of the insert. Rat SNAP-25B (1–206, entire coding sequence) was subcloned viaNcoI and EcoRI into the vector pHO2d. The SNAP-25B sequence was first amplified by PCR from the SNAP-25B (rat) cDNA (kindly provided by T. C. Südhof, University of Texas Southwestern Medical Center) (17Blasi J. Chapman E.R. Link E. Binz T. Yamasaki S. De Camilli P. Südhof T.C. Niemann H. Jahn R. Nature. 1993; 365: 160-163Crossref PubMed Scopus (1050) Google Scholar, 18Chapman E.R. An S. Barton N. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar) using the PCR primers 5′-CATGCCATGGCCGAAGACGCGGAT-3′ and 5′-CGAATTCCCCCCACTGCCCAGCATCTTTGTTGC-3′ and subcloned into theNcoI and EcoRI sites, resulting in the additional C-terminal sequence GNSHHHHHH in the expressed protein. The cDNA encoding for rat syntaxin 1A (19Bennett M.K. Garcia-Arraras J.E. Elferink L.A. Peterson K. Fleming A.M. Hazuka C.D. Scheller R.H. Cell. 1993; 74: 863-873Abstract Full Text PDF PubMed Scopus (591) Google Scholar) was kindly provided by R. H. Scheller. Rat syntaxin 1A-(1–265) (i.e. without the transmembrane region) was subcloned either into TrcHisA (Invitrogen) with an N-terminal His6 tag as described previously (18Chapman E.R. An S. Barton N. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar) or into the vector pHO2c with a C-terminal His6 tag. For subcloning into the vector pHO2c, the coding region corresponding to amino acid residues 1–265 was amplified using the primers 5′-GGGATTCCATATGAAGGACCGAACCCAG-3′ and 5′-GCGAATTCCCCTTCTTCCTGCGTGCCTT-3′. The resulting product was subcloned into the NdeI and EcoRI sites of the vector pHO2c resulting in the additional C-terminal sequence GNSHHHHHH. This syntaxin construct was used for nondenaturing gel electrophoresis (Figs. 1 and 2). However, the location of the His6 tag showed little effect on the properties of syntaxin.Figure 2Stoichiometry of SNAP-25 and syntaxin in the ternary complex with synaptobrevin, monitored by nondenaturing electrophoresis. Synaptobrevin, SNAP-25, and syntaxin (concentrations as indicated) were incubated overnight in standard buffer prior to separation by nondenaturing gel electrophoresis. Note that due to an isoelectric point of 8.5, monomeric synaptobrevin is not detectable in the nondenaturing gel. To achieve complete transition to the ternary complex in each case, an excess of synaptobrevin was used.A, constant amounts of SNAP-25 were incubated with increasing amounts of syntaxin; B, constant amounts of syntaxin were incubated with increasing amounts of SNAP-25.View Large Image Figure ViewerDownload Hi-res image Download (PPT) His6-tagged fusion proteins were first purified by Ni2+-Sepharose chromatography (18Chapman E.R. An S. Barton N. Jahn R. J. Biol. Chem. 1994; 269: 27427-27432Abstract Full Text PDF PubMed Google Scholar). Proteins were eluted by increasing the imidazole concentration stepwise to 40, 80, 120, or 240 mm (in 20 mm Tris, pH 7.4, 500 mm NaCl). Fractions were analyzed for purity by SDS-PAGE (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) and staining with Coomassie Blue. Fractions containing recombinant proteins were dialyzed against standard buffer (20 mm Tris, pH 7.4, 100 mmNaCl, 1 mm EDTA, 1 mm dithiothreitol). SNAP-25 and syntaxin were further purified by anion exchange chromatography on a Mono-Q column and synaptobrevin by cation exchange chromatography on a Mono-S column (Pharmacia Biotech Inc.). After loading, the proteins were eluted with a linear gradient of NaCl in standard buffer. The peak fractions were pooled and dialyzed against standard buffer. The His6 tag of synaptobrevin was cleaved with thrombin followed by another chromatographic step on a Mono-S column. The eluted protein was 95% pure as determined by gel electrophoresis and mass spectrometry. The syntaxin-SNAP-25 and ternary complexes were purified using a Mono-Q column (Pharmacia). The ternary complex was disassembled when incubated with NSF, α-SNAP, and ATP (not shown), demonstrating that the proteins are functional with respect to NSF-mediated disassembly (12Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar, 13Hanson P.I. Otto H. Barton N. Jahn R. J. Biol. Chem. 1995; 270: 16955-16961Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). After purification, the proteins and the protein complexes were dialyzed against standard buffer and concentrated by ultrafiltration to final concentrations of 1–10 mg/ml. Protein at were by amino acid acid by the W. M. at University). CD were by with a step of on an CD with a All were performed in with a of or For thermal the at were as a of with the in the were in the of mm or effect on the CD was observed. All CD were an overnight