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

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment receptors) represent an evolutionarily conserved protein family that mediates membrane fusion in the secretory and endocytic pathways of eukaryotic cells 1–3. Upon membrane contact, these proteins assemble in trans between the membranes as a bundle of four α-helices, with the energy released upon assembly being thought to drive fusion 4–6. However, it is unclear how the energy is transferred to the membranes and whether assembly is conformationally linked to fusion. Here, we report the X-ray structure of the neuronal SNARE complex, consisting of syntaxin 1A, SNAP-25 and synaptobrevin 2, with the C-terminal linkers and transmembrane regions at a resolution of 3.4 Å. The structure shows that assembly proceeds beyond the already known core-SNARE complex 7, resulting in a continuous helical bundle that is further stabilized by side-chain interactions in the linker region. Our results suggest that the final phase of SNARE assembly is directly coupled to membrane merger.

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.

Synaptotagmin-1 is the calcium sensor for neuronal exocytosis, but the mechanism by which it triggers membrane fusion is not fully understood. Here we show that synaptotagmin accelerates SNARE-dependent fusion of liposomes by interacting with neuronal Q-SNARES in a Ca 2+ -independent manner. Ca 2+ -dependent binding of synaptotagmin to its own membrane impedes the activation. Preventing this cis interaction allows Ca 2+ to trigger synaptotagmin binding in trans, accelerating fusion. However, when an activated SNARE acceptor complex is used, synaptotagmin has no effect on fusion kinetics, suggesting that synaptotagmin operates upstream of SNARE assembly in this system. Our results resolve major discrepancies concerning the effects of full-length synaptotagmin and its C2AB fragment on liposome fusion and shed new light on the interactions of synaptotagmin with SNAREs and membranes. However, our findings also show that the action of synaptotagmin on the fusionarrested state of docked vesicles in vivo is not fully reproduced in vitro.Neurotransmitters are stored in synaptic vesicles that undergo Ca 2+ -dependent exocytosis upon stimulation. Fusion of synaptic vesicles with the presynaptic plasma membrane is mediated by the neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins, including synaptobrevin-2 (also referred to as VAMP2) in the membrane of synaptic vesicles and syntaxin-1 and SNAP-25 in the plasma membrane. SNAREs are characterized by conserved stretches of 60-70 amino acid residues, referred to as SNARE motifs. Syntaxin-1 and synaptobrevin-2 each possess a single SNARE motif adjacent to the C-terminal transmembrane domain, whereas SNAP-25 contains two SNARE motifs that are separated by a palmitoylated linker 1,2 . The SNARE motifs are unstructured as monomers 3 but assemble into a tight bundle of four a-helices 4 . SNARE motifs are divided into four conserved subfamilies, referred to as Qa-, Qb-, Qc-and R-SNARE motifs. Each SNARE complex contains one member of each subfamily 5 . The assembly of SNARE complexes is currently believed to be the essential reaction in driving membrane fusion. According to this model, the formation of the SNARE complex is initiated in a trans configuration at the N-terminal ends of the SNARE motifs, forming a bridge between the membranes. Assembly then proceeds toward the C-terminal membrane anchor domains, clamping the membranes together and thus overcoming the energy barrier for fusion [6][7][8] .In contrast to several other SNARE-dependent fusion reactions, neuronal exocytosis is strongly upregulated by calcium 9 . The fast component of Ca 2+ -dependent release, which is essential for synchronous, action potential-coupled release, is mediated by the proteins synaptotagmin-1, synaptotagmin-2 and probably synaptotagmin-9, which reside in the membrane of synaptic vesicles 2 . Synaptotagmins constitute a family of type I membrane proteins with widespread tissue distribution 10 . The cytoplasmic part of the synaptotagmins contains t...

