SMART MODE:

NORMAL GENOMIC

• Scott G, Zhao Q
• Rab3a and SNARE proteins: potential regulators of melanosome movement.
• J Invest Dermatol. 2001; 116: 296-304
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• Melanosomes are specialized organelles that undergo a dynamic process of transport along the melanocyte dendrite to the dendrite tip and transfer to keratinocytes. We hypothesized that soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE), which are involved in membrane fusion, and rab3a, a GTP-binding protein involved in exocytosis in neuronal cells and in SNARE complex assembly, may play a part in melanosome transport and transfer. By reverse transcription-polymerase chain reaction we identified transcripts for rab3a, vesicle-associated membrane protein-2, synaptosome-associated proteins of 23 kDa and 25 kDa, and syntaxin-4 in murine melanocytic cells. We also showed that purified melanosome preparations contain rab3a and SNARE, including vesicle-associated membrane protein-2, syntaxin-4, synaptosome-associated proteins 23 kDa and 25 kDa, and the SNARE accessory protein, alpha-soluble N-ethylmaleimide-sensitive factor attachment protein. Ultraviolet radiation is a potent stimulus for melanosome transport and transfer. We show that ultraviolet radiation rapidly suppresses melanosome-associated rab3a expression and that this occurs at the protein and mRNA level. Finally, we show that vesicle-associated membrane protein-2 and synaptosome-associated protein 23 kDa coimmunoprecipitate from purified melanocytic cell membranes, suggesting that they form complexes. The presence of rab3a and SNARE on melanosomes, and of SNARE complexes in melanocytic cell membranes suggests that these proteins play a part in targeting melanosomes to the plasma membrane, to melanosome transfer to keratinocytes, or both.

• Marash M, Gerst JE
• t-SNARE dephosphorylation promotes SNARE assembly and exocytosis in yeast.
• EMBO J. 2001; 20: 411-21
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• The role of protein phosphorylation in secretion is not well understood. Here we show that yeast lacking the Snc1,2 v-SNAREs, or bearing a temperature-sensitive mutation in the Sso2 t-SNARE, are rescued at restrictive conditions by the addition of ceramide precursors and analogs to the growth medium. Rescue results from dephosphorylation of the Sso t-SNAREs by a ceramide-activated type 2A protein phosphatase (Sit4) involved in cell cycle control. Sso t-SNARE dephosphorylation correlated with its assembly into complexes with the Sec9 t-SNARE, both in vitro and in vivo, and with an increase in protein trafficking and secretion in cells. SNARE complexes isolated under these conditions contained only Sso and Sec9, suggesting that a t-t-SNARE fusion complex is sufficient to confer exocytosis. Mutation of a single PKA site (Ser79 to Ala79) in Sso1 resulted in a decrease in phosphorylation and was sufficient to confer growth to snc cells at restrictive conditions. Thus, modulation of t-SNARE phosphorylation regulates SNARE complex assembly and membrane fusion in vivo.

• Carr CM
• The taming of the SNARE.
• Nat Struct Biol. 2001; 8: 186-8

• Xiao W, Poirier MA, Bennett MK, Shin YK
• The neuronal t-SNARE complex is a parallel four-helix bundle.
• Nat Struct Biol. 2001; 8: 308-11
• Display abstract

• Assembly of the soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) complex is an essential step for neurotransmitter release in synapses. The presynaptic plasma membrane associated proteins (t-SNAREs), SNAP-25 (synaptosome-associated protein of 25,000 Da) and syntaxin 1A may form an intermediate complex that later binds to vesicle-associated membrane protein 2 (VAMP2). Using spin labeling electron paramagnetic resonance (EPR), we found that the two t-SNARE proteins assemble into a parallel four-helix bundle that consists of two identical syntaxin 1A components and the N-terminal and C-terminal domains of SNAP-25. Although the structure is generally similar to that of the final SNARE complex, the middle region of the helical bundle appears more flexible in the t-SNARE complex. Such flexibility might facilitate interactions between VAMP2 and the t-SNARE complex.

• Wagner ML, Tamm LK
• Reconstituted syntaxin1a/SNAP25 interacts with negatively charged lipids as measured by lateral diffusion in planar supported bilayers.
• Biophys J. 2001; 81: 266-75
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• According to the soluble N-ethylmaleimide-sensitive factor (NSF)-attachment protein (SNAP) receptor hypothesis (SNARE hypothesis), interactions between target SNAREs and vesicle SNAREs (t- and v-SNAREs) are required for membrane fusion in intracellular vesicle transport and exocytosis. The precise role of the SNAREs in tethering, docking, and fusion is still disputed. Biophysical measurements of SNARE interactions in planar supported membranes could potentially resolve some of the key questions regarding the mechanism of SNARE-mediated membrane fusion. As a first step toward this goal, recombinant syntaxin1A/SNAP25 (t-SNARE) was reconstituted into polymer-supported planar lipid bilayers. Reconstituted t-SNAREs in supported bilayers bound soluble green fluorescent protein/vesicle-associated membrane protein (v-SNARE), and the SNARE complexes could be specifically dissociated by NSF/alpha-SNAP in the presence of ATP. The physiological activities of SNARE complex formation were thus well reproduced in this reconstituted planar model membrane system. A large fraction (~75%) of the reconstituted t-SNARE was laterally mobile with a lateral diffusion coefficient of 7.5 x 10(-9) cm(2)/s in a phosphatidylcholine lipid background. Negatively charged lipids reduced the mobile fraction of the t-SNARE and the lipids themselves. Phosphatidylinositol-4,5-bisphosphate was more effective than phosphatidylserine in reducing the lateral mobility of the complexes. A model of how acidic lipid-SNARE interactions might alter lipid fluidity is discussed.

• Tokumaru H et al.
• SNARE complex oligomerization by synaphin/complexin is essential for synaptic vesicle exocytosis.
• Cell. 2001; 104: 421-32
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• Synaphin/complexin is a cytosolic protein that preferentially binds to syntaxin within the SNARE complex. We find that synaphin promotes SNAREs to form precomplexes that oligomerize into higher order structures. A peptide from the central, syntaxin binding domain of synaphin competitively inhibits these two proteins from interacting and prevents SNARE complexes from oligomerizing. Injection of this peptide into squid giant presynaptic terminals inhibited neurotransmitter release at a late prefusion step of synaptic vesicle exocytosis. We propose that oligomerization of SNARE complexes into a higher order structure creates a SNARE scaffold for efficient, regulated fusion of synaptic vesicles.

• Bryant NJ, James DE
• Vps45p stabilizes the syntaxin homologue Tlg2p and positively regulates SNARE complex formation.
• EMBO J. 2001; 20: 3380-8
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• Sec1p-like/Munc-18 (SM) proteins bind to t-SNAREs and inhibit ternary complex formation. Paradoxically, the absence of SM proteins does not result in constitutive membrane fusion. Here, we show that in yeast cells lacking the SM protein Vps45p, the t-SNARE Tlg2p is down-regulated, to undetectable levels, by rapid proteasomal degradation. In the absence of Vps45p, Tlg2p can be stabilized through abolition of proteasome activity. Surprisingly, the stabilized Tlg2p was targeted to the correct intracellular location. However, the stabilized Tlg2p is non-functional and unable to bind its cognate SNARE binding partners, Tlg1p and Vti1p, in the absence of Vps45p. A truncation mutant lacking the first 230 residues of Tlg2p no longer bound Vps45p but was able to form complexes with Tlg1p and Vti1p in the absence of the SM protein. These data provide us with two valuable insights into the function of SM proteins. First, SM proteins act as chaperone-like molecules for their cognate t-SNAREs. Secondly, SM proteins play an essential role in the activation process allowing their cognate t-SNARE to participate in ternary complex formation.

• Misura KM, Scheller RH, Weis WI
• Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly.
• J Biol Chem. 2001; 276: 13273-82
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• Intracellular membrane fusion requires SNARE proteins found on the vesicle and target membranes. SNAREs associate by formation of a parallel four-helix bundle, and it has been suggested that formation of this complex promotes membrane fusion. The membrane proximal region of the cytoplasmic domain of the SNARE syntaxin 1A, designated H3, contributes one of the four helices to the SNARE complex. In the crystal structure of syntaxin 1A H3, four molecules associate as a homotetramer composed of two pairs of parallel helices that are anti-parallel to each other. The H3 oligomer observed in the crystals is also found in solution, as assessed by gel filtration and chemical cross-linking studies. The crystal structure reveals that the highly conserved Phe-216 packs against conserved Gln-226 residues present on the anti-parallel pair of helices. Modeling indicates that Phe-216 prevents parallel tetramer formation. Mutation of Phe-216 to Leu appears to allow formation of parallel tetramers, whereas mutation to Ala destabilizes the protein. These results indicate that Phe-216 has a role in preventing formation of stable parallel helical bundles, thus favoring the interaction of the H3 region of syntaxin 1a with other proteins involved in membrane fusion.

• Hua Y, Scheller RH
• Three SNARE complexes cooperate to mediate membrane fusion.
• Proc Natl Acad Sci U S A. 2001; 98: 8065-70
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• Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins of the syntaxin, SNAP-25, and VAMP families mediate intracellular membrane fusion through the formation of helical bundles that span opposing membranes. Soluble SNARE domains that lack their integral membrane anchors inhibit membrane fusion by forming nonfunctional complexes with endogenous SNARE proteins. In this study we investigate the dependence of membrane fusion on the concentration of a soluble SNARE coil domain derived from VAMP2. The increase in the inhibition of fusion observed with increasing concentration of inhibitor is best fit to a function that suggests three SNARE complexes cooperate to mediate fusion of a single vesicle. These three complexes likely contribute part of a protein and lipidic fusion pore.

• Wendler F, Page L, Urbe S, Tooze SA
• Homotypic fusion of immature secretory granules during maturation requires syntaxin 6.
• Mol Biol Cell. 2001; 12: 1699-709
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• Homotypic fusion of immature secretory granules (ISGs) gives rise to mature secretory granules (MSGs), the storage compartment in endocrine and neuroendocrine cells for hormones and neuropeptides. With the use of a cell-free fusion assay, we investigated which soluble N-ethylmaleimide-sensitive fusion protein attachment receptor (SNARE) molecules are involved in the homotypic fusion of ISGs. Interestingly, the SNARE molecules mediating the exocytosis of MSGs in neuroendocrine cells, syntaxin 1, SNAP-25, and VAMP2, were not involved in homotypic ISG fusion. Instead, we have identified syntaxin 6 as a component of the core machinery responsible for homotypic ISG fusion. Subcellular fractionation studies and indirect immunofluorescence microscopy show that syntaxin 6 is sorted away during the maturation of ISGs to MSGs. Although, syntaxin 6 on ISG membranes is associated with SNAP-25 and SNAP-29/GS32, we could not find evidence that these target (t)-SNARE molecules are involved in homotypic ISG fusion. Nor could we find any involvement for the vesicle (v)-SNARE VAMP4, which is known to be associated with syntaxin 6. Importantly, we have shown that homotypic fusion requires the function of syntaxin 6 on both donor as well as acceptor membranes, which suggests that t-t-SNARE interactions, either direct or indirect, may be required during fusion of ISG membranes.

• Washbourne P, Cansino V, Mathews JR, Graham M, Burgoyne RD, Wilson MC
• Cysteine residues of SNAP-25 are required for SNARE disassembly and exocytosis, but not for membrane targeting.
• Biochem J. 2001; 357: 625-34
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• The release of neurotransmitter at a synapse occurs via the regulated fusion of synaptic vesicles with the plasma membrane. The fusion of the two lipid bilayers is mediated by a protein complex that includes the plasma membrane target soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (t-SNAREs), syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25), and the vesicle SNARE (v-SNARE), vesicle-associated membrane protein (VAMP). Whereas syntaxin 1A and VAMP are tethered to the membrane by a C-terminal transmembrane domain, SNAP-25 has been suggested to be anchored to the membrane via four palmitoylated cysteine residues. We demonstrate that the cysteine residues of SNAP-25 are not required for membrane localization when syntaxin 1A is present. Analysis of the 7 S and 20 S complexes formed by mutants that lack cysteine residues demonstrates that the cysteines are required for efficient SNARE complex dissociation. Furthermore, these mutants are unable to support exocytosis, as demonstrated by a PC12 cell secretion assay. We hypothesize that syntaxin 1A serves to direct newly synthesized SNAP-25 through the Golgi transport pathway to the axons and synapses, and that palmitoylation of cysteine residues is not required for targeting, but to optimize interactions required for SNARE complex dissociation.

• Margittai M, Fasshauer D, Pabst S, Jahn R, Langen R
• Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling.
• J Biol Chem. 2001; 276: 13169-77
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• SNARE (soluble NSF acceptor protein receptor) proteins are thought to mediate membrane fusion by assembling into heterooligomeric complexes that connect the fusing membranes and initiate the fusion reaction. Here we used site-directed spin labeling to map conformational changes that occur upon homo- and heterooligomeric complex formation of neuronal SNARE proteins. We found that the soluble domains of synaptobrevin, SNAP-25, and syntaxin 1 are unstructured. At higher concentrations, the SNARE motif of syntaxin 1 forms homooligomeric helical bundles with at least some of the alpha-helices aligned in parallel. In the assembled SNARE complex, mapping of thirty side chain positions yielded spectra which are in good agreement with the recently published crystal structure. The loop region of SNAP-25 that connects the two SNARE motifs is largely unstructured. C-terminal truncation of synaptobrevin resulted in complexes that are completely folded N-terminal of the truncation but become unstructured at the C-terminal end. The binary complex of syntaxin and SNAP-25 consists of a parallel four helix-bundle with properties resembling that of the ternary complex.

• Charest A, Lane K, McMahon K, Housman DE
• Association of a novel PDZ domain-containing peripheral Golgi protein with the Q-SNARE (Q-soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor) protein syntaxin 6.
• J Biol Chem. 2001; 276: 29456-65
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• PDZ domains are involved in the scaffolding and assembly of multi-protein complexes at various subcellular sites. We describe here the isolation and characterization of a novel PDZ domain-containing protein that localizes to the Golgi apparatus. Using an in silico cloning approach, we have identified and isolated a cDNA encoding a ubiquitously expressed 59-kDa protein that we call FIG. It is composed of two coiled coil regions, a leucine zipper, and a single PDZ domain. Cytological studies using indirect immunofluorescence microscopy revealed that FIG is a peripheral protein that uses one of its coiled coil domains to localize to the Golgi apparatus. To ascertain the modalities of this Golgi localization, the same coiled coil region was tested for its ability to interact with a panel of coiled coil domain-containing integral membrane Golgi proteins. Using a series of GST fusion protein binding assays, co-immunofluorescence and co-immunoprecipitation experiments, we show that FIG specifically binds to the coiled coil domain-containing Q-SNARE (Q-soluble NSF attachment protein receptor) protein syntaxin 6 both in vitro and in vivo. The structural features of FIG and its interaction with a SNARE protein suggest that FIG may play a role in membrane vesicle trafficking. This is the first example of a PDZ domain-containing peripheral protein that localizes to the Golgi through a coiled coil-mediated interaction with a resident membrane protein. Our results broaden the scope of PDZ domain-mediated functions.

• Littleton JT et al.
• synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo.
• J Neurosci. 2001; 21: 1421-33
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• Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

• Wendler F, Tooze S
• Syntaxin 6: the promiscuous behaviour of a snare protein.
• Traffic. 2001; 2: 606-11
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• Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.