at in standard buffer. To changes of the CD to complex the were with the of the using the are the respective concentrations of protein are the respective of amino and are the of the individual proteins. In the of several CD the by the concentration of each in the of the and ternary the complexes have a effect on the SDS-PAGE was as described by (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar). for were in buffer mm Tris, pH and incubated at or for analyzed on a gel. Nondenaturing were and in an as (20Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (207538) Google Scholar) that was from all To for with the the were incubated overnight in standard buffer at the concentrations in the After buffer mm Tris, pH the were on a pH and a separation gel pH chromatography was performed on a column in standard buffer with the NaCl concentration to and with mm at a of The were monitored by at light scattering at and scattering and were using the and of was as described by P. 1993; 272: Scopus Google Scholar). For each 100 of protein was The protein concentrations were by amino acid M. at University). The of with respect to a in concentration of the is constant for proteins J. T. 1996; PubMed Scopus Google Scholar) and was to for the of the In we that of CD of the purified complex with that of a showed a in with CD and in Jahn R. J. Biol. Chem. 272: Full Text Full Text PDF PubMed Scopus Google Scholar). is that the complex has a we that of the of the SNAREs and their complexes. reactions at protein concentrations amino acid were and each was analyzed using nondenaturing gel electrophoresis. This for the separation of the individual proteins from provided that the latter are of stability. In the first of the formation of the syntaxin-SNAP-25 complex was amounts of SNAP-25 were incubated with increasing amounts of syntaxin and 1 Both showed that about 2 of syntaxin were required for complete of 1 of A complex of synaptobrevin with either SNAP-25 or syntaxin was not by nondenaturing gel electrophoresis (not shown), in with that these are the of syntaxin with SNAP-25 (11Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar, N. Bennett M.K. Peterson Scheller R.H. Science. 1994; PubMed Scopus Google Scholar, J. J.E. Calakos N. Bennett M.K. Scheller R.H. 1994; 13: Full Text PDF PubMed Scopus Google Scholar, Y. Scheller R.H. 1995; 14: Full Text PDF PubMed Scopus Google Scholar, N. Bennett M.K. J. Neurosci. PubMed Google Scholar). the described was in the of an excess of individual proteins and the ternary complex were observed. This suggests that any complex of syntaxin and SNAP-25 in the formation of a ternary complex 2). 2 that the between syntaxin and SNAP-25 changes from 2:1 in the complex to in the ternary complex. To the of synaptobrevin to syntaxin and SNAP-25 in the ternary amounts of syntaxin and SNAP-25 were incubated with increasing amounts of In the of synaptobrevin, all syntaxin was in the whereas a of SNAP-25 was in with a 2:1 stoichiometry of the complex. increasing amounts of synaptobrevin were the complex as as SNAP-25 and ternary complex was formed not The three proteins were in the ternary complex in amounts The described that in the syntaxin-SNAP-25 one of the syntaxin as a that the site for synaptobrevin and that is when synaptobrevin is To synaptobrevin was to the purified syntaxin-SNAP-25 complex. In to nondenaturing gel electrophoresis, the was analyzed by SDS-PAGE without the a to the ternary but of the complexes (11Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). in syntaxin is from complex with SNAP-25 when synaptobrevin is that the of syntaxin and SNAP-25 changes upon transition from the to the ternary complex. synaptobrevin, SNAP-25, and syntaxin and their complexes eluted at of mass when with mass (not To the we used MALLS, a that is of the of P. 1993; 272: Scopus Google Scholar). Synaptobrevin eluted as a monomeric with a mass of in with the mass determined by mass spectroscopy not of synaptobrevin at showed demonstrating that synaptobrevin does not at and A. T. Both SNAP-25 and syntaxin eluted as monomeric with of and in with the of 25 and The syntaxin-SNAP-25 complex eluted as a peak in the which is to the mass of the 2:1 complex The purified ternary complex eluted as a peak in the which is to the mass of the ternary complex The that both the and the ternary complex are with a stoichiometry that can be purified by chromatographic complex formation is when the are in the Therefore, of CD from the individual proteins with of or purified complexes structural changes associated with complex formation. The CD of syntaxin showed significant as by the at and In SNAP-25 a structure in with on the SNAP-25 from Jahn R. J. Biol. Chem. 272: Full Text Full Text PDF PubMed Scopus Google Scholar). synaptobrevin showed little structure to as by effect suggesting a or an with little To investigate structural changes associated with CD of each and of the ternary complex were by the proteins at the The CD of these were with the of the individual and For the the is that for a similar to on the Jahn R. J. Biol. Chem. 