SNAREs mediate membrane fusion in intracellular vesicle traffic and neuronal exocytosis. Reconstitution of membrane fusion in vitro proved that SNAREs constitute the minimal fusion machinery. However, the slow fusion rates observed in these systems are incompatible with those required in neurotransmission. Here we present a single vesicle fusion assay that records individual SNARE-mediated fusion events with millisecond time resolution. Docking and fusion of reconstituted synaptobrevin vesicles to target SNARE complex-containing planar membranes are distinguished by total internal reflection fluorescence microscopy as separate events. Docking and fusion are SNAP-25-dependent, require no Ca 2؉ , and are efficient at room temperature. Analysis of the stochastic data with sequential and parallel multi-particle activation models reveals six to nine fast-activating steps. Of all the tested models, the kinetic model consisting of eight parallel reaction rates statistically fits the data best. This might be interpreted by fusion sites consisting of eight SNARE complexes that each activate in a single ratelimiting step in 8 ms.Neurotransmitter release in synaptic transmission by fusion of synaptic vesicles with the presynaptic membrane is tightly regulated and is probably the fastest membrane fusion event in mammalian cells. Synaptic vesicles are primed and docked to the plasma membrane but do not fuse until triggered by an influx of Ca 2ϩ from opened Ca 2ϩ channels. After electrical stimulation, neurotransmitter release is observed in less than 1 ms (1-3). The neuronal fusion and disassembly machinery is composed of the soluble N-ethylmaleimide-sensitive factor, N-ethylmaleimide-sensitive factor attachment proteins (SNAPs), 2 and the SNAP receptors (SNAREs) syntaxin1a (Syx1a), SNAP-25, and synaptobrevin2 (Syb). Proteins such as the Ca 2ϩ sensor synaptotagmin, complexin, Sec1/Munc18 homologs, Munc13, and synaptophysin are involved in regulating the fusion process, and Rab GTPases function as upstream tethering factors (4 -6). SNARE proteins, which assemble during fusion with equimolar stoichiometry into a parallel four-helix coiled-coil structure with their C termini oriented toward their respective membranes (7-9), play the most essential role in this machinery (10 -12). Energy released from a proposed N 3 C folding process pulls the two membranes together, deforms them, and eventually fuses them in a process that is mechanistically still poorly understood. SNARE-mediated fusion between target (t)-SNARE (Syx1a and SNAP-25) and vesicle (v)-SNARE (Syb) liposomes has been reconstituted in vitro (12). This and many subsequent similar studies were initially criticized because the reaction was very slow (minutes to hours). Adding a C-terminal fragment (residues 49 -96) of Syb to the Syx1a/SNAP-25 heterodimer, resulting in a ternary acceptor-SNARE complex, increased the rate of fusion with Syb liposomes by more than an order of magnitude, presumably by preventing the formation of a nonproductive 2:1 Syx1a⅐SNAP-25 complex (13)...

The synaptic vesicle protein synaptotagmin 1 is thought to convey the calcium signal onto the core secretory machinery. Its cytosolic portion mainly consists of two C2 domains, which upon calcium binding are enabled to bind to acidic lipid bilayers. Despite major advances in recent years, it is still debated how synaptotagmin controls the process of neurotransmitter release. In particular, there is disagreement with respect to its calcium binding properties and lipid preferences. To investigate how the presence of membranes influences the calcium affinity of synaptotagmin, we have now measured these properties under equilibrium conditions using isothermal titration calorimetry and fluorescence resonance energy transfer. Our data demonstrate that the acidic phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ), but not phosphatidylserine, markedly increases the calcium sensitivity of synaptotagmin. PI(4,5)P 2 binding is confined to the C2B domain but is not affected significantly by mutations of a lysine-rich patch. Together, our findings lend support to the view that synaptotagmin functions by binding in a trans configuration whereby the C2A domain binds to the synaptic vesicle and the C2B binds to the PI(4,5)P 2 -enriched plasma membrane.Calcium-dependent secretion of neurotransmitter-loaded synaptic vesicles is at the heart of synaptic transmission. The underlying membrane fusion reaction between vesicle and plasma membrane has been intensively studied and found to be promoted by both protein-protein as well as protein-lipid interactions. From the multitude of proteins involved in this membrane fusion event, the Ca 2ϩ -binding protein synaptotagmin 1 is one of its central regulating factors (for review, see Refs. 1-6). Synaptotagmin 1 is anchored in the membrane of synaptic vesicles via a single transmembrane region. Its N-terminal region comprises a short luminal domain, whereas the larger cytoplasmic C-terminal region consists of tandem C2 domains, termed C2A and C2B, tethered to each other via a short linker (7) (a schematic outline of the structural features of synaptotagmin 1 is given in Fig. 1A). Several isoforms with similar domain structure have been identified (8).C2 domains are Ca 2ϩ binding modules of ϳ130 amino acids, first described as the second conserved region of protein kinase C (PKC) 2 (9). The C2A domain of synaptotagmin 1 was the first C2 domain structure to be determined (10). In subsequent studies other C2 domains, including the C2B domain of synaptotagmin, were shown to exhibit very similar three-dimensional structures. They have a conserved eight-stranded anti-parallel ␤-sandwich connected by surface loops. C2 modules are most commonly found in enzymes involved in lipid modifications and signal transduction (PKC, phospholipases, phosphatidylinositol 3-kinases, etc.) and proteins involved in membrane trafficking (synaptotagmins, rabphilin, DOC2, etc.) (11).Calcium ions bind in a cup-shaped depression formed by the N-and C-terminal loops of the C2 key motifs of C2 domains. Nota...

Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT͞E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin͞VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step. E xocytosis of synaptic vesicles requires the N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP-25 on the synaptic plasma membrane, and synaptobrevin (also referred to as VAMP) on the vesicle membrane. Syntaxin 1 and synaptobrevin possess a single transmembrane domain at the C-terminal end, whereas SNAP-25 is membrane-anchored by palmitoyl side chains attached in the middle of the molecule. Although the essential function of the SNAREs for neurotransmitter release is well established, it is still debated whether these proteins operate as fusion catalysts or whether they act upstream of the actual fusion reaction (for review, see refs. 1-3).Each SNARE-protein contains one (syntaxin, synaptobrevin) or two (SNAP-25) characteristic stretches of 60-70 aa arranged in heptad repeats, referred to as SNARE motifs (4). Although isolated SNARE motifs are unstructured, they spontaneously assemble into stable core complexes consisting of four helix bundles. Each helix is contributed by a different SNARE motif (5, 6) representing a separate subfamily, referred to as Qa-, Qb-, Qc-, and R-SNARE motif (4, 7). Disassembly requires ATP and the action of the AAA-ATPase the N-ethylmaleimide-sensitive factor in conjunction with cofactors (8). Because membrane fusion requires that SNAREs are initially present on both membranes, assembly of the core complex would pull the membranes closely together, resulting in fusion, with the energy being provided by the assembly reaction (9, 10).Although studies on soluble recombinant SNAREs have been instrumental in developing our current thinking about how SNAREs fuse membranes, it still needs to be clarified how the speed and efficiency of biological fusion reactions is brought about at the molecular level. As a step toward this goal, it is necessary to reconstitute exocytotic membrane fusion by using purified proteins and artificial membranes. Recently, R...

Summary Misfolded proteins of the endoplasmic reticulum (ER) are retro-translocated into the cytosol, poly-ubiquitinated, and degraded by the proteasome, a process called ER-associated protein degradation (ERAD). Here, we have used purified components from Saccharomyces cerevisiae to analyze the mechanism of retro-translocation of luminal substrates (ERAD-L), recapitulating key steps in a basic process where the ubiquitin ligase Hrd1p is the only required membrane protein. We show that Hrd1p interacts with substrate through its membrane-spanning domain, discriminating misfolded from folded polypeptides. Both Hrd1p and substrate are poly-ubiquitinated, resulting in the binding of the Cdc48p ATPase complex. Subsequently, ATP hydrolysis by Cdc48p releases substrate from Hrd1p. Finally, ubiquitin chains are trimmed by the de-ubiquitinating enzyme Otu1p, which is recruited and activated by the Cdc48p complex. Cdc48p-dependent membrane extraction of poly-ubiquitinated proteins can be reproduced with reconstituted proteoliposomes. Our results suggest a model for retro-translocation in which Hrd1p forms a membrane conduit for misfolded proteins.