• Wimmer C et al.
• Molecular mass, stoichiometry, and assembly of 20 S particles.
• J Biol Chem. 2001; 276: 29091-7
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• N-Ethylmaleimide-sensitive factor (NSF), soluble NSF attachment proteins (SNAPs), and SNAP receptor (neuronal SNARE) complexes form 20 S particles with a mass of 788 +/- 122 kDa as judged by scanning transmission electron microscopy. A single NSF hexamer and three alpha SNAP monomers reside within a 20 S particle as determined by quantitative amino acid analysis. In order to study the binding of alpha SNAP and NSF in solution, to define their binding domains, and to specify the role of oligomerization in their interaction, we fused domains of alpha SNAP and NSF to oligomerization modules derived from thrombospondin-1, a trimer, and cartilage oligomeric matrix protein, a pentamer, respectively. Binding studies with these fusion proteins reproduced the interaction of alpha SNAP and NSF N domains in the absence of the hexamerization domain of NSF (D2). Trimeric alpha SNAP (or its C-terminal half) is sufficient to recruit NSF even in the absence of SNARE complexes. Furthermore, pentameric NSF N domains are able to bind alpha SNAP in complex with SNAREs, whereas monomeric N domains do not. Our results demonstrate that the oligomerization of both NSF N domains and alpha SNAP provides a critical driving force for their interaction and the assembly of 20 S particles.

• Wang Y, Dulubova I, Rizo J, Sudhof TC
• Functional analysis of conserved structural elements in yeast syntaxin Vam3p.
• J Biol Chem. 2001; 276: 28598-605
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• Vam3p, a syntaxin-like SNARE protein involved in yeast vacuole fusion, is composed of a three-helical N-terminal domain, a canonical SNARE motif, and a C-terminal transmembrane region (TMR). Surprisingly, we find that the N-terminal domain of Vam3p is not essential for fusion, although analogous domains in other syntaxins are indispensible for fusion and/or protein-protein interactions. In contrast to the N-terminal domain, mutations in the SNARE motif of Vam3p or replacement of the SNARE motif of Vam3p with the SNARE motif from other syntaxins inhibited fusion. Furthermore, the precise distance between the SNARE motif and the TMR was critical for fusion. Insertion of only three residues after the SNARE motif significantly impaired fusion and insertion of 12 residues abolished fusion. As judged by co-immunoprecipitation experiments, the SNARE motif mutations and the insertions did not alter the association of Vam3p with Vam7p, Vti1p, Nyv1p, and Ykt6p, other vacuolar SNARE proteins implicated in fusion. In contrast, the SNARE motif substitutions interfered with the stable formation of Vam3p complexes with Nyv1p and Vti1p, although Vam3p complexes with Vam7p and Ykt6p were still present. Our data suggest that in contrast to previously characterized syntaxins, Vam3p contains only two domains essential for fusion, the SNARE motif and the TMR, and these domains have to be closely coupled to function in fusion.

• Dulubova I, Yamaguchi T, Wang Y, Sudhof TC, Rizo J
• Vam3p structure reveals conserved and divergent properties of syntaxins.
• Nat Struct Biol. 2001; 8: 258-64
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• Syntaxins and Sec1/munc18 proteins are central to intracellular membrane fusion. All syntaxins comprise a variable N-terminal region, a conserved SNARE motif that is critical for SNARE complex formation, and a transmembrane region. The N-terminal region of neuronal syntaxin 1A contains a three-helix domain that folds back onto the SNARE motif forming a 'closed' conformation; this conformation is required for munc18-1 binding. We have examined the generality of the structural properties of syntaxins by NMR analysis of Vam3p, a yeast syntaxin essential for vacuolar fusion. Surprisingly, Vam3p also has an N-terminal three-helical domain despite lacking apparent sequence homology with syntaxin 1A in this region. However, Vam3p does not form a closed conformation and its N-terminal domain is not required for binding to the Sec1/munc18 protein Vps33p, suggesting that critical distinctions exist in the mechanisms used by syntaxins to govern different types of membrane fusion.

• Bracher A, Weissenhorn W
• Crystal structures of neuronal squid Sec1 implicate inter-domain hinge movement in the release of t-SNAREs.
• J Mol Biol. 2001; 306: 7-13
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• Sec1 molecules associate with t-SNAREs from the syntaxin family in a heterodimeric complex that plays an essential role in vesicle transport and membrane fusion. Neuronal rat n-Sec1 has an arch-shaped three-domain structure, which binds syntaxin 1a through contacts in domains 1 and 3. In both rat nSec1 and homologous squid s-Sec1, a potential effector-molecule binding-pocket is shaped by residues from domains 1 and 2 and is localized on the opposite side of the syntaxin 1a interaction site. Comparison of several crystal forms of unliganded neuronal squid Sec1 indicates a hinge region between domains 1 and 2 which allows domain 1 to rotate along a central axis. This movement could release syntaxin 1a upon interaction with a yet unspecified Sec1 effector molecule(s). The binding of an effector protein may also directly affect the conformation of the helical hairpin of domain 3, which contributes the other significant syntaxin 1a binding sites in the rat nSec1/syntaxin 1a complex structure but adopts multiple conformations in the unliganded s-Sec1 structures reported here. Copyright 2001 Academic Press.

• Misura KM, May AP, Weis WI
• Protein-protein interactions in intracellular membrane fusion.
• Curr Opin Struct Biol. 2000; 10: 662-71
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• The fusion of intracellular vesicles with their target membranes is an essential feature of the compartmental structure of eukaryotic cells. This process requires proteins that dictate the targeting of a vesicle to the correct cellular location, mediate bilayer fusion and, in some systems, regulate the precise time at which fusion occurs. Recent biophysical and structural studies of these proteins have begun to provide a foundation for understanding their functions at a molecular level.

• Lao G et al.
• Syntaphilin: a syntaxin-1 clamp that controls SNARE assembly.
• Neuron. 2000; 25: 191-201
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• Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that forms the SNARE complex with VAMP/synaptobrevin and SNAP-25. Identifying proteins that modulate SNARE complex formation is critical for understanding the molecular mechanisms underlying neurotransmitter release and its modulation. We have cloned and characterized a protein called syntaphilin that is selectively expressed in brain. Syntaphilin competes with SNAP-25 for binding to syntaxin-1 and inhibits SNARE complex formation by absorbing free syntaxin-1. Transient overexpression of syntaphilin in cultured hippocampal neurons significantly reduces neurotransmitter release. Furthermore, introduction of syntaphilin into presynaptic superior cervical ganglion neurons in culture inhibits synaptic transmission. These findings suggest that syntaphilin may function as a molecular clamp that controls free syntaxin-1 availability for the assembly of the SNARE complex, and thereby regulates synaptic vesicle exocytosis.

• Quetglas S, Leveque C, Miquelis R, Sato K, Seagar M
• Ca2+-dependent regulation of synaptic SNARE complex assembly via a calmodulin- and phospholipid-binding domain of synaptobrevin.
• Proc Natl Acad Sci U S A. 2000; 97: 9695-700
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• Synaptic core complex formation is an essential step in exocytosis, and assembly into a superhelical structure may drive synaptic vesicle fusion. To ascertain how Ca(2+) could regulate this process, we examined calmodulin binding to recombinant core complex components. Surface plasmon resonance and pull-down assays revealed Ca(2+)-dependent calmodulin binding (K(d) = 500 nM) to glutathione S-transferase fusion proteins containing synaptobrevin (VAMP 2) domains but not to syntaxin 1 or synaptosomal-associated protein of 25 kDa (SNAP-25). Deletion mutations, tetanus toxin cleavage, and peptide synthesis localized the calmodulin-binding domain to VAMP(77-94), immediately C-terminal to the tetanus toxin cleavage site (Q(76)-F(77)). In isolated synaptic vesicles, Ca(2+)/calmodulin protected native membrane-inserted VAMP from proteolysis by tetanus toxin. Assembly of a (35)S-SNAP-25, syntaxin 1 GST-VAMP(1-96) complex was inhibited by Ca(2+)/calmodulin, but assembly did not mask subsequent accessibility of the calmodulin-binding domain. The same domain contains a predicted phospholipid interaction site. SPR revealed calcium-independent interactions between VAMP(77-94) and liposomes containing phosphatidylserine, which blocked calmodulin binding. Circular dichroism spectroscopy demonstrated that the calmodulin/phospholipid-binding peptide displayed a significant increase in alphahelical content in a hydrophobic environment. These data provide insight into the mechanisms by which Ca(2+) may regulate synaptic core complex assembly and protein interactions with membrane bilayers during exocytosis.

• Ishizuka T, Abe T
• [Synaphins/complexins, cytosolic proteins associated for neurotransmitter release]
• Tanpakushitsu Kakusan Koso. 2000; 45: 449-55

• Paz Y, Elazar Z, Fass D
• Structure of GATE-16, membrane transport modulator and mammalian ortholog of autophagocytosis factor Aut7p.
• J Biol Chem. 2000; 275: 25445-50
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• The GATE-16 protein participates in intra-Golgi transport and can associate with the N-ethylmaleimide-sensitive fusion protein and with Golgi SNAREs. The yeast ortholog of GATE-16 is the autophagocytosis factor Aut7p. GATE-16 is also closely related to the GABA receptor-associated protein (GABARAP), which has been proposed to cluster neurotransmitter receptors by mediating interaction with the cytoskeleton, and to the light chain-3 subunit of the neuronal microtubule-associated protein complex. Here, we present the crystal structure of GATE-16 refined to 1.8 A resolution. GATE-16 contains a ubiquitin fold decorated by two additional N-terminal helices. Proteins with strong structural similarity but no detectable sequence homology to GATE-16 include Ras effectors that mediate diverse downstream functions, but each interacts with Ras by forming pseudo-continuous beta-sheets. The GATE-16 surface suggests that it binds its targets in a similar manner. Moreover, a second potential protein-protein interaction site on GATE-16 may explain the adapter activity observed for members of the GATE-16 family.

• Laage R, Rohde J, Brosig B, Langosch D
• A conserved membrane-spanning amino acid motif drives homomeric and supports heteromeric assembly of presynaptic SNARE proteins.
• J Biol Chem. 2000; 275: 17481-7
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• Assembly of the SNARE proteins synaptobrevin/VAMP, syntaxin, and SNAP-25 to binary and ternary complexes is important for docking and/or fusion of presynaptic vesicles to the neuronal plasma membrane prior to regulated neurotransmitter release. Despite the well characterized structure of their cytoplasmic assembly domains, little is known about the role of the transmembrane segments in SNARE protein assembly and function. Here, we identified conserved amino acid motifs within the transmembrane segments that are required for homodimerization of synaptobrevin II and syntaxin 1A. Minimal motifs of 6-8 residues grafted onto an otherwise monomeric oligoalanine host sequence were sufficient for self-interaction of both transmembrane segments in detergent solution or membranes. These motifs constitute contiguous areas of interfacial residues assuming alpha-helical secondary structures. Since the motifs are conserved, they also contributed to heterodimerization of synaptobrevin II and syntaxin 1A and therefore appear to constitute interaction domains independent of the cytoplasmic coiled coil regions. Interactions between the transmembrane segments may stabilize the SNARE complex, cause its multimerization to previously observed multimeric superstructures, and/or be required for the fusogenic activity of SNARE proteins.

• Hanson PI
• Sec1 gets a grip on syntaxin.
• Nat Struct Biol. 2000; 7: 347-9

• Neiman AM, Katz L, Brennwald PJ
• Identification of domains required for developmentally regulated SNARE function in Saccharomyces cerevisiae.
• Genetics. 2000; 155: 1643-55
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• Saccharomyces cerevisiae cells contain two homologues of the mammalian t-SNARE protein SNAP-25, encoded by the SEC9 and SPO20 genes. Although both gene products participate in post-Golgi vesicle fusion events, they cannot substitute for one another; Sec9p is active primarily in vegetative cells while Spo20p functions only during sporulation. We have investigated the basis for the developmental stage-specific differences in the function of these two proteins. Localization of the other plasma membrane SNARE subunits, Ssop and Sncp, in sporulating cells suggests that these proteins act in conjunction with Spo20p in the formation of the prospore membrane. In vitro binding studies demonstrate that, like Sec9p, Spo20p binds specifically to the t-SNARE Sso1p and, once bound to Sso1p, can complex with the v-SNARE Snc2p. Therefore, Sec9p and Spo20p interact with the same binding partners, but developmental conditions appear to favor the assembly of complexes with Spo20p in sporulating cells. Analysis of chimeric Sec9p/Spo20p molecules indicates that regions in both the SNAP-25 domain and the unique N terminus of Spo20p are required for activity during sporulation. Additionally, the N terminus of Spo20p is inhibitory in vegetative cells. Deletion studies indicate that activation and inhibition are separable functions of the Spo20p N terminus. Our results reveal an additional layer of regulation of the SNARE complex, which is necessary only in sporulating cells.

• Antonin W, Holroyd C, Fasshauer D, Pabst S, Von Mollard GF, Jahn R
• A SNARE complex mediating fusion of late endosomes defines conserved properties of SNARE structure and function.
• EMBO J. 2000; 19: 6453-64
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• Sets of SNARE proteins mediate membrane fusion by assembling into core complexes. Multiple SNAREs are thought to function in different intracellular trafficking steps but it is often unclear which of the SNAREs cooperate in individual fusion reactions. We report that syntaxin 7, syntaxin 8, vti1b and endobrevin/VAMP-8 form a complex that functions in the fusion of late endosomes. Antibodies specific for each protein coprecipitate the complex, inhibit homotypic fusion of late endosomes in vitro and retard delivery of endocytosed epidermal growth factor to lysosomes. The purified proteins form core complexes with biochemical and biophysical properties remarkably similar to the neuronal core complex, although each of the four proteins carries a transmembrane domain and three have independently folded N-terminal domains. Substitution experiments, sequence and structural comparisons revealed that each protein occupies a unique position in the complex, with syntaxin 7 corresponding to syntaxin 1, and vti1b and syntaxin 8 corresponding to the N- and C-terminal domains of SNAP-25, respectively. We conclude that the structure of core complexes and their molecular mechanism in membrane fusion is highly conserved between distant SNAREs.

• Yang B, Steegmaier M, Gonzalez LC Jr, Scheller RH
• nSec1 binds a closed conformation of syntaxin1A.
• J Cell Biol. 2000; 148: 247-52
• Display abstract

• The Sec1 family of proteins is proposed to function in vesicle trafficking by forming complexes with target membrane SNAREs (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein [SNAP] receptors) of the syntaxin family. Here, we demonstrate, by using in vitro binding assays, nondenaturing gel electrophoresis, and specific neurotoxin treatment, that the interaction of syntaxin1A with the core SNARE components, SNAP-25 (synaptosome-associated protein of 25 kD) and VAMP2 (vesicle-associated membrane protein 2), precludes the interaction with nSec1 (also called Munc18 and rbSec1). Inversely, association of nSec1 and syntaxin1A prevents assembly of the ternary SNARE complex. Furthermore, using chemical cross-linking of rat brain membranes, we identified nSec1 complexes containing syntaxin1A, but not SNAP-25 or VAMP2. These results support the hypothesis that Sec1 proteins function as syntaxin chaperons during vesicle docking, priming, and membrane fusion.

• Fukuda R et al.
• Functional architecture of an intracellular membrane t-SNARE.
• Nature. 2000; 407: 198-202
• Display abstract

• Lipid bilayer fusion is mediated by SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) located on the vesicle membrane (v-SNAREs) and the target membrane (t-SNAREs). The assembled v-SNARE/t-SNARE complex consists of a bundle of four helices, of which one is supplied by the v-SNARE and the other three by the t-SNARE. For t-SNAREs on the plasma membrane, the protein syntaxin supplies one helix and a SNAP-25 protein contributes the other two. Although there are numerous homologues of syntaxin on intracellular membranes, there are only two SNAP-25-related proteins in yeast, Sec9 and Spo20, both of which are localized to the plasma membrane and function in secretion and sporulation, respectively. What replaces SNAP-25 in t-SNAREs of intracellular membranes? Here we show that an intracellular t-SNARE is built from a 'heavy chain' homologous to syntaxin and two separate non-syntaxin 'light chains'. SNAP-25 may thus be the exception rather than the rule, having been derived from genes that encoded separate light chains that fused during evolution to produce a single gene encoding one protein with two helices.