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In a in was when SNAP-25 and synaptobrevin were and was when synaptobrevin was with syntaxin in with the that complexes were by nondenaturing gel electrophoresis. ternary complex an in was for the syntaxin-SNAP-25 complex was between the of the and the purified complexes (not the CD has a more the the formation of the ternary complex is associated with a dramatic in is due to of structure in the unstructured proteins synaptobrevin The and of SNARE proteins suggests that assembly is an energetically that in complexes. a first step complex thermal have been by as a of The thermal of syntaxin a with a first transition point at and a more transition at In to syntaxin, significant changes in were for synaptobrevin and SNAP-25, in with the of structure the thermal of the purified syntaxin-SNAP-25 complex. The a more but in thermal stability with In formation of the ternary complex resulted in a dramatic in thermal stability The thermal was with a transition at were detectable at In addition, thermal was largely with about (not A thermal of the ternary complex was in This was by the that the ternary complex is to with without (11Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar). has been used for the stability of ternary complexes formed from proteins (11Hayashi T. McMahon H. Yamasaki S. Binz T. Hata Y. Südhof T.C. Niemann H. EMBO J. 1994; 13: 5051-5061Crossref PubMed Scopus (664) Google Scholar, Y. Scheller R.H. 1995; 14: Full Text PDF PubMed Scopus Google Scholar, N. Bennett M.K. J. Neurosci. PubMed Google Scholar). in as a of with that and stability of the ternary complex. Synaptobrevin, syntaxin, and SNAP-25 form a ternary complex with 1:1:1 stoichiometry and a to This complex can be purified by chromatographic and as a single on a nondenaturing gel. The of the complex is about as as that of the of similar structural changes were upon assembly of the the domain of and P. FEBS Lett. PubMed Scopus Google Scholar). We that the structural of the assembly reactions between the and SNARE proteins are by their sequence A of assembly be essential for elucidating the mechanism by which these proteins membrane fusion. The 2:1 of the syntaxin-SNAP-25 complex is However, a of on the Jahn R. J. Biol. Chem. 272: Full Text Full Text PDF PubMed Scopus Google Scholar) the suggesting that is to complexes. In the complex formed from the and a stoichiometry P. FEBS Lett. PubMed Scopus Google Scholar). This a structural between and SNAREs. In to syntaxin, both SNAP-25 and synaptobrevin have little However, both proteins are in the that can form the ternary complex with (Figs. 2 and we which of the three proteins is for the in is that both SNAP-25 and synaptobrevin a more and in the complex. syntaxin and the syntaxin-SNAP-25 the ternary complex is to In addition, the for the assembly to be on the side of complex (12Hayashi T. Yamasaki S. Nauenburg S. Binz T. Niemann H. EMBO J. 1995; 14: 2317-2325Crossref PubMed Scopus (227) Google Scholar, 13Hanson P.I. Otto H. Barton N. Jahn R. J. Biol. Chem. 1995; 270: 16955-16961Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). is that amounts of energy are upon assembly and that the complex a of can these be integrated into a coherent that assembly and disassembly of complex in the of membrane studies from have that a ternary complex from native proteins can assemble with all three proteins as in a single membrane H. P.I. Jahn R. S. A. PubMed Scopus Google Scholar). However, the of syntaxin and SNAP-25 is to the membrane and synaptobrevin is to the membrane of synaptic vesicles (2Südhof T.C. Nature. 1995; 375: 645-653Crossref PubMed Scopus (1770) Google Scholar). Therefore, is to that complex assembly in a of the proteins that the membrane anchors and the two membranes P.I. J.E. Jahn R. PubMed Scopus Google Scholar). This is further by from the suggesting significant between SNAREs to the transmembrane domain of P. FEBS Lett. PubMed Scopus Google Scholar). The proteins achieve their final energy by the membranes and the energy assembly be to energy barriers for membrane fusion. We Barton for with the construction of the pHO for the SNAP-25 Fleming for with the for with for with mass and and for