A lipid-anchored SNARE supports membrane fusion

Intracellular membrane fusion requires R-SNAREs and Q-SNAREs to assemble into a four-helical parallel coiled-coil, with their hydrophobic anchors spanning the two apposed membranes. Based on the fusion properties of chemically defined SNARE- proteoliposomes, it has been proposed that the assembly of this helical bundle transduces force through the entire bilayer via the transmembrane SNARE anchor domains to drive fusion. However, an R-SNARE, Nyv1p, with a genetically engineered lipid anchor that spans half of the bilayer suffices for the fusion of isolated vacuoles, although this organelle has other R-SNAREs. To demonstrate unequivocally the fusion activity of lipid-anchored Nyv1p, we reconstituted proteoliposomes with purified lipid-anchored Nyv1p as the only protein. When these proteoliposomes were incubated with those bearing cognate Q-SNAREs, there was trans-SNARE complex assembly but, in accord with prior studies of the neuronal SNAREs, little lipid mixing. However, the addition of physiological fusion accessory proteins (HOPS, Sec17p, and Sec18p) allows lipid-anchored Nyv1p to support fusion, suggesting that trans-SNARE complex function is not limited to force transduction across the bilayers through the transmembrane domains.     

Trans-SNARE complex assembly and yeast vacuole membrane fusion

cis-SNARE complexes (anchored in one membrane) are disassembled by Sec17p (alpha-SNAP) and Sec18p (NSF), permitting the unpaired SNAREs to assemble in trans. We now report a direct assay of trans-SNARE complex formation during yeast vacuole docking. SNARE complex assembly and fusion is promoted by high concentrations of the SNARE Vam7p or Nyv1p or by addition of HOPS (homotypic fusion and vacuole protein sorting), a Ypt7p (Rab)-effector complex with a Sec1/Munc18-family subunit. Inhibitors that target Ypt7p, HOPS, or key regulatory lipids prevent trans-SNARE complex assembly and ensuing fusion. Strikingly, the lipid ligand MED (myristoylated alanine-rich C kinase substrate effector domain) or elevated concentrations of Sec17p, which can displace HOPS from SNARE complexes, permit full trans-SNARE pairing but block fusion. These findings suggest that efficient fusion requires trans-SNARE complex associations with factors such as HOPS and subsequent regulated lipid rearrangements.     

Phosphoinositides and SNARE chaperones synergistically assemble and remodel SNARE complexes for membrane fusion