• Scales SJ, Chen YA, Yoo BY, Patel SM, Doung YC, Scheller RH
• SNAREs contribute to the specificity of membrane fusion.
• Neuron. 2000; 26: 457-64
• Display abstract

• Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.

• Foletti DL, Lin R, Finley MA, Scheller RH
• Phosphorylated syntaxin 1 is localized to discrete domains along a subset of axons.
• J Neurosci. 2000; 20: 4535-44
• Display abstract

• Syntaxin 1 is a SNARE protein that plays a central role in synaptic vesicle (SV) exocytosis. We generated an antibody that specifically recognizes a casein kinase II-mediated phosphorylation on serine-14 of syntaxin 1. In this report we show that this phosphorylation occurs in vivo and is developmentally regulated in the rat brain, rising to a level of 40% of the total syntaxin in adult animals. Phosphorylated syntaxin is preferentially associated with SNAP-25 and localizes to discrete domains of the axonal plasma membrane that do not colocalize with pools of synaptic vesicles. These phosphosyntaxin domains may define fusion sites for a novel class of vesicles outside classical active zones.

• Bracher A, Perrakis A, Dresbach T, Betz H, Weissenhorn W
• The X-ray crystal structure of neuronal Sec1 from squid sheds new light on the role of this protein in exocytosis.
• Structure Fold Des. 2000; 8: 685-94
• Display abstract

• BACKGROUND: Sec1-like molecules have been implicated in a variety of eukaryotic vesicle transport processes including neurotransmitter release by exocytosis. They regulate vesicle transport by binding to a t-SNARE from the syntaxin family. This process is thought to prevent SNARE complex formation, a protein complex required for membrane fusion. Whereas Sec1 molecules are essential for neurotransmitter release and other secretory events, their interaction with syntaxin molecules seems to represent a negative regulatory step in secretion. RESULTS: Here we report the X-ray crystal structure of a neuronal Sec1 homologue from squid, s-Sec1, at 2.4 A resolution. Neuronal s-Sec1 is a modular protein that folds into a V-shaped three-domain assembly. Peptide and mutagenesis studies are discussed with respect to the mechanism of Sec1 regulation. Comparison of the structure of squid s-Sec1 with the previously determined structure of rat neuronal Sec1 (n-Sec1) bound to syntaxin-1a indicates conformational rearrangements in domain III induced by syntaxin binding. CONCLUSIONS: The crystal structure of s-Sec1 provides the molecular scaffold for a number of molecular interactions that have been reported to affect Sec1 function. The structural differences observed between s-Sec1 and the structure of a rat n-Sec1-syntaxin-1a complex suggest that local conformational changes are sufficient to release syntaxin-1a from neuronal Sec1, an active process that is thought to involve additional effector molecule(s).

• Woodbury DJ, Rognlien K
• The t-SNARE syntaxin is sufficient for spontaneous fusion of synaptic vesicles to planar membranes.
• Cell Biol Int. 2000; 24: 809-18
• Display abstract

• Vesicular trafficking and exocytosis are directed by the complementary interaction of membrane proteins that together form the SNARE complex. This complex is composed of proteins in the vesicle membrane (v-SNAREs) that intertwine with proteins of the target membrane (t-SNAREs). Here we show that modified synaptic vesicles (mSV), containing v-SNAREs, spontaneously fuse to planar membranes containing the t-SNARE, syntaxin 1A. Fusion was Ca(2+)-independent and did not occur with vesicles lacking v-SNAREs. Therefore, syntaxin alone forms a functional fusion complex with v-SNAREs. Our functional fusion assay uses synaptic vesicles that are modified, so each fusion event results in an observable transient current. The mSV do not fuse with protein-free membranes. Additionally, artificial vesicles lacking v-SNAREs do not fuse with membranes containing syntaxin. This technique can be adapted to measure fusion in other SNARE systems and should enable the identification of proteins critical to vesicle-membrane fusion. This will further our understanding of exocytosis and may improve targeting and delivery of therapeutic agents packaged in vesicles.

• Tsujimoto S, Bean AJ
• Distinct protein domains are responsible for the interaction of Hrs-2 with SNAP-25. The role of Hrs-2 in 7 S complex formation.
• J Biol Chem. 2000; 275: 2938-42
• Display abstract

• Regulated secretion of neurotransmitter at the synapse is likely to be mediated by dynamic protein interactions involving components of the vesicle (vesicle-associated membrane protein; VAMP) and plasma membrane (syntaxin and synaptosomal associated protein of 25 kDa (SNAP-25)) along with additional molecules that allow for the regulation of this process. Recombinant Hrs-2 interacts with SNAP-25 in a calcium-dependent manner (they dissociate at elevated calcium levels) and inhibits neurotransmitter release. Thus, Hrs-2 has been hypothesized to serve a negative regulatory role in secretion through its interaction with SNAP-25. In this report, we show that Hrs-2 and SNAP-25 interact directly through specific coiled-coil domains in each protein. The presence of syntaxin enhances the binding of Hrs-2 to SNAP-25. Moreover, while both Hrs-2 and VAMP can separately bind to SNAP-25, they cannot bind simultaneously. Additionally, the presence of Hrs-2 reduces the incorporation of VAMP into the syntaxin.SNAP-25.VAMP (7 S) complex. These findings suggest that Hrs-2 may modulate exocytosis by regulating the assembly of a protein complex implicated in membrane fusion.

• Ybe JA, Wakeham DE, Brodsky FM, Hwang PK
• Molecular structures of proteins involved in vesicle fusion.
• Traffic. 2000; 1: 474-9
• Display abstract

• We present a summary of the structures of 13 proteins involved in the docking and fusion of intracellular transport vesicles to their target membranes.

• Wright MD, Ni J, Rudy GB
• The L6 membrane proteins--a new four-transmembrane superfamily.
• Protein Sci. 2000; 9: 1594-600
• Display abstract

• L6, IL-TMP, and TM4SF5 are cell surface proteins predicted to have four transmembrane domains. Previous sequence analysis led to their assignment as members of the tetraspanin superfamily. In this paper, we identify a new sequence (L6D) that is strikingly similar to L6, IL-TMP, and TM4SF5. Analyses of these four sequences indicate that they are not significantly related to genuine tetraspanins, but instead constitute their own L6 superfamily.

• Pabst S et al.
• Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions.
• J Biol Chem. 2000; 275: 19808-18
• Display abstract

• Complexins are evolutionarily conserved proteins that specifically bind to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes and thus may regulate SNARE function. Using purified proteins, we have performed a detailed analysis of the structure of complexin and of its interaction with SNARE proteins. NMR spectroscopy revealed that isolated complexins have no tertiary structure but contain an unusual alpha-helical middle domain of approximately 58 amino acids that overlaps with the most highly conserved region of the molecules. Complexins form a stable stoichiometric complex with the central domain of the ternary SNARE complex, whereas no binding was observed to monomeric SNAREs. Using a combination of limited proteolysis, deletion mutagenesis, and NMR spectroscopy, we found that the helical middle region of complexin is responsible for binding to the SNARE complex. Binding was highly sensitive to substitution of syntaxin 1 or synaptobrevin 2 with other SNARE homologs but less sensitive to substitution of SNAP-25. In addition, a stretch of 12 amino acids in the middle of the SNARE motif of syntaxin 1A was able to confer binding activity to the non-binding relative syntaxin 4. Furthermore, disassembly of ternary complexes is not affected by complexins. We conclude that complexins are specific ligands of the neuronal core complex that bind with a central alpha-helical domain, probably to the middle of the surface groove formed by synaptobrevin and syntaxin. Complexins may regulate the function of ternary complexes and control membrane fusion through this interaction.

• Misura KM, Scheller RH, Weis WI
• Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex.
• Nature. 2000; 404: 355-62
• Display abstract

• Syntaxin 1a and neuronal Sec1 (nSec1) form an evolutionarily conserved heterodimer that is essential for vesicle trafficking and membrane fusion. The crystal structure of the nSec1-syntaxin 1a complex, determined at 2.6 A resolution, reveals that major conformational rearrangements occur in syntaxin relative to both the core SNARE complex and isolated syntaxin. We identify regions of the two proteins that seem to determine the binding specificity of particular Sec1 proteins for syntaxin isoforms, which is likely to be important for the fidelity of membrane trafficking. The structure also indicates mechanisms that might couple the action of upstream effector proteins to conformational changes in syntaxin 1a and nSec1 that lead to core complex formation and membrane fusion.

• McNew JA et al.
• Close is not enough: SNARE-dependent membrane fusion requires an active mechanism that transduces force to membrane anchors.
• J Cell Biol. 2000; 150: 105-17
• Display abstract

• Is membrane fusion an essentially passive or an active process? It could be that fusion proteins simply need to pin two bilayers together long enough, and the bilayers could do the rest spontaneously. Or, it could be that the fusion proteins play an active role after pinning two bilayers, exerting force in the bilayer in one or another way to direct the fusion process. To distinguish these alternatives, we replaced one or both of the peptidic membrane anchors of exocytic vesicle (v)- and target membrane (t)-SNAREs (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor) with covalently attached lipids. Replacing either anchor with a phospholipid prevented fusion of liposomes by the isolated SNAREs, but still allowed assembly of trans-SNARE complexes docking vesicles. This result implies an active mechanism; if fusion occurred passively, simply holding the bilayers together long enough would have been sufficient. Studies using polyisoprenoid anchors ranging from 15-55 carbons and multiple phospholipid-containing anchors reveal distinct requirements for anchors of v- and t-SNAREs to function: v-SNAREs require anchors capable of spanning both leaflets, whereas t-SNAREs do not, so long as the anchor is sufficiently hydrophobic. These data, together with previous results showing fusion is inhibited as the length of the linker connecting the helical bundle-containing rod of the SNARE complex to the anchors is increased (McNew, J.A., T. Weber, D.M. Engelman, T.H. Sollner, and J.E. Rothman, 1999. Mol. Cell. 4:415-421), suggests a model in which one activity of the SNARE complex promoting fusion is to exert force on the anchors by pulling on the linkers. This motion would lead to the simultaneous inward movement of lipids from both bilayers, and in the case of the v-SNARE, from both leaflets.

• Smith SK et al.
• Identification of syntaxin 1A as a novel binding protein for presenilin-1.
• Brain Res Mol Brain Res. 2000; 78: 100-7
• Display abstract

• Mutations in the presenilin 1 gene have been shown to result in Alzheimer's disease. Presenilin 1 is a multi-transmembrane protein with a large hydrophilic loop near the C-terminus. This region is required for known functions of presenilin 1. We have constrained this loop within the active site of the bacterial protein, thioredoxin, to mimic its native conformational state. This hybrid protein was used as bait in a yeast two hybrid screen in an attempt to identify presenilin binding proteins. By this method syntaxin 1A, a synaptic plasma membrane protein, was identified as a novel binding protein for presenilin 1. In vitro experiments confirm the two-hybrid results suggesting that PS1 binds syntaxin under physiological conditions.

• Ossig R et al.
• Exocytosis requires asymmetry in the central layer of the SNARE complex.
• EMBO J. 2000; 19: 6000-10
• Display abstract

• Assembly of SNAREs (soluble N:-ethylmaleimide- sensitive factor attachment protein receptors) mediates membrane fusions in all eukaryotic cells. The synaptic SNARE complex is represented by a twisted bundle of four alpha-helices. Leucine zipper-like layers extend through the length of the complex except for an asymmetric and ionic middle layer formed by three glutamines (Q) and one arginine (R). We have examined the functional consequences of Q-R exchanges in the conserved middle layer using the exocytotic SNAREs of yeast as a model. Exchanging Q for R in Sso2p drastically reduces cell growth and protein secretion. When a 3Q/1R ratio is restored by a mirror R-->Q substitution in the R-SNARE Snc2p, wild-type functionality is observed. Secretion is near normal when all four helices contain Q, but defects become apparent when additional mutations are present in other layers. Using molecular dynamics free energy perturbation simulations, these findings are rationalized in structural and energetic terms. We conclude that the asymmetric arrangement of the polar amino acids in the central layer is essential for normal function of SNAREs in membrane fusion.

• Jahn R
• Sec1/Munc18 proteins: mediators of membrane fusion moving to center stage.
• Neuron. 2000; 27: 201-4

• Lerman JC, Robblee J, Fairman R, Hughson FM
• Structural analysis of the neuronal SNARE protein syntaxin-1A.
• Biochemistry. 2000; 39: 8470-9
• Display abstract

• Intracellular trafficking depends on the docking and fusion of transport vesicles with cellular membranes. Central to docking and fusion is the pairing of SNARE proteins (soluble NSF attachment protein receptors) associated with the vesicle and target membranes (v- and t-SNAREs, respectively). Here, the X-ray structure of an N-terminal conserved domain of the neuronal t-SNARE syntaxin-1A was determined to a resolution of 1.9 A using multiwavelength anomalous diffraction. This X-ray structure, which is in general agreement with an NMR structure of a similar fragment, provides new insight into the interaction surface between the N-terminal domain and the remainder of the protein. In vitro characterization of the intact cytoplasmic domain of syntaxin revealed that it forms dimers, and probably tetramers, at low micromolar concentrations, with concomitant structural changes that can be detected by limited proteolysis. These observations suggest that the promiscuity characteristic of pairing between v-SNAREs and t-SNAREs extends to the formation of homo-oligomeric t-SNARE complexes as well. They also suggest a potential role for the neuronal Sec1 protein (nSec1) in preventing the formation of syntaxin multimers.

• Gonzalo S, Greentree WK, Linder ME
• SNAP-25 is targeted to the plasma membrane through a novel membrane-binding domain.
• J Biol Chem. 1999; 274: 21313-8
• Display abstract

• SNAP-25, syntaxin, and synaptobrevin are SNARE proteins that mediate fusion of synaptic vesicles with the plasma membrane. Membrane attachment of syntaxin and synaptobrevin is achieved through a C-terminal hydrophobic tail, whereas SNAP-25 association with membranes appears to depend upon palmitoylation of cysteine residues located in the center of the molecule. This process requires an intact secretory pathway and is inhibited by brefeldin A. Here we show that the minimal plasma membrane-targeting domain of SNAP-25 maps to residues 85-120. This sequence is both necessary and sufficient to target a heterologous protein to the plasma membrane. Palmitoylation of this domain is sensitive to brefeldin A, suggesting that it uses the same membrane-targeting mechanism as the full-length protein. As expected, the palmitoylated cysteine cluster is present within this domain, but surprisingly, membrane anchoring requires an additional five-amino acid sequence that is highly conserved among SNAP-25 family members. Significantly, the membrane-targeting module coincides with the protease-sensitive stretch (residues 83-120) that connects the two alpha-helices that SNAP-25 contributes to the four-helix bundle of the synaptic SNARE complex. Our results demonstrate that residues 85-120 of SNAP-25 represent a protein module that is physically and functionally separable from the SNARE complex-forming domains.

• Davis AF, Bai J, Fasshauer D, Wolowick MJ, Lewis JL, Chapman ER
• Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
• Neuron. 1999; 24: 363-76
• Display abstract

• The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.

• Chen YA, Scales SJ, Patel SM, Doung YC, Scheller RH
• SNARE complex formation is triggered by Ca2+ and drives membrane fusion.
• Cell. 1999; 97: 165-74
• Display abstract

• Neurotransmitter exocytosis, a process mediated by a core complex of syntaxin, SNAP-25, and VAMP (SNAREs), is inhibited by SNARE-cleaving neurotoxins. Botulinum neurotoxin E inhibition of norepinephrine release in permeabilized PC12 cells can be rescued by adding a 65 aa C-terminal fragment of SNAP-25 (S25-C). Mutations along the hydrophobic face of the S25-C helix result in SNARE complexes with different thermostabilities, and these mutants rescue exocytosis to different extents. Rescue depends on the continued presence of both S25-C and Ca2+ and correlates with complex formation. The data suggest that Ca2+ triggers S25-C binding to a low-affinity site, initiating trans-complex formation. Pairing of SNARE proteins on apposing membranes leads to bilayer fusion and results in a high-affinity cis-SNARE complex.

• Margittai M, Otto H, Jahn R
• A stable interaction between syntaxin 1a and synaptobrevin 2 mediated by their transmembrane domains.
• FEBS Lett. 1999; 446: 40-4
• Display abstract

• The proteins synaptobrevin (VAMP), SNAP-25 and syntaxin 1 are essential for neuronal exocytosis. They assemble into a stable ternary complex which is thought to initiate membrane fusion. In vitro, the transmembrane domains of syntaxin and synaptobrevin are not required for association. Here we report a novel interaction between synaptobrevin and syntaxin that requires the presence of the transmembrane domains. When co-reconstituted into liposomes, the proteins form a stable binary complex that cannot be disassembled by NSF and that is resistant to denaturation by SDS. Cleavage of synaptobrevin with tetanus toxin does not affect the interaction. Furthermore, the complex is formed when a truncated version of syntaxin is used that contains only 12 additional amino acid residues outside the membrane anchor. We conclude that the interaction is mediated by the transmembrane domains.

• Fasshauer D, Antonin W, Margittai M, Pabst S, Jahn R
• Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.
• J Biol Chem. 1999; 274: 15440-6
• Display abstract

• Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.

• McNew JA, Weber T, Engelman DM, Sollner TH, Rothman JE
• The length of the flexible SNAREpin juxtamembrane region is a critical determinant of SNARE-dependent fusion.
• Mol Cell. 1999; 4: 415-21
• Display abstract

• The topology of a SNARE complex bridging two docked vesicles could act as a mechanical couple to do work on the lipid bilayer resulting in fusion. To test this, we prepared a series of modified SNARE proteins and determined their effects on SNARE-dependent membrane fusion. When two helix-breaking proline residues are introduced into the juxtamembrane region of VAMP, there is little or no effect on fusion, and the same change in syntaxin 1A only reduced the extent and rate of fusion by half. The insertion of a flexible linker between the transmembrane domain and the conserved coiled-coil domain only moderately affected fusion; however, fusion efficiency systematically decreased with increasing length of the linker. Together, these results rule out a requirement for helical continuity and suggest that distance is a critical factor for membrane fusion.

• Fiebig KM, Rice LM, Pollock E, Brunger AT
• Folding intermediates of SNARE complex assembly.
• Nat Struct Biol. 1999; 6: 117-23
• Display abstract

• SNARE (soluble NSF attachment protein receptor) proteins assemble into a stable complex essential for vesicle-membrane fusion. To further understand SNARE function we have used solution nuclear magnetic resonance (NMR) spectroscopy to characterize three assembly states of a yeast SNARE complex: first, the 'closed' conformation of Sso1; second, the binary complex of Sso1 and Sec9; and third, the ternary complex of Sso1, Sec9 and Snc1. Sec9 and Snc1 are unstructured in isolation. Sso1 likely consists of a four helix bundle formed by part of the C-terminal Hcore domain and the N-terminal H(A)H(B)H(C) domain, and this bundle is flanked on both sides by large flexible regions. Sso1 switches to an 'open' state when its Hcore domain binds Sec9. Conformational switching of the Hcore domain, via H(A)H(B)H(C), may provide a key regulatory mechanism in SNARE assembly. Formation of binary and ternary complexes induces additional alpha-helical structure in previously unstructured regions. Our data suggest a directed assembly process beginning distal to the membrane surfaces and proceeding toward them, bringing membranes into close proximity and possibly leading to membrane fusion.

• Traub LM, Downs MA, Westrich JL, Fremont DH
• Crystal structure of the alpha appendage of AP-2 reveals a recruitment platform for clathrin-coat assembly.
• Proc Natl Acad Sci U S A. 1999; 96: 8907-12
• Display abstract

• AP-2 adaptors regulate clathrin-bud formation at the cell surface by recruiting clathrin trimers to the plasma membrane and by selecting certain membrane proteins for inclusion within the developing clathrin-coat structure. These functions are performed by discrete subunits of the adaptor heterotetramer. The carboxyl-terminal appendage of the AP-2 alpha subunit appears to regulate the translocation of several endocytic accessory proteins to the bud site. We have determined the crystal structure of the alpha appendage at 1.4-A resolution by multiwavelength anomalous diffraction phasing. It is composed of two distinct structural modules, a beta-sandwich domain and a mixed alpha-beta platform domain. Structure-based mutagenesis shows that alterations to the molecular surface of a highly conserved region on the platform domain differentially affect associations of the appendage with amphiphysin, eps15, epsin, and AP180, revealing a common protein-binding interface.

• Gerst JE
• SNAREs and SNARE regulators in membrane fusion and exocytosis.
• Cell Mol Life Sci. 1999; 55: 707-34
• Display abstract

• Eukaryotes have a remarkably well-conserved apparatus for the trafficking of proteins between intracellular compartments and delivery to their target organelles. This apparatus comprises the secretory (or 'protein export') pathway, which is responsible for the proper processing and delivery of proteins and lipids, and is essential for the derivation and maintenance of those organelles. Protein transport between intracellular compartments is mediated by carrier vesicles that bud from one organelle and fuse selectively with another. Therefore, organelle-specific trafficking of vesicles requires specialized proteins that regulate vesicle transport, docking and fusion. These proteins are generically termed SNAREs and comprise evolutionarily conserved families of membrane-associated proteins (i.e. the synaptobrevin/VAMP, syntaxin and SNAP-25 families) which mediate membrane fusion. SNAREs act at all levels of the secretory pathway, but individual family members tend to be compartment-specific and, thus, are thought to contribute to the specificity of docking and fusion events. In this review, we describe the different SNARE families which function in exocytosis, as well as discuss the role of possible negative regulators (e.g. 'SNARE-masters') in mediating events leading to membrane fusion. A model to illustrate the dynamic cycling of SNAREs between fusion-incompetent and fusion-competent states, called the SNARE cycle, is presented.

• Vance CL et al.
• N-type calcium channel/syntaxin/SNAP-25 complex probed by antibodies to II-III intracellular loop of the alpha1B subunit.
• Neuroscience. 1999; 90: 665-76
• Display abstract

• Neuronal voltage-dependent calcium channels are integral components of cellular excitation and neurosecretion. In addition to mediating the entry of calcium across the plasma membrane, both N-type and P/Q-type voltage-dependent calcium channels have been shown to form stable complexes with synaptic vesicle and presynaptic membrane proteins, indicating a structural role for the voltage-dependent calcium channels in secretion. Recently, detailed structural analyses of N-type calcium channels have identified residues amino acids 718-963 as the site in the rat alpha1B subunit that mediates binding to syntaxin, synaptosome-associated protein of 25,000 mol. wt and synaptotagmin [Sheng et al. (1996) Nature 379, 451-454]. The purpose of this study was to employ site-directed antibodies to target domains within and outside of the interaction site on the rat alpha1B to probe potential binding sites for syntaxin/SNAP-25/synaptotagmin. Our results demonstrate that both antibodies employed in this study have access to their epitopes on the alpha1B as evidenced by equivalent immunoprecipitation of native [125I]omega-conotoxin GVIA-labeled alpha1B protein from CHAPS-solubilized preparations. The N-type voltage-dependent calcium channel immunoprecipitated by Ab CW14, the antibody directed to a domain outside of the synprint site, is associated with syntaxin and SNAP-25 with the recovery of these proteins, increasing in parallel to the recovery of alpha1B. However, when we used the antibody raised to an epitope within the synprint site (Ab CW8) to immunoprecipitate N-type calcium channels, the alpha1B was depleted of more than 65% of syntaxin and 80% of SNAP-25 when compared to the recovery of these proteins using Ab CW14. This is the first report of a defined epitope on the alpha1B subunit II-III loop (amino acids 863-875) whose perturbation by a site-directed antibody influences the dissociation of SNAP-25 and syntaxin.

• Sutton RB, Ernst JA, Brunger AT
• Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.
• J Cell Biol. 1999; 147: 589-98
• Display abstract

• Synaptotagmins are synaptic vesicle-associated, phospholipid-binding proteins most commonly associated with Ca(+2)-dependent exocytotic and Ca(+2)- independent endocytotic events. Synaptotagmin III is a 63.2-kD member of the synaptotagmin homology group; one of its characteristic properties is the ability to bind divalent cations and accessory proteins promiscuously. In the cytosolic portion of this protein, a flexible seven-amino acid linker joins two homologous C2 domains. The C2A domain binds to phospholipid membranes and other accessory proteins in a divalent cation-dependent fashion. The C2B domain promotes binding to other C2B domains, as well as accessory proteins independent of divalent cations. The 3.2 A crystal structure of synaptotagmin III, residues 295-566, which includes the C2A and C2B domains, exhibits differences in the shape of the Ca(+2)-binding pocket, the electrostatic surface potential, and the stoichiometry of bound divalent cations for the two domains. These observations may explain the disparate binding properties of the two domains. The C2A and the C2B domains do not interact; synaptotagmin, therefore, covalently links two independent C2 domains, each with potentially different binding partners. A model of synaptotagmin's involvement in Ca(+2)-dependent regulation of membrane fusion through its interaction with the SNARE complex is presented.

• Hazzard J, Sudhof TC, Rizo J
• NMR analysis of the structure of synaptobrevin and of its interaction with syntaxin.
• J Biomol NMR. 1999; 14: 203-7
• Display abstract

• Synaptobrevin is a synaptic vesicle protein that has an essential role in exocytosis and forms the SNARE complex with syntaxin and SNAP-25. We have analyzed the structure of isolated synaptobrevin and its binary interaction with syntaxin using NMR spectroscopy. Our results demonstrate that isolated synaptobrevin in largely unfolded in solution. The entire SNARE motif of synaptobrevin is capable of interacting with the isolated C-terminal SNARE motif of syntaxin but only a few residues bind to the full-length cytoplasmic region of syntaxin. This result suggests an interaction between the N- and C-terminal regions of syntaxin that competes with core complex assembly.

• Carr CM, Grote E, Munson M, Hughson FM, Novick PJ
• Sec1p binds to SNARE complexes and concentrates at sites of secretion.
• J Cell Biol. 1999; 146: 333-44
• Display abstract

• Proteins of the Sec1 family have been shown to interact with target-membrane t-SNAREs that are homologous to the neuronal protein syntaxin. We demonstrate that yeast Sec1p coprecipitates not only the syntaxin homologue Ssop, but also the other two exocytic SNAREs (Sec9p and Sncp) in amounts and in proportions characteristic of SNARE complexes in yeast lysates. The interaction between Sec1p and Ssop is limited by the abundance of SNARE complexes present in sec mutants that are defective in either SNARE complex assembly or disassembly. Furthermore, the localization of green fluorescent protein (GFP)-tagged Sec1p coincides with sites of vesicle docking and fusion where SNARE complexes are believed to assemble and function. The proposal that SNARE complexes act as receptors for Sec1p is supported by the mislocalization of GFP-Sec1p in a mutant defective for SNARE complex assembly and by the robust localization of GFP-Sec1p in a mutant that fails to disassemble SNARE complexes. The results presented here place yeast Sec1p at the core of the exocytic fusion machinery, bound to SNARE complexes and localized to sites of secretion.

• Montal M
• Electrostatic attraction at the core of membrane fusion.
• FEBS Lett. 1999; 447: 129-30
• Display abstract

• SNARE proteins appear to be involved in homotypic and heterotypic membrane fusion events [Sollner et al. (1993) Nature 362, 318-324]. The crystal structure of the synaptic SNARE complex exhibits a parallel four-helical bundle fold with two helices contributed by SNAP-25, a target SNARE (t-SNARE), and the other two by a different t-SNARE, syntaxin, and a donor vesicle SNARE (v-SNARE), synaptobrevin. The carboxy-terminal boundary of the complex, predicted to occur at the closest proximity between the apposed membranes, displays a high density of positively charged residues. This feature combined with the enrichment of negatively charged phospholipids in the cytosolic exposed leaflet of the membrane bilayer suggest that electrostatic attraction between oppositely charged interfaces may be sufficient to induce dynamic and discrete micellar discontinuities of the apposed membranes with the transient breakdown at the junction and subsequent reformation. Thus, the positively charged end of the SNARE complex in concert with Ca2+ may be sufficient to generate a transient 'fusion pore'.

• Dulubova I et al.
• A conformational switch in syntaxin during exocytosis: role of munc18.
• EMBO J. 1999; 18: 4372-82
• Display abstract

• Syntaxin 1, an essential protein in synaptic membrane fusion, contains a helical autonomously folded N-terminal domain, a C-terminal SNARE motif and a transmembrane region. The SNARE motif binds to synaptobrevin and SNAP-25 to assemble the core complex, whereas almost the entire cytoplasmic sequence participates in a complex with munc18-1, a neuronal Sec1 homolog. We now demonstrate by NMR spectroscopy that, in isolation, syntaxin adopts a 'closed' conformation. This default conformation of syntaxin is incompatible with core complex assembly which requires an 'open' syntaxin conformation. Using site-directed mutagenesis, we find that disruption of the closed conformation abolishes the ability of syntaxin to bind to munc18-1 and to inhibit secretion in PC12 cells. These results indicate that syntaxin binds to munc18-1 in a closed conformation and suggest that this conformation represents an essential intermediate in exocytosis. Our data suggest a model whereby, during exocytosis, syntaxin undergoes a large conformational switch that mediates the transition between the syntaxin-munc18-1 complex and the core complex.

• Perez-Branguli F, Ruiz-Montasell B, Blasi J
• Differential interaction patterns in binding assays between recombinant syntaxin 1 and synaptobrevin isoforms.
• FEBS Lett. 1999; 458: 60-4
• Display abstract

• Syntaxin 1 and synaptobrevin play an essential role in synaptic vesicle exocytosis. Two isoforms for each of these proteins, syntaxin 1A and 1B and synaptobrevin 1 and 2, have been found in nerve endings. Previous morphological studies have revealed a characteristic co-localization of syntaxin 1 and synaptobrevin isoforms in nervous and endocrine systems; however, the physiological significance of differential distribution is not known. In the present study an in vitro assay has been used to study a possible isoform specific interaction between syntaxin and synaptobrevin isoforms. The results show that although both syntaxin 1A and 1B may interact with synaptobrevin 1 and 2, this interaction is not uniform, showing different affinity patterns depending on the syntaxin 1/synaptobrevin isoform combination. The addition of SNAP-25 increased the binding capacity of syntaxin and synaptobrevin isoforms without affecting specific interactions.

• Tishgarten T et al.
• Structures of yeast vesicle trafficking proteins.
• Protein Sci. 1999; 8: 2465-73
• Display abstract

• In protein transport between organelles, interactions of v- and t-SNARE proteins are required for fusion of protein-containing vesicles with appropriate target compartments. Mammalian SNARE proteins have been observed to interact with NSF and SNAP, and yeast SNAREs with yeast homologues of NSF and SNAP proteins. This observation led to the hypothesis that, despite low sequence homology, SNARE proteins are structurally similar among eukaryotes. SNARE proteins can be classified into two groups depending on whether they interact with SNARE binding partners via conserved glutamine (Q-SNAREs) or arginine (R-SNAREs). Much of the published structural data available is for SNAREs involved in exocytosis (either in yeast or synaptic vesicles). This paper describes circular dichroism, Fourier transform infrared spectroscopy, and dynamic light scattering data for a set of yeast v- and t-SNARE proteins, Vti1p and Pep12p, that are Q-SNAREs involved in intracellular trafficking. Our results suggest that the secondary structure of Vti1p is highly alpha-helical and that Vti1p forms multimers under a variety of solution conditions. In these respects, Vti1p appears to be distinct from R-SNARE proteins characterized previously. The alpha-helicity of Vti1p is similar to that of Q-SNARE proteins characterized previously. Pep12p, a Q-SNARE, is highly alpha-helical. It is distinct from other Q-SNAREs in that it forms dimers under many of the solution conditions tested in our experiments. The results presented in this paper are among the first to suggest heterogeneity in the functioning of SNARE complexes.

• Hughson FM
• Membrane fusion: structure snared at last.
• Curr Biol. 1999; 9: 4952-4952
• Display abstract

• The structure of the core of the neuronal 'SNARE complex', involved in neurotransmitter release, has been determined recently. Its topological similarity to viral fusion proteins suggests how the SNARE complex might facilitate membrane fusion.

• Yang B, Gonzalez L Jr, Prekeris R, Steegmaier M, Advani RJ, Scheller RH
• SNARE interactions are not selective. Implications for membrane fusion specificity.
• J Biol Chem. 1999; 274: 5649-53
• Display abstract

• The SNARE hypothesis proposes that membrane trafficking specificity is mediated by preferential high affinity interactions between particular v (vesicle membrane)- and t (target membrane)-SNARE combinations. The specificity of interactions among a diverse set of SNAREs, however, is unknown. We have tested the SNARE hypothesis by analyzing potential SNARE complexes between five proteins of the vesicle-associated membrane protein (VAMP) family, three members of the synaptosome-associated protein-25 (SNAP-25) family and three members of the syntaxin family. All of the 21 combinations of SNAREs tested formed stable complexes. Sixteen were resistant to SDS denaturation, and most complexes thermally denatured between 70 and 90 degreesC. These results suggest that the specificity of membrane fusion is not encoded by the interactions between SNAREs.

• Kosodo Y, Noda Y, Yoda K
• Protein-protein interactions of the yeast Golgi t-SNARE Sed5 protein distinct from its neural plasma membrane cognate syntaxin 1.
• Biochem Biophys Res Commun. 1998; 250: 212-6
• Display abstract

• Targeting of vesicles to the acceptor membrane in protein transport depends on membrane proteins called SNAREs. Saccharomyces cerevisiae Golgi t-SNARE Sed5 protein and its neural cognate syntaxin 1 have similar three alpha-helices which are predicted to form coiled coils. We dissected the helices of Sed5 and found several characteristics unexpectedly distinct from those of syntaxin 1. Most importantly, only the N-terminal helix is responsible for the binding of Sly1 protein while almost the entire molecule of syntaxin is necessary for the binding of the cognate, Munc-18. The N-terminal region of Sed5 protein also binds to the C-terminal helix and Sly1 protein interfered this binding.

• Advani RJ et al.
• Seven novel mammalian SNARE proteins localize to distinct membrane compartments.
• J Biol Chem. 1998; 273: 10317-24
• Display abstract

• Soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) proteins of the vesicle-associated membrane protein (VAMP) and syntaxin families play a central role in vesicular trafficking through the formation of complexes between proteins present on vesicle and target membranes. Formation of these complexes is proposed to mediate aspects of the specificity of vesicle trafficking and to promote fusion of the lipid bilayers. In order to further understand the molecular mechanisms that organize membrane compartments, we have characterized seven new mammalian proteins of the VAMP and syntaxin families. The proteins are broadly expressed; however, syntaxin 13 is enriched in brain and VAMP 8 in kidney. The seven novel SNAREs localize in distinct patterns overlapping with Golgi, endosomal, or lysosomal markers. Our studies support the hypothesis that evolutionary radiation of these two gene families gave rise to sets of proteins whose differential expression and combinatorial associations define and organize the membrane compartments of cells.

• Hohl TM, Parlati F, Wimmer C, Rothman JE, Sollner TH, Engelhardt H
• Arrangement of subunits in 20 S particles consisting of NSF, SNAPs, and SNARE complexes.
• Mol Cell. 1998; 2: 539-48
• Display abstract

• The structure of 20 S particles, consisting of NSF, SNAPs, and SNARE complexes, was analyzed by electron microscopy and fluorescence resonance energy transfer. Structural changes associated with the binding of alpha-SNAP and NSF to SNARE complexes define the contribution of each component to the 20 S particle structure. The synaptic SNARE complex forms a 2.5 x 15 nm rod. alpha-SNAP binds laterally to the rod, increasing its width but not its length. NSF binds to one end of the SNAP/SNARE complex; the resulting 20 S particles measure 22 nm in length and vary in width from 6 nm at their narrowest point to 13.5 nm at their widest. The transmembrane domains of VAMP and syntaxin emerge together at the NSF-distal end of 20 S particles, adjacent to the amino terminus of alpha-SNAP.

• Schulz JR, Sasaki JD, Vacquier VD
• Increased association of synaptosome-associated protein of 25 kDa with syntaxin and vesicle-associated membrane protein following acrosomal exocytosis of sea urchin sperm.
• J Biol Chem. 1998; 273: 24355-9
• Display abstract

• Synaptosomal-associated protein of 25 kDa (SNAP-25) is a palmitoylated integral membrane protein expressed almost exclusively in neuronal and neuroendocrine tissues. This protein forms a ternary complex with vesicle-associated membrane protein (VAMP) and syntaxin, which is thought to regulate the fusion of plasma and vesicle membranes during exocytosis. We report the identification of SNAP-25 expressed in sea urchin sperm. Sea urchin SNAP-25 shares greater identity with mammalian SNAP-25 than with mammalian SNAP-23, a ubiquitously expressed homologue believed to regulate membrane fusion in non-neuronal tissues. Sea urchin sperm contain a single exocytotic vesicle, the acrosomal vesicle, whose contents are exposed during the acrosome reaction. Fusion of the plasma membrane with the acrosomal vesicle membrane at multiple points (vesiculation) results in the release of SNAP-25 with the shed acrosome reaction vesicles. A complex containing SNAP-25, syntaxin, and VAMP is present in sperm, as detected by affinity chromatography and immunoprecipitation. Although this complex is present prior to the acrosome reaction, the amount of complex increases over 4-fold following acrosomal exocytosis. These findings support the involvement of SNAP-25 in the invertebrate sperm acrosome reaction, possibly through increased association with VAMP and syntaxin driving the fusion of plasma and acrosomal membranes.

• Lill H, Nelson N
• Homologies and family relationships among Na+/Cl- neurotransmitter transporters.
• Methods Enzymol. 1998; 296: 425-36

• Xu YK, Nusse R
• The Frizzled CRD domain is conserved in diverse proteins including several receptor tyrosine kinases.
• Curr Biol. 1998; 8: 4056-4056

• Canaves JM, Montal M
• Assembly of a ternary complex by the predicted minimal coiled-coil-forming domains of syntaxin, SNAP-25, and synaptobrevin. A circular dichroism study.
• J Biol Chem. 1998; 273: 34214-21
• Display abstract

• The assembly of target (t-SNARE) and vesicle-associated SNAP receptor (v-SNARE) proteins is a critical step for the docking of synaptic vesicles to the plasma membrane. Syntaxin-1A, SNAP-25, and synaptobrevin-2 (also known as vesicle-associated membrane protein, or VAMP-2) bind to each other with high affinity, and their binding regions are predicted to form a trimeric coiled-coil. Here, we have designed three peptides, which correspond to sequences located in the syntaxin-1A H3 domain, the C-terminal domain of SNAP-25, and a conserved central domain of synaptobrevin-2, that exhibit a high propensity to form a minimal trimeric coiled-coil. The peptides were synthesized by solid phase methods, and their interactions were studied by CD spectroscopy. In aqueous solution, the peptides were unstructured and showed no interactions with each other. In contrast, upon the addition of moderate amounts of trifluoroethanol (30%), the peptides adopted an alpha-helical structure and displayed both homomeric and heteromeric interactions. The interactions observed in ternary mixtures induce a stabilization of peptide structure that is greater than that predicted from individual binary interactions, suggesting the formation of a higher order structure compatible with the assembly of a trimeric coiled-coil.

• Matveeva E, Whiteheart SW
• The effects of SNAP/SNARE complexes on the ATPase of NSF.
• FEBS Lett. 1998; 435: 211-4
• Display abstract

• The ATPase of the N-ethylmaleimide sensitive factor (NSF) appears to be central to the events that culminate in vesicle-target membrane fusion. Complexes containing different combinations of NSF, alpha-SNAP, Vamp-2 (synaptobrevin 2), syntaxin 1, and SNAP-25 were reconstituted and then tested for their effect on the ATPase of NSF. While NSF interacts with all alpha-SNAP-containing complexes, only the alpha-SNAP/t-SNARE complex significantly stimulated ATPase activity. This stimulation was dependent on increasing SNAP/t-SNARE complex and was saturable. The apparent stimulation of ATPase activity is due to a 10-fold increase in initial hydrolysis rate. Complex containing both v- and t-SNAREs bound significantly more alpha-SNAP but did not stimulate the ATPase of NSF.

• Steegmaier M et al.
• Three novel proteins of the syntaxin/SNAP-25 family.
• J Biol Chem. 1998; 273: 34171-9
• Display abstract

• Intracellular membrane traffic is thought to be regulated in part by soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) through the formation of complexes between these proteins present on vesicle and target membranes. All known SNARE-mediated fusion events involve members of the syntaxin and vesicle-associated membrane protein families. The diversity of mammalian membrane compartments predicts the existence of a large number of different syntaxin and vesicle-associated membrane protein genes. To further investigate the spectrum of SNAREs and their roles in membrane trafficking we characterized three novel members of the syntaxin and SNAP-25 (synaptosome-associated protein of 25 kDa) subfamilies. The proteins are broadly expressed, suggesting a general role in vesicle trafficking, and localize to distinct membrane compartments. Syntaxin 8 co-localizes with markers of the endoplasmic reticulum. Syntaxin 17, a divergent member of the syntaxin family, partially overlaps with endoplasmic reticulum markers, and SNAP-29 is broadly localized on multiple membranes. SNAP-29 does not contain a predicted membrane anchor characteristic of other SNAREs. In vitro studies established that SNAP-29 is capable of binding to a broad range of syntaxins.

• Abeliovich H, Grote E, Novick P, Ferro-Novick S
• Tlg2p, a yeast syntaxin homolog that resides on the Golgi and endocytic structures.
• J Biol Chem. 1998; 273: 11719-27
• Display abstract

• Intracellular membrane fusion events in eukaryotic cells are thought to be mediated by protein-protein interactions between soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex proteins. We have identified and analyzed a new yeast syntaxin homolog, Tlg2p. Tlg2p is unique among known syntaxin family proteins in possessing a sizeable hydrophilic domain of 63 amino acids that is C-terminal to the membrane spanning region and nonessential for Tlg2p function. Tlg2p resides on the endosome and late Golgi by co-localization with an endocytic intermediate and co-fractionation with markers for both endosomes and late Golgi. Cells depleted for Tlg2p missort a portion of carboxypeptidase Y and are defective in endocytosis. In addition, we report that Tlg2p forms a SEC18-dependent SNARE complex with Snc2p, a vesicle SNARE known to function in Golgi to plasma membrane trafficking. Based on these findings we propose that Tlg2p is a t-SNARE that functions in transport from the endosome to the late Golgi and on the endocytic pathway.

• Hodel A
• SNAP-25.
• Int J Biochem Cell Biol. 1998; 30: 1069-73
• Display abstract

• SNAP-25 belongs to a family of evolutionarily conserved proteins whose members are essential for exocytosis. Neurons and neuroendocrine cells differentially express two SNAP-25 isoforms in a developmentally regulated manner, and related homologues have been detected in most eukaryotic cells. SNAP-25 is localised on the cytoplasmic face of the plasma membrane and on secretory vesicles. It forms a stable ternary complex with two other exocytotic proteins: syntaxin and the synaptic vesicle protein synaptobrevin. A cytosolic ATPase dissociates this complex during priming of the exocytotic apparatus. Subsequent reassembly is promoted by SNAP-25 and may drive Ca(2+)-triggered vesicle-plasma membrane fusion. A mutant mouse that lacks the SNAP-25 gene is defective in neuronal dopamine signalling and exhibits similar behaviour as sufferers from hyperactivity disorders. Use of this animal model thus provides a promising avenue for the development of therapeutic treatments. Additionally, SNAP-25-based peptides that mimic the effect of botulinum neurotoxin A may be used for the treatment of involuntary muscle spasms.

• Poirier MA et al.
• Protease resistance of syntaxin.SNAP-25.VAMP complexes. Implications for assembly and structure.
• J Biol Chem. 1998; 273: 11370-7
• Display abstract

• A stable ternary complex formed with vesicle-associated membrane protein 2 (VAMP2) and plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) is proposed to function in synaptic vesicle exocytosis. To analyze the structural characteristics of this synaptic protein complex, recombinant binary (syntaxin 1A.SNAP-25), recombinant ternary, and native ternary complexes were subjected to limited trypsin proteolysis. The protected fragments, defined by amino-terminal sequencing and mass spectrometry, included a carboxyl-terminal region of syntaxin 1A, the cytoplasmic domain of VAMP2, and amino- and carboxyl-terminal regions of SNAP-25. Furthermore, separate amino- and carboxyl-terminal fragments of SNAP-25, when combined with VAMP2 and syntaxin 1A, were sufficient for stable complex assembly. Analysis of ternary complexes formed with full-length proteins revealed that the carboxyl-terminal transmembrane anchors of both syntaxin 1A and VAMP2 were protected from trypsin digestion. Moreover, the stability of ternary complexes was increased by inclusion of these transmembrane domains. These results suggest that the transmembrane domains of VAMP2 and syntaxin 1A contribute to complex assembly and stability and that amino- and carboxyl-terminal regions of SNAP-25 may function as independent domains.

• Ungermann C, Wickner W
• Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion.
• EMBO J. 1998; 17: 3269-76
• Display abstract

• The vacuole v-t-SNARE complex is disassembled by Sec17p/alpha-SNAP and Sec18p/NSF prior to vacuole docking and fusion. We now report a functional characterization of the vacuolar SNARE Vam7p, a SNAP-25 homolog. Although Vam7p has no hydrophobic domains, it is tightly associated with the vacuolar membrane. Vam7p is a constituent of the vacuole SNARE complex and is released from this complex by the Sec17p/Sec18p/ATP-mediated priming of the vacuoles. Even in the absence of the vacuolar v-SNARE Nyv1p, a subcomplex which includes Vam7p and the t-SNARE Vam3p is preserved. Vam7p is necessary for the stability of the vacuolar SNARE complex, since vacuoles from mutants deleted in VAM7 do not have a Vam3p-Nyv1p complex. Furthermore, Vam7p alone, in the absence of Nyv1p and Vam3p, cannot mediate fusion with wild-type vacuoles, whereas vacuoles with only Nyv1p or Vam3p alone can fuse with wild-type vacuoles in the absence of the other two SNAREs. Thus, Vam7p is important for the stable assembly and efficient function of the vacuolar SNARE complex and maintenance of the vacuolar morphology. This functional characterization of Vam7p suggests a general role for SNAP-25 homologs, not only on the plasma membrane but along the secretory pathway.

• Fischer von Mollard G, Stevens TH
• A human homolog can functionally replace the yeast vesicle-associated SNARE Vti1p in two vesicle transport pathways.
• J Biol Chem. 1998; 273: 2624-30
• Display abstract

• Membrane traffic in eukaryotic cells requires the interaction of a vesicle-associated soluble NSF attachment protein receptor (v-SNARE) on transport vesicles with a SNARE on the target membrane (t-SNARE). Recently, we identified the yeast protein Vti1p as a v-SNARE that is involved in two transport reactions. Vti1p interacts with the prevacuolar t-SNARE Pep12p in Golgi to prevacuolar transport and with the cis-Golgi t-SNARE Sed5p in traffic to the cis-Golgi. Here we describe a human Vti1p homolog, hVti1. Whereas vti1Delta cells are inviable, expression of hVti1 allows vti1Delta cells to grow at nearly the wild-type growth rate. When expressed in yeast hVti1 can replace Vti1p in both Golgi to prevacuolar transport and in traffic to the cis-Golgi. Sequence comparisons with a Schizosaccharomyces pombe and two different mouse Vti1 homologs led to the identification of a very conserved predicted alpha-helix. Amino acid exchanges in vti1 mutant alleles defective either in one or both trafficking steps cluster in this domain, suggesting that this structure is probably the binding site for effector proteins.

• Jeong EH, Webster P, Khuong CQ, Abdus Sattar AK, Satchi M, Jena BP
• The native membrane fusion machinery in cells.
• Cell Biol Int. 1998; 22: 657-70
• Display abstract

• Recombinant SNAREs have been demonstrated as the minimal membrane fusion machinery. The participation of additional proteins in the regulation of membrane fusion has been suggested. In this study we provide nanometer-resolution images of native NSF oligomers and SNARE complexes isolated from neurons and the pancreas. Our study reveals the presence of new coiled rod-like structures in association with the SNARE complex only in neuronal tissue. Neuronal SNAREs were found coiled and super-coiled with these structures. The existence of NSF as pentamers in its native state is also demonstrated. The extent of coiling and super-coiling of SNAREs may regulate the potency and efficacy of membrane fusion in cells.

• Coorssen JR, Blank PS, Tahara M, Zimmerberg J
• Biochemical and functional studies of cortical vesicle fusion: the SNARE complex and Ca2+ sensitivity.
• J Cell Biol. 1998; 143: 1845-57
• Display abstract

• Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Here we show that Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation were comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, we have analyzed the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+, the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions which disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. We suggest that this complex modulates the Ca2+ sensitivity of fusion.

• Gotte M, von Mollard GF
• A new beat for the SNARE drum.
• Trends Cell Biol. 1998; 8: 215-8
• Display abstract

• Eukaryotic cells contain membrane-bound compartments that are connected by trafficking of vesicular intermediates. To maintain compartmental organization, proper targeting of transport vesicles is achieved by specific evolutionarily conserved transmembrane proteins that reside on vesicles and target membranes. According to the original SNARE hypothesis, the formation of a complex of an NEM-sensitive fusion protein (NSF), soluble NSF attachment proteins (SNAPs) and membrane-bound SNAP receptor proteins (SNAREs) ensures docking specificity and leads to membrane fusion driven by the ATPase activity of NSF. Recent results have challenged some aspects of this hypothesis and led to a reassessment of models of SNARE interactions and the events leading to vesicle docking and fusion.

• Katz L, Hanson PI, Heuser JE, Brennwald P
• Genetic and morphological analyses reveal a critical interaction between the C-termini of two SNARE proteins and a parallel four helical arrangement for the exocytic SNARE complex.
• EMBO J. 1998; 17: 6200-9
• Display abstract

• In a screen for suppressors of a temperature-sensitive mutation in the yeast SNAP-25 homolog, Sec9, we have identified a gain-of-function mutation in the yeast synaptobrevin homolog, Snc2. The genetic properties of this suppression point to a specific interaction between the C-termini of Sec9 and Snc2 within the SNARE complex. Biochemical analysis of interactions between the wild-type and mutant proteins confirms this prediction, demonstrating specific effects of these mutations on interactions between the SNAREs. The location of the mutations suggests that the C-terminal H2 helical domain of Sec9 is likely to be aligned in parallel with Snc2 in the SNARE complex. To test this prediction, we examined the structure of the yeast exocytic SNARE complex by deep-etch electron microscopy. Like the neuronal SNARE complex, it is a rod approximately 14 nm long. Using epitope tags, antibodies and maltose-binding protein markers, we find that the helical domains of Sso, Snc and both halves of Sec9 are all aligned in parallel within the SNARE complex, suggesting that the yeast exocytic SNARE complex consists of a parallel four helix bundle. Finally, we find a similar arrangement for SNAP-25 in the neuronal SNARE complex. This provides strong evidence that the exocytic SNARE complex is a highly conserved structure composed of four parallel helical domains whose C-termini must converge in order to bring about membrane fusion.

• Swanton E, Sheehan J, Bishop N, High S, Woodman P
• Formation and turnover of NSF- and SNAP-containing "fusion" complexes occur on undocked, clathrin-coated vesicle-derived membranes.
• Mol Biol Cell. 1998; 9: 1633-47
• Display abstract

• Specificity of vesicular transport is determined by pair-wise interaction between receptors (SNAP receptors or SNAREs) associated with a transport vesicle and its target membrane. Two additional factors, N-ethylmaleimide-sensitive fusion protein (NSF) and soluble NSF attachment protein (SNAP) are ubiquitous components of vesicular transport pathways. However, the precise role they play is not known. On the basis that NSF and SNAP can be recruited to preformed SNARE complexes, it has been proposed that NSF- and SNAP-containing complexes are formed after SNARE-dependent docking of transport vesicles. This would enable ATPase-dependent complex disassembly to be coupled directly to membrane fusion. Alternatively, binding and release of NSF/SNAP may occur before vesicle docking, and perhaps be involved in the activation of SNAREs. To gain more information about the point at which so-called 20S complexes form during the transport vesicle cycle, we have examined NSF/SNAP/SNARE complex turnover on clathrin-coated vesicle-derived membranes in situ. This has been achieved under conditions in which the extent of membrane docking can be precisely monitored. We demonstrate by UV-dependent cross-linking experiments, coupled to laser light-scattering analysis of membranes, that complexes containing NSF, SNAP, and SNAREs will form and dissociate on the surface of undocked transport vesicles.

• Littleton JT, Chapman ER, Kreber R, Garment MB, Carlson SD, Ganetzky B
• Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.
• Neuron. 1998; 21: 401-13
• Display abstract

• The neuronal SNARE complex is formed via the interaction of synaptobrevin with syntaxin and SNAP-25. Purified SNARE proteins assemble spontaneously, while disassembly requires the ATPase NSF. Cycles of assembly and disassembly have been proposed to drive lipid bilayer fusion. However, this hypothesis remains to be tested in vivo. We have isolated a Drosophila temperature-sensitive paralytic mutation in syntaxin that rapidly blocks synaptic transmission at nonpermissive temperatures. This paralytic mutation specifically and selectively decreases binding to synaptobrevin and abolishes assembly of the 7S SNARE complex. Temperature-sensitive paralytic mutations in NSF (comatose) also block synaptic transmission, but over a much slower time course and with the accumulation of syntaxin and SNARE complexes on synaptic vesicles. These results provide in vivo evidence that cycles of assembly and disassembly of SNARE complexes drive membrane trafficking at synapses.

• Fasshauer D, Eliason WK, Brunger AT, Jahn R
• Identification of a minimal core of the synaptic SNARE complex sufficient for reversible assembly and disassembly.
• Biochemistry. 1998; 37: 10354-62
• Display abstract

• Assembly of the three neuronal membrane proteins synaptobrevin, syntaxin, and SNAP-25 is thought to be one of the key steps in mediating exocytosis of synaptic vesicles. In vivo and in vitro, these proteins form a tight complex. Assembly is associated with a large increase in alpha-helical content, suggesting that major structural and conformational changes are associated with the assembly reaction. Limited proteolysis by trypsin, chymotrypsin, and proteinase K of the ternary complex formed from recombinant proteins lacking their membrane anchors revealed a SDS-resistant minimal core. The components of this core complex were purified and characterized by N-terminal sequencing and mass spectrometry. They include a slightly shortened synaptobrevin fragment, C- and N-terminal fragments of SNAP-25, and a C-terminal fragment of syntaxin that is slightly larger than the previously characterized H3 domain. Recombinant proteins corresponding to these fragments are sufficient for assembly and disassembly. In addition, each of the two SNAP-25 fragments can individually form complexes with syntaxin and synaptobrevin, suggesting that they both contribute to the assembly of the SNARE complex. Upon complex assembly, a large increase in alpha-helical content is observed along with a significantly increased melting temperature (Tm). Like the full-length complex, the minimal complex tends to form an oligomeric species; global analysis of equilibrium ultracentrifugation data suggests a monomer-trimer equilibrium exists. These conserved biophysical properties may thus be of fundamental importance in the mechanism of membrane fusion.

• Jahn R, Hanson PI
• Membrane fusion. SNAREs line up in new environment.
• Nature. 1998; 393: 14-5

• Shao X, Li C, Fernandez I, Zhang X, Sudhof TC, Rizo J
• Synaptotagmin-syntaxin interaction: the C2 domain as a Ca2+-dependent electrostatic switch.
• Neuron. 1997; 18: 133-42
• Display abstract

• Synaptotagmin I is a synaptic vesicle protein that is thought to act as a Ca2+ sensor in neurotransmitter release. The first C2 domain of synaptotagmin I (C2A domain) contains a bipartite Ca2+-binding motif and interacts in a Ca2+-dependent manner with syntaxin, a central component of the membrane fusion complex. Analysis by nuclear magnetic resonance spectroscopy and site-directed mutagenesis shows that this interaction is mediated by the cooperative action of basic residues surrounding the Ca2+-binding sites of the C2A domain and is driven by a change in the electrostatic potential of the C2A domain induced by Ca2+ binding. A model is proposed whereby synaptotagmin acts as an electrostatic switch in Ca2+-triggered synaptic vesicle exocytosis, promoting a structural rearrangement in the fusion machinery that is effected by its interaction with syntaxin.

• Igarashi M, Tagaya M, Komiya Y
• The soluble N-ethylmaleimide-sensitive factor attached protein receptor complex in growth cones: molecular aspects of the axon terminal development.
• J Neurosci. 1997; 17: 1460-70
• Display abstract

• Soluble N-ethylmaleimide-sensitive factor attached protein (SNAP) receptor (SNARE) mechanisms are thought to be involved in two important processes in axonal growth cones: (1) membrane expansion for axonal growth and (2) vesicular membrane fusion for mature synaptic transmission. We investigated the localization and interactions among the proteins involved in SNARE complex formation in isolated growth cone particles (GCP) from forebrain. We demonstrated that the SNARE complex is present in GCPs morphologically without synaptic vesicles (SVs) and associated with growth cone vesicles. However, the apparently SV-free GCP was lacking in the regulatory mechanisms inhibiting SNARE complex formation proposed in SV fusion, i.e., the association of synaptotagmin with the SNARE complex, and vesicle-associated membrane protein (VAMP)-synaptophysin complex formation. The core components of the SNARE complex (syntaxin, SNAP-25, and VAMP) accumulated for several days before postnatal day 7, when SVs first appeared, and preceded the accumulation of marker proteins such as synaptophysin, SV2, and V-ATPase. Our present results suggest that the SNARE mechanism for vesicular transmitter release is not fully functional in growth cones before the appearance of SVs, but the SNARE mechanism is working for membrane expansion in growth cones, which supports our recent report. We concluded that the regulation of the SNARE complex in growth cones is different from that in mature presynaptic terminals and that this switching may be one of the key steps in development from the growth cone to the presynaptic terminal.

• Peles E, Joho K, Plowman GD, Schlessinger J
• Close similarity between Drosophila neurexin IV and mammalian Caspr protein suggests a conserved mechanism for cellular interactions.
• Cell. 1997; 88: 745-6

• Stone S et al.
• Bet1p activates the v-SNARE Bos1p.
• Mol Biol Cell. 1997; 8: 1175-81
• Display abstract

• Bet1p is a type II membrane protein that is required for vesicular transport between the endoplasmic reticulum and Golgi complex in the yeast Saccharomyces cerevisiae. A domain of Bet1p, that shows potential to be involved in a coiled-coil interaction, is homologous to a region of the neuronal protein SNAP-25. Here, we used in vitro binding studies to demonstrate that Bet1p plays a role in potentiating soluble NSF attachment protein receptor (SNARE) interactions. Mutational analysis points to the coiled-coil region as necessary for Bet1p function, and circular dichroism experiments support this theory. In vitro binding studies were also used to demonstrate that a direct interaction between Bet1p and Bos1p is required for the efficient interaction of the vesicle SNARE with its SNARE target. Genetic studies suggest that the interactions of Bet1p with Bos1p are regulated by the small GTP-binding protein Ypt1p.

• Fasshauer D, Bruns D, Shen B, Jahn R, Brunger AT
• A structural change occurs upon binding of syntaxin to SNAP-25.
• J Biol Chem. 1997; 272: 4582-90
• Display abstract

• The highly conserved proteins syntaxin and SNAP-25 are part of a protein complex that is thought to play a key role in exocytosis of synaptic vesicles. Previous work demonstrated that syntaxin and SNAP-25 bind to each other with high affinity and that their binding regions are predicted to form coiled coils. Circular dichroism spectroscopy was used here to study the alpha-helicity of the individual proteins and to gain insight into structural changes associated with complex formation. Syntaxin displayed approximately 43% alpha-helical content. In contrast, the alpha-helical content of SNAP-25 was low under physiological conditions. Formation of the SNAP-25-syntaxin complex was associated with a dramatic increase in alpha-helicity. Interaction of a 90-residue NH2-terminal fragment of SNAP-25 comprising the minimal syntaxin binding domain lead to a similar but less pronounced increase in alpha-helicity. Single amino acid replacements in the putative hydrophobic core of this fragment with hydrophilic amino acids abolished the induced structural change and disrupted the interaction monitored by binding assays. Replacements with hydrophobic residues had no effect. Our findings are consistent with induced coiled coil formation upon binding of syntaxin and SNAP-25.

• Lin RC, Scheller RH
• Structural organization of the synaptic exocytosis core complex.
• Neuron. 1997; 19: 1087-94
• Display abstract

• Syntaxin, vesicle-associated membrane protein (VAMP), and synaptosome-associated protein of 25 kDa (SNAP-25) form a ternary "core complex" central to the process of synaptic vesicle docking and fusion. Several lines of evidence support the hypothesis that the proteins assemble in a coiled-coil structure, but the alignment of alpha helices in this coil and the overall conformation of the coil are unknown. We employ the technique of fluorescence resonance energy transfer (FRET) to investigate the alignment between syntaxin and VAMP. With the acceptor probe coupled to the amino-terminal end of the VAMP coiled-coil domain, the donor probe fluorescence is quenched to a greater extent when it is on the amino-terminal end of the syntaxin H3 domain than when it is on the carboxy-terminal end. The data indicate that syntaxin and VAMP bind primarily in a parallel arrangement and suggest a coiled-coil structure that is bent rather than fully extended. We propose a model in which binding of SNAP receptor (SNARE) protein coiled-coil domains helps drive vesicle fusion.

• Nichols BJ, Ungermann C, Pelham HR, Wickner WT, Haas A
• Homotypic vacuolar fusion mediated by t- and v-SNAREs.
• Nature. 1997; 387: 199-202
• Display abstract

• Membrane fusion is necessary both in the eukaryotic secretory pathway and for the inheritance of organelles during the cell cycle. In the secretory pathway, heterotypic fusion takes place between small transport vesicles and organelles. It requires N-ethylmaleimide-sensitive fusion protein (NSF/Sec18p), soluble NSF attachment proteins (SNAPs/Sec17p) and SNAP receptors (SNAREs). SNAREs are integral membrane proteins (v-SNAREs on vesicles, t-SNAREs on the target organelles) and are thought to provide specificity to the fusion process. It has been suggested that Sec17p and Sec18p bind to v-SNARE/t-SNARE complexes and mediate the membrane fusion event. Homotypic fusion of yeast vacuoles also requires Sec17p and Sec18p (ref. 6), but in vitro they are needed only to 'prime' the vacuoles, not for subsequent docking or fusion. It has been unclear whether these reactions involve SNAREs that are similar to those previously identified in heterotypic fusion systems and, hence, whether the actions of Sec18p/NSF and Sec17p/alpha SNAP in these systems can be compared. Here we identify typical v- and t-SNAREs on the yeast vacuolar membrane. Although both are normally present, vacuoles containing only the v-SNARE can fuse with those containing only the t-SNARE. Vacuoles containing neither SNARE cannot fuse with those containing both, demonstrating that docking is mediated by cognate SNAREs on the two organelle membranes. Even when t- and v-SNAREs are on separate membranes, Sec17p and Sec18p act at the priming stage. Their action is not required at the point of assembly of the SNARE complex, nor for the fusion event itself.

• Bazan JF, Goodman CS
• Modular structure of the Drosophila Beat protein.
• Curr Biol. 1997; 7: 3389-3389

• Takuma T, Tagaya M, Ichida T
• Evidence for the putative docking/fusion complex of exocytosis in parotid acinar cells.
• FEBS Lett. 1997; 404: 34-6
• Display abstract

• In lysates of the rat brain, the SNARE complex, a putative membrane fusion machinery of synaptic exocytosis, is extremely stable and is detected after SDS-PAGE. Applying this technique to parotid acinar cells, however, we could only detect the monomeric VAMP-2, but not the high molecular forms associated with other components of the SNARE complex. Parotid acini did not contain brain-type t-SNAREs, but contained NSF and alpha SNAP. When VAMP-2 was immunoprecipitated from parotid acinar cell lysates, NSF and alpha SNAP were coprecipitated with it. Since NSF and alpha SNAP are unable to bind directly to VAMP-2 but indirectly bind via t-SNAREs, the immunoprecipitate very likely contained unidentified t-SNAREs.

• Terrian DM, White MK
• Phylogenetic analysis of membrane trafficking proteins: a family reunion and secondary structure predictions.
• Eur J Cell Biol. 1997; 73: 198-204
• Display abstract

• The realization that a highly conserved family of membrane proteins are localized to transport vesicles and selectively interact with proteins anchored at appropriate target sites of membrane fusion inspired a simple and compelling explanation of how proteins might be transferred and segregated within the cell, the "SNARE hypothesis". This model holds that vesicle and target membrane proteins (designated as v-SNARE and t-SNARE proteins, respectively) wind around one another to form a three-stranded coiled coil structure, termed the prefusion complex. While the molecular topology of the prefusion complex has not been established, the concept that phylogenetically diverse SNARE proteins may become interlocked in a stable coiled coil is particularly attractive, because such a tertiary fold would only be permitted between strictly matched binding partners. For this reason, we have performed a phenetic analysis of all known SNARE sequences to assess the evolutionary and structural relatedness of these ancient protein families. Our phylogenetic analysis and consensus structure predictions revealed that syntaxin and SNAP-25 homologs are significantly related and constitute a superfamily of t-SNARE proteins that fall naturally into four major classes with distinct architectural motifs. The synaptobrevins sorted into three different classes of v-SNARE proteins. Comparison of the consensus structure predictions within each lineage or class of SNARE proteins strongly implied that coiled coil domains may not be required for fusion complex assembly in simple eukaryotic cells. It is our hypothesis that SNARE proteins in the late secretory pathway of mammalian cells may have elaborated more complex secondary structures (coiled coils), at about the time metazoan organisms diverged from yeast, that provide a sterically rigid foundation for positioning a conserved binding domain, the amphipathic alpha-helix.

• Rice LM, Brennwald P, Brunger AT
• Formation of a yeast SNARE complex is accompanied by significant structural changes.
• FEBS Lett. 1997; 415: 49-55
• Display abstract

• The evolutionarily conserved SNARE (SNAP receptor) proteins and their complexes are key players in the docking and fusion of secretory vesicles with their target membrane. Biophysical techniques were used to characterize structural and energetic properties of the cytoplasmic domains of the yeast SNAREs Snc1 and Sso1, of the SNAP-25-like domain of Sec9, and of the Sso1:Sec9 and Sso1:Sec9:Snc1 complexes. Individually, all three SNAREs are monomeric; Sso1 shows significant secondary structure while Snc1 and Sec9 are largely unstructured. Ternary SNARE complex formation (KD <50 nM) is accompanied by a more than two-fold increase in secondary structure. This binding induced structure, the large increase in thermal stability, and the self-association of the ternary complex represent conserved properties of SNAREs that are probably important in vesicle docking and fusion.

• Hanson PI, Roth R, Morisaki H, Jahn R, Heuser JE
• Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy.
• Cell. 1997; 90: 523-35
• Display abstract

• Using quick-freeze/deep-etch electron microscopy of recombinant proteins adsorbed to mica, we show that NSF, the oligomeric ATPase involved in membrane fusion, is a hollow 10 x 16 nm cylinder whose conformation depends upon nucleotide binding. Depleted of nucleotide, NSF converts to a "splayed" protease-sensitive conformation that reveals its subunit composition. NSF's synaptic membrane substrate, the ternary SNARE complex containing syntaxin, SNAP-25, and synaptobrevin, is a 4 x 14 nm rod with a "tail" at one end, corresponding to the N-terminus of syntaxin. Using epitope tags, antibodies, and maltose-binding protein markers, we find that syntaxin and synaptobrevin are aligned in parallel in the complex, with their membrane anchors located at the same end of the rod. This SNARE rod binds with alpha-SNAP to one end of the NSF cylinder to form an asymmetric "20S" complex. Together, these images suggest how NSF could dissociate the SNARE complex and how association and dissociation of the complex could be related to membrane fusion.

• Verhage M, de Vries KJ, Roshol H, Burbach JP, Gispen WH, Sudhof TC
• DOC2 proteins in rat brain: complementary distribution and proposed function as vesicular adapter proteins in early stages of secretion.
• Neuron. 1997; 18: 453-61
• Display abstract

• DOC2 proteins constitute a novel protein family that may function in secretion and contain a double C2 domain. We have cloned and characterized two DOC2 isoforms in rat brain and studied their interactions with other proteins implicated in secretion. DOC2A was virtually brain specific, DOC2B ubiquitous. Within brain, the isoforms were expressed nonuniformly and complementary within neurons, not astroglia, and copurified with synaptic vesicles. Affinity purification, yeast two-hybrid analysis, and coimmunoprecipitation revealed that DOC2 binds munc18, a protein also implicated in secretion. The first DOC2 C2 domain and most of munc18 are involved in direct interactions. Munc18 may regulate formation of 'core complexes' during vesicle docking, by interacting with syntaxin. We show that DOC2 and syntaxin compete for munc18. Other core complex components shifted the equilibrium between syntaxin-munc18 versus DOC2-munc18. These data suggest that DOC2 proteins are vesicular adapter proteins regulating munc18-syntaxin complexes and herewith synaptic vesicle docking.

• El-Husseini AE, Guthrie H, Snutch TP, Vincent SR
• Molecular cloning of a mammalian homologue of the yeast vesicular transport protein vps45.
• Biochim Biophys Acta. 1997; 1325: 8-12
• Display abstract

• We have identified the rat homologue (rvps45) of the yeast vps45 protein, a member of the Sec1 family of proteins involved in intracellular vesicle trafficking. Sequence analysis of the full-length rvps45 cDNA obtained from a rat brain library predicts a protein of 570 amino acids which shares 36% identity with the yeast vps45 protein. The sequence shows less homology with other mammalian Sec1 family proteins. Northern blotting identified a 2.3 kb mRNA highly expressed in brain and testis. RT-PCR analysis showed that the rvps45 gene product is expressed throughout the brain. The homology of this protein with the yeast vps45 together with its high expression in brain suggests a role for rvps45 in transport from the Golgi complex to synaptic vesicles.

• Zhong P, Chen YA, Tam D, Chung D, Scheller RH, Miljanich GP
• An alpha-helical minimal binding domain within the H3 domain of syntaxin is required for SNAP-25 binding.
• Biochemistry. 1997; 36: 4317-26
• Display abstract

• The interaction between the proteins syntaxin 1A and SNAP-25 is a key step in synaptic vesicle docking and fusion. To define the SNAP-25 binding domain on syntaxin, we have prepared peptides that span the syntaxin H3 domain (residues 191-266), the region previously shown to be important for binding to SNAP-25, and then determined the affinities of these peptides for binding to SNAP-25. A minimal binding domain was identified within a region of 32 amino acids (residues 189-220). Its affinity for SNAP-25 is substantially enhanced by C-terminal extension (residues 221-266). Circular dichroism revealed the presence of substantial alpha-helicity in the H3 domain and in the 32-mer minimal binding domain, but not in H3 peptides that do not bind to SNAP-25. At temperatures that denature the alpha-helix of the minimal binding domain peptide, SNAP-25 binding is lost. Selected mutations in evolutionarily conserved residues of the amphiphilic alpha-helix within the minimal binding domain (e.g., residues 205 and 209) greatly reduce the affinity for SNAP-25 but have no major effect on secondary structure, suggesting that these residues may interact directly with SNAP-25. The H3 domain peptide and the minimal binding domain peptide inhibit norepinephrine release from PC12 cells. These results suggest that specific amino acid residues in the H3 domain, positioned by the underlying alpha-helical structure, are important for its binding to SNAP-25 and support the notion that this interaction is important for presynaptic vesicular exocytosis.

• Hao JC, Salem N, Peng XR, Kelly RB, Bennett MK
• Effect of mutations in vesicle-associated membrane protein (VAMP) on the assembly of multimeric protein complexes.
• J Neurosci. 1997; 17: 1596-603
• Display 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.

• Morgans CW, Brandstatter JH, Kellerman J, Betz H, Wassle H
• A SNARE complex containing syntaxin 3 is present in ribbon synapses of the retina.
• J Neurosci. 1996; 16: 6713-21
• Display abstract

• In contrast to conventional synapses, which release neurotransmitter transiently, ribbon synapses formed by photoreceptors and bipolar cells of the retina release neurotransmitter continuously and modulate the rate in response to light. Both modes of release are mediated by synaptic vesicles but probably differ in the regulation of docking and fusion of synaptic vesicles with the plasma membrane. We have found that syntaxin 1, an essential component of the core fusion complex in conventional synapses, is absent from ribbon synapses of the retina, raising the possibility that these synapses contain a different type of syntaxin or syntaxin-like protein. By immunoprecipitating syntaxin 1-depleted retina and brain extracts with a SNAP-25 antibody and microsequencing the precipitated proteins, syntaxin 3 was detected in retina complexed with SNAP-25, synaptobrevin, and complexin. Using an anti-syntaxin 3 antiserum, syntaxin 3 was demonstrated to be present at high levels in retina compared to brain. Immunofluorescent staining of rat retina sections confirmed that syntaxin 3 is expressed by photoreceptor and bipolar cells in the retina. Thus, in the retina, expression of syntaxin 3 is correlated with ribbon synapses and may play a role in the tonic release of neurotransmitter.

• Sudhof TC, Rizo J
• Synaptotagmins: C2-domain proteins that regulate membrane traffic.
• Neuron. 1996; 17: 379-88

• Rettig J, Sheng ZH, Kim DK, Hodson CD, Snutch TP, Catterall WA
• Isoform-specific interaction of the alpha1A subunits of brain Ca2+ channels with the presynaptic proteins syntaxin and SNAP-25.
• Proc Natl Acad Sci U S A. 1996; 93: 7363-8
• Display abstract

• Presynaptic Ca2+ channels are crucial elements in neuronal excitation-secretion coupling. In addition to mediating Ca2+ entry to initiate transmitter release, they are thought to interact directly with proteins of the synaptic vesicle docking/fusion machinery. Here we report isoform-specific, stoichiometric interaction of the BI and rbA isoforms of the alpha1A subunit of P/Q-type Ca2+ channels with the presynaptic membrane proteins syntaxin and SNAP-25 in vitro and in rat brain membranes. The BI isoform binds to both proteins, while only interaction with SNAP-25 can be detected in vitro for the rbA isoform. The synaptic protein interaction ("synprint") site involves two adjacent segments of the intracellular loop connecting domains II and III between amino acid residues 722 and 1036 of the BI sequence. This interaction is competitively blocked by the corresponding region of the N-type Ca2+ channel, indicating that these two channels bind to overlapping regions of syntaxin and SNAP-25. Our results provide a molecular basis for a physical link between Ca2+ influx into nerve terminals and subsequent exocytosis of neurotransmitters at synapses that have presynaptic Ca2+ channels containing alpha1A subunits.

• Li JY, Jahn R, Dahlstrom A
• Axonal transport and targeting of the t-SNAREs SNAP-25 and syntaxin 1 in the peripheral nervous system.
• Eur J Cell Biol. 1996; 70: 12-22
• Display abstract

• Axonal transport and targeting of the t-SNAREs SNAP-25 and syntaxin 1 were investigated in the rat peripheral nervous system using a stop-flow (crush) technique. In crush-operated sciatic nerves, accumulations of SNAP-25 and syntaxin 1 immunoreactivities were detected as early as 1 h after operation, indicating fast axonal transport. The amounts increased on the proximal side of the crush with time after crushing. Distal accumulations of SNAP-25, representing recycling to the cell body, were less than 10% of the proximal accumulations, but 40% for syntaxin 1, 50% for synaptobrevin II and 70% for synaptophysin. Immunoelectron microscopic studies demonstrated that SNAP-25 and syntaxin 1 are present on pleiotropic membranes within a diameter of 50 to 100 nm in axons proximal to a crush. Distal to the crush, labeling for syntaxin 1 and SNAP-25 were sparse and barely detectable, respectively. In addition, the two proteins were found in the axolemma. In nerve terminals of the spinal cord, both proteins were concentrated around small synaptic vesicles (about 50 nm in diameter), whereas only very few gold particles were observed near the presynaptic membrane or the active zones.

• Zimmermann H, Volknandt W, Hausinger A, Herrmann C
• Molecular properties and cellular distribution of cholinergic synaptic proteins.
• Prog Brain Res. 1996; 109: 31-40

• Hackam DJ, Rotstein OD, Bennett MK, Klip A, Grinstein S, Manolson MF
• Characterization and subcellular localization of target membrane soluble NSF attachment protein receptors (t-SNAREs) in macrophages. Syntaxins 2, 3, and 4 are present on phagosomal membranes.
• J Immunol. 1996; 156: 4377-83
• Display abstract

• Phagosomes formed during ingestion of microorganisms by leukocytes undergo a rapid maturation, generating an acidic, microbicidal organelle. Maturation requires interactions with intracellular vesicles that dock and fuse preferentially with the phagosomal membrane. The basis of specificity of vesiculo-phagosomal interaction has not been elucidated. By contrast, the molecular basis of vesicular fusion in other systems is better understood. At neural synapses, vesicular docking and fusion to the plasma membrane are mediated by a protein complex including syntaxin 1. We explored whether macrophages contain syntaxins, and whether selective fusion of vesicles with the phagosome results from the accumulation of syntaxins in the phagosomal membrane. Isoform-specific Abs were utilized to demonstrate utilized to demonstrate that syntaxins 2, 3, and 4, but not syntaxin 1, are present in murine and human macrophages. Biochemical characterization demonstrated the presence of these syntaxins on microsomes, where they are integral membrane proteins. Subcellular localization using confocal immunofluorescence microscopy demonstrated that syntaxins 3 and 4 are present on the plasma membrane as well as on intracellular vesicles. Importantly, phagosomes isolated by fractionation were shown by immunoblotting to contain syntaxins 2, 3, and 4, suggesting that they may participate in phagosomal maturation. The density of the syntaxins on the phagosomal membrane was found to be comparable with that on the surface membrane. This suggests that preferential fusion of vesicles with the phagosomal membrane is not the result of segregation of the syntaxins to this organelle. Instead, local generation of second messengers in the vicinity of the phagosomal membrane may trigger focal fusion.

• Colombo MI
• Role for NSF on vesicular transport: insights from in vitro endosome fusion.
• Biocell. 1996; 20: 317-23

• Hanson PI, Otto H, Barton N, Jahn R
• The N-ethylmaleimide-sensitive fusion protein and alpha-SNAP induce a conformational change in syntaxin.
• J Biol Chem. 1995; 270: 16955-61
• Display 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 alpha-SNAP and NSF with individual components of this complex and have identified syntaxin as a primary substrate for NSF/alpha-SNAP. We find that alpha-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 alpha-SNAP and in the presence of ATP catalyzes a conformational rearrangement which abolishes binding of itself and alpha-SNAP. This reaction leads to the previously described disassembly of the fusion complex, since synaptobrevin binding to syntaxin is also reduced. alpha-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 alpha-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.

• Parfitt K et al.
• Drosophila genetics and the functions of synaptic proteins.
• Cold Spring Harb Symp Quant Biol. 1995; 60: 371-7

• Grote E, Hao JC, Bennett MK, Kelly RB
• A targeting signal in VAMP regulating transport to synaptic vesicles.
• Cell. 1995; 81: 581-9
• Display abstract

• VAMP is a synaptic vesicle membrane protein required for fusion. Synaptic vesicle targeting was measured for mutants of an epitope-tagged form of VAMP in transfected PC12 cells. A signal within a predicted amphipathic alpha helix is essential for targeting to synaptic vesicles. Cellubrevin, a nonneural VAMP homolog, contains this signal and is also targeted to synaptic vesicles. Amino acid substitutions within the synaptic vesicle targeting signal either enhance or inhibit sorting of VAMP to synaptic vesicles, but do not affect the ability of VAMP to form complexes with syntaxin and SNAP-25.

• Haltia T, Freire E
• Forces and factors that contribute to the structural stability of membrane proteins.
• Biochim Biophys Acta. 1995; 1241: 295-322

• McMahon HT, Missler M, Li C, Sudhof TC
• Complexins: cytosolic proteins that regulate SNAP receptor function.
• Cell. 1995; 83: 111-9
• Display abstract

• A family of proteins called complexins was discovered that compete with alpha-SNAP, but not synaptotagmin, for SNAP receptor binding. Complexins I and II are highly homologous hydrophilic proteins that are tightly conserved, with 100% identity among mouse, rat, and human complexin II. They are enriched in neurons where they colocalize with syntaxin and SNAP-25; in addition, complexin II is expressed ubiquitously at low levels. Complexins bind weakly to syntaxin alone and not at all to synaptobrevin and SNAP-25, but strongly to the SNAP receptor-core complex composed of these three molecules. They compete with alpha-SNAP for binding to the core complex but not with other interacting molecules, including synaptotagmin I, suggesting that the complexins regulate the sequential interactions of alpha-SNAP and synaptotagmins with the SNAP receptor during exocytosis.

• Hayashi T, Yamasaki S, Nauenburg S, Binz T, Niemann H
• Disassembly of the reconstituted synaptic vesicle membrane fusion complex in vitro.
• EMBO J. 1995; 14: 2317-25
• Display abstract

• The interaction of the presynaptic membrane proteins SNAP-25 and syntaxin with the synaptic vesicle protein synaptobrevin (VAMP) plays a key role in the regulated exocytosis of neurotransmitters. Clostridial neurotoxins, which proteolyze these polypeptides, are potent inhibitors of neurotransmission. The cytoplasmic domains of the three membrane proteins join into a tight SDS-resistant complex (Hayashi et al., 1994). Here, we show that this reconstituted complex, as well as heterodimers composed of syntaxin and SNAP-25, can be disassembled by the concerted action of the N-ethylmaleimide-sensitive factor, NSF, and the soluble NSF attachment protein, alpha-SNAP. alpha-SNAP binds to predicted alpha-helical coiled-coil regions of syntaxin and SNAP-25, shown previously to be engaged in their direct interaction. Synaptobrevin, although incapable of binding alpha-SNAP individually, induced a third alpha-SNAP binding site when associated with syntaxin and SNAP-25 into heterotrimers. NSF released prebound alpha-SNAP from full-length syntaxin but not from a syntaxin derivative truncated at the N-terminus. Disassembly of complexes containing this syntaxin mutant was impaired, indicating a critical role for the N-terminal domain in the alpha-SNAP/NSF-mediated dissociation process. Complexes containing C-terminally deleted SNAP-25 derivatives, as generated by botulinal toxins type A and E, were dissociated more efficiently. In contrast, the N-terminal fragment generated from synaptobrevin by botulinal toxin type F produced an SDS-sensitive complex that was poorly dissociated.

• Hunt JM et al.
• A post-docking role for synaptobrevin in synaptic vesicle fusion.
• Neuron. 1994; 12: 1269-79
• Display abstract

• We have used the squid giant synapse to determine the role of synaptobrevin, integral membrane proteins of small synaptic vesicles, in neurotransmitter release. The sequence of squid synaptobrevin, deduced by cDNA cloning, is 65%-68% identical to mammalian isoforms and includes the conserved cleavage site for tetanus and botulinum B toxins. Injection of either toxin into squid nerve terminals caused a slow, irreversible inhibition of release without affecting the Ca2+ signal which triggers release. Microinjection of a recombinant protein corresponding to the cytoplasmic domain of synaptobrevin produced a more rapid and reversible inhibition of release, whereas two smaller peptide fragments were without effect. Electron microscopy of tetanus-injected terminals revealed an increased number of both docked and undocked synaptic vesicles. These data indicate that synaptobrevin participates in neurotransmitter release at a step between vesicle docking and fusion.

• Rothman JE
• Intracellular membrane fusion.
• Adv Second Messenger Phosphoprotein Res. 1994; 29: 81-96
• Display abstract

• The NSF, SNAP, and SNAP receptors are key elements of the intracellular membrane fusion machinery. We use an affinity purification scheme, based on the function of SNAP receptor in assembling 20S fusion particles from NSF and SNAP proteins, to purify SNAP receptors from brain. Remarkably, each of the four SNAP receptors (or, SNAREs) thus delineated resides in synapses, with one receptor originating in the synaptic vesicle and another in the presynaptic plasma membrane that is targeted for fusion. This suggests a simple mechanism in which the general NSF/SNAP fusion machinery can assemble to bridge partner membranes in a complex containing elements of both vesicle and target membranes, and implies that similar fusion machines drive both constitutive fusion (ER-->Golgi-->surface and endocytosis) and regulated exocytosis. The vesicle (v-SNARE) and the target-associated t-SNAREs from the synapse are each members of compartmentally-specific families of membrane proteins found in yeast, animal cells, and neurons, thus raising the possibility that v-SNAREs and t-SNAREs encode specificity in membrane fusion processes that utilize a common mechanism.

• Chapman ER, An S, Barton N, Jahn R
• SNAP-25, a t-SNARE which binds to both syntaxin and synaptobrevin via domains that may form coiled coils.
• J Biol Chem. 1994; 269: 27427-32
• Display abstract

• The membrane proteins SNAP-25, syntaxin, and synaptobrevin (vesicle-associated membrane protein) have recently been implicated as central elements of an exocytotic membrane fusion complex in neurons. Here we report that SNAP-25 binds directly to both syntaxin and synaptobrevin. The SNAP-25-binding domain of syntaxin lies between residues 199 and 243, within the region previously shown to mediate synaptobrevin binding (Calakos, N., Bennett, M. K., Peterson, K. E., and Scheller, R. H. (1994) Science 263, 1146-1149). The syntaxin-binding domain of SNAP-25 encompasses most of the amino-terminal half of SNAP-25, including its putative palmitoylation sites. Truncation of the carboxyl-terminal 9 residues of SNAP-25, which yields a fragment corresponding to that generated by botulinum neurotoxin A, diminishes the interaction of SNAP-25 with synaptobrevin, but not with syntaxin. Sequence analysis revealed that the regions that mediate the interaction between SNAP-25 and syntaxin contain heptad repeats characteristic of certain classes of alpha-helices. Similar repeats are also present at the carboxyl terminus of SNAP-25 and in synaptobrevin. These domains have a moderate to high probability of forming coiled coils. We conclude that SNAP-25 can interact with both syntaxin and synaptobrevin and that binding may be mediated by alpha-helical domains that form intermolecular coiled-coil structures.

• Calakos N, Bennett MK, Peterson KE, Scheller RH
• Protein-protein interactions contributing to the specificity of intracellular vesicular trafficking.
• Science. 1994; 263: 1146-9
• Display abstract

• Intracellular vesicles destined to fuse with the plasma membrane and secrete their contents must have a mechanism for specifically interacting with the appropriate target membrane. Such a mechanism is now suggested by the demonstration of specific interaction between vesicular proteins and plasma membrane proteins. The vesicle-associated membrane proteins (VAMPs) 1 and 2 specifically bind the acceptor membrane proteins syntaxin 1A and 4 but not syntaxin 2 or 3. The binding site is within amino acids 194 to 267 of syntaxin 1A, and the approximate equilibrium dissociation constants is 4.7 x 10(-6) molar. These data suggest a physical basis for the specificity of intracellular vesicular transport.

• Rossetto O et al.
• SNARE motif and neurotoxins.
• Nature. 1994; 372: 415-6

• Pevsner J et al.
• Specificity and regulation of a synaptic vesicle docking complex.
• Neuron. 1994; 13: 353-61
• Display abstract

• Synaptic vesicles are proposed to dock at the presynaptic plasma membrane through the interaction of two integral membrane proteins of synaptic vesicles, VAMP and synaptotagmin, and two plasma membrane proteins, syntaxin and SNAP-25. We have characterized the binding properties of these proteins and observed SNAP-25 potentiation of VAMP 2 binding to syntaxins 1a and 4 but not syntaxins 2 or 3. n-sec1, a neuron-specific syntaxin-binding protein, bound syntaxin with nanomolar affinity, forming a complex that is distinct from the previously identified 7S and 20S syntaxin-containing complexes. This suggests that syntaxin exists in at least three states: bound to n-sec1, in a 7S particle, and in a 20S particle. Recombinant n-sec1 inhibited VAMP or SNAP-25 binding to syntaxin. We propose that the specific associations of VAMP, SNAP-25, and syntaxin mediate vesicle docking and that a syntaxin/n-sec1 complex precedes and/or regulates formation of these complexes.

• Trimble WS
• Analysis of the structure and expression of the VAMP family of synaptic vesicle proteins.
• J Physiol Paris. 1993; 87: 107-15
• Display abstract

• VAMP/synaptobrevin proteins were first discovered as small integral membrane proteins in synaptic vesicles of vertebrates and invertebrates. At least two isoforms are expressed in the central nervous system of mammals in non-overlapping patterns. Biochemical studies have revealed that the VAMP synaptic vesicle proteins are the specific target in the presynaptic nerve terminal of botulinum B neurotoxin and tetanus toxin metalloendoprotease activities. The fact that these toxins rapidly and completely abrogate neurotransmission suggests that VAMP proteins play an essential role in this process. More recently, immunologically related proteins have been identified in non-neuronal cells such as adipocytes. In addition, molecular genetic studies of yeast secretion have identified VAMP-related proteins as playing important roles in vesicular transport between the endoreticulum and Golgi. Taken together, these results suggest that the VAMP proteins found on synaptic vesicles might represent specialized forms of proteins which participate in general aspects of cell membrane trafficking.

• Hurtley SM
• Membrane proteins involved in targetted membrane fusion.
• Trends Biochem Sci. 1993; 18: 453-5

• Sollner T, Bennett MK, Whiteheart SW, Scheller RH, Rothman JE
• A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion.
• Cell. 1993; 75: 409-18
• Display abstract

• The SNARE hypothesis holds that a transport vesicle chooses its target for fusion when a soluble NSF attachment protein (SNAP) receptor on the vesicle (v-SNARE) pairs with its cognate t-SNARE at the target membrane. Three synaptosomal membrane proteins have previously been identified: syntaxin, SNAP-25 (t-SNAREs), and vesicle-associated membrane protein (VAMP) (v-SNARE); all assemble with SNAPs and NSF into 20S fusion particles. We now report that in the absence of SNAP and NSF, these three SNAREs form a stable complex that can also bind synaptotagmin. Synaptotagmin is displaced by alpha-SNAP, suggesting that these two proteins share binding sites on the SNARE complex and implying that synaptotagmin operates as a "clamp" to prevent fusion from proceeding in the absence of a signal. The alpha-SNAP-SNARE complex can bind NSF, and NSF-dependent hydrolysis of ATP dissociates the complex, separating syntaxin, SNAP-25, and VAMP. ATP hydrolysis by NSF may provide motion to initiate bilayer fusion.

• Hata Y, Slaughter CA, Sudhof TC
• Synaptic vesicle fusion complex contains unc-18 homologue bound to syntaxin.
• Nature. 1993; 366: 347-51
• Display abstract

• Three synaptic proteins, syntaxin, SNAP-25 and synaptobrevin, were recently identified as targets of clostridial neurotoxins that irreversibly inhibit synaptic vesicle fusion. Experiments searching for membrane receptors for N-ethylmaleimide-sensitive fusion protein (NSF), which has an important role in membrane fusion, revealed an ATP-dependent interaction of the same three synaptic proteins with NSF and its soluble attachment proteins. Thus, two independent approaches identify syntaxin, synaptobrevin and SNAP-25 as components of the synaptic vesicle fusion machinery, but their mode of action is unclear. We have now discovered a brain protein of relative molecular mass 67,000 (67K) which binds stably to syntaxin. Amino-acid sequencing and complementary DNA cloning revealed that the 67K protein is encoded by the mammalian homologue of the Caenorhabditis elegans gene unc-18. In C. elegans, unc-18 belongs to a group of genes defined by mutations with a paralytic phenotype and accumulations of acetylcholine, suggesting a defect in neurotransmitter release. The binding of the mammalian homologue of unc-18 (Munc-18) to syntaxin requires the N terminus of syntaxin whereas that of SNAP-25 involves a more C-terminal sequence. Our data suggest that Munc-18 is a previously unidentified essential component of the synaptic vesicle fusion protein complex.

• Sudhof TC, De Camilli P, Niemann H, Jahn R
• Membrane fusion machinery: insights from synaptic proteins.
• Cell. 1993; 75: 1-4

• Linial M, Levius O
• VAT-1 from Torpedo is a membranous homologue of zeta crystallin.
• FEBS Lett. 1993; 315: 91-4
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• VAT-1 is a major protein from Torpedo synaptic vesicles. A protein data-base search revealed a striking homology to zeta crystallin from guinea pig lens. The overall amino-acid identity is 27%, and 58% similarity is reached by including conserved substitutions. The highest similarity (60% to 85%) between the two proteins is observed in five discrete domains, which are also conserved in zinc-dependent dehydrogenases, particularly in the alcohol dehydrogenase family. The cofactor-binding domain of oxidoreductases is conserved in VAT-1 and in zeta crystallin. VAT-1 preferably binds NADPH in the presence of zinc. In contrast with its homologous proteins, VAT-1 is an integral membrane protein of synaptic vesicles.

• Sollner T et al.
• SNAP receptors implicated in vesicle targeting and fusion.
• Nature. 1993; 362: 318-24
• Display abstract

• The N-ethylmaleimide-sensitive fusion protein (NSF) and the soluble NSF attachment proteins (SNAPs) appear to be essential components of the intracellular membrane fusion apparatus. An affinity purification procedure based on the natural binding of these proteins to their targets was used to isolate SNAP receptors (SNAREs) from bovine brain. Remarkably, the four principal proteins isolated were all proteins associated with the synapse, with one type located in the synaptic vesicle and another in the plasma membrane, suggesting a simple mechanism for vesicle docking. The existence of numerous SNARE-related proteins, each apparently specific for a single kind of vesicle or target membrane, indicates that NSF and SNAPs may be universal components of a vesicle fusion apparatus common to both constitutive and regulated fusion (including neurotransmitter release), in which the SNAREs may help to ensure vesicle-to-target specificity.

• Ngsee JK et al.
• Molecular analysis of proteins associated with the synaptic vesicle membrane.
• Cold Spring Harb Symp Quant Biol. 1990; 55: 111-8

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