Yeast vacuole fusion requires 4 SNAREs, 2 SNARE chaperone systems (Sec17p/Sec18p/ATP and the HOPS complex), and 2 phosphoinositides, phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂]. By reconstituting proteoliposomal fusion with purified components, we now show that phosphoinositides have 4 distinct roles: PI(3)P is recognized by the PX domain of the SNARE Vam7p; PI(3)P enhances the capacity of membrane-bound SNAREs to drive fusion in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ can activate SNARE chaperones for the recruitment of Vam7p into fusion-competent SNARE complexes; and either PI(3)P or PI(4,5)P₂ strikingly promotes synergistic SNARE complex remodeling by Sec17p/Sec18p/ATP and HOPS. This ternary synergy of phosphoinositides and 2 SNARE chaperone systems is required for rapid fusion.Intracellular membrane fusion is a conserved reaction, vital for vesicle trafficking, hormone secretion, and neurotransmission. Fusion is regulated by NSF (N-ethylmaleimide-sensitive factor)/Sec18p, αSNAP (soluble NSF attachment protein)/Sec17p, SNAREs (SNAP receptors), Sec1p/Munc18–1p family (SM) proteins, Rab GTPases, and Rab:GTP-binding proteins, termed “Rab effectors” (1–3). Lipids, including phosphoinositides, sterols, diacylglycerol (DAG), and phosphatidic acid (PA), have specific roles in fusion (4–14). Proteins and lipids cooperate for their enrichment in membrane fusion microdomains (6, 8, 15, 16).SNARE proteins are integral or peripheral membrane proteins required for membrane fusion. SNAREs have either a Q or R residue at the center of their SNARE domain and associate in 4-helical QabcR complexes in cis (anchored to one membrane) or in trans (anchored to apposed membranes), where a, b, and c are families of related Q-SNAREs (2, 17, 18). Reconstituted proteoliposomes (RPLs) bearing Q-SNAREs fuse with RPLs bearing an R-SNARE through trans-SNARE-complex assembly (19, 20). This fusion has slow kinetics, requires nonphysiologically high SNARE densities, and causes substantial leakage of luminal contents of the RPLs (21–24).We study membrane fusion with yeast vacuoles (lysosomes). Vacuole fusion (25) requires 3 Q-SNAREs (Vam3p, Vti1p, and Vam7p) and 1R-SNARE (Nyv1p) (26, 27), two SNARE chaperone systems, Sec17p/Sec18p/ATP (28), and the HOPS (homotypic fusion and vacuole protein sorting)/Vps Class C complex (29, 30), the Rab-GTPase Ypt7p (31), and chemically minor but functionally vital “regulatory lipids”: ergosterol (ERG), DAG, PI(3)P, and PI(4,5)P₂ (8). Inactive 4SNARE cis-complexes on isolated organelles are disassembled by Sec17p/Sec18p/ATP (27). The heterohexameric HOPS complex, containing the SM protein Vps33p as a subunit, promotes and proofreads SNARE-complex assembly (32–34). HOPS can physically interact with the Q-SNAREs [Vam7p (35) and Vam3p (36, 37)], 4SNARE cis-complexes (32), GTP-bound Ypt7p (29), and phosphoinositides (35). PI(3)P supports the membrane association of the Qc-SNARE Vam7p, which has no transmembrane domain, through binding its PX domain (38). SNAREs, HOPS, Ypt7p, and regulatory lipids assemble in an interdependent fashion to form a fusion-competent membrane microdomain, the “vertex ring” (8, 16, 39). Trans-SNARE complexes are essential for fusion (26), yet fusion can be accelerated by SNARE-associating factors such as HOPS (14, 35) and by cycles of SNARE complex disassembly and reassembly, termed “remodeling” (40).Membrane fusion has been reconstituted with all purified yeast vacuolar components, including 4SNAREs, vacuolar lipids, 2 SNARE chaperone systems, and phosphoinositides (14). We now show distinct functions of phosphoinositides in RPL fusion: the PX-domain of the SNARE Vam7p recognizes PI(3)P, as reported (38); PI(3)P activates the 3Q-SNAREs to be more fusogenic in the absence of SNARE chaperones; either PI(3)P or PI(4,5)P₂ accelerates fusion by promoting the synergy between Sec17p/Sec18p and HOPS, although this synergy is not a function of the membrane recruitments of these SNARE chaperones. This ternary synergy between phosphoinositides and SNARE chaperones is essential for the assembly and remodeling of SNARE complexes.     

From the Cover: Feature Article: Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

Rab GTPases and their effectors mediate docking, the initial contact of intracellular membranes preceding bilayer fusion. However, it has been unclear whether Rab proteins and effectors are sufficient for intermembrane interactions. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, lipids, and the homotypic fusion and vacuole protein sorting (HOPS)/class C Vps complex, an effector and guanine nucleotide exchange factor for the yeast vacuolar Rab GTPase Ypt7p. We now report reconstitution of lysis-free membrane fusion that requires purified GTP-bound Ypt7p, HOPS complex, vacuolar SNAREs, ATP hydrolysis, and the SNARE disassembly catalysts Sec17p and Sec18p. We use this reconstituted system to show that SNAREs and Sec17p/Sec18p, and Ypt7p and the HOPS complex, are required for stable intermembrane interactions and that the three vacuolar Q-SNAREs are sufficient for these interactions.     

A Ypt/Rab effector complex containing the Sec1 homolog Vps33p is required for homotypic vacuole fusion

Yeast vacuoles undergo priming, docking, and homotypic fusion, although little has been known of the connections between these reactions. Vacuole-associated Vam2p and Vam6p (Vam2/6p) are components of a 65S complex containing SNARE proteins. Upon priming by Sec18p/NSF and ATP, Vam2/6p is released as a 38S subcomplex that binds Ypt7p to initiate docking. We now report that the 38S complex consists of both Vam2/6p and the class C Vps proteins [Reider, S. E. and Emr, S. D. (1997) Mol. Biol. Cell 8, 2307-2327]. This complex includes Vps33p, a member of the Sec1 family of proteins that bind t-SNAREs. We term this 38S complex HOPS, for homotypic fusion and vacuole protein sorting. This unexpected finding explains how Vam2/6p associates with SNAREs and provides a mechanistic link of the class C Vps proteins to Ypt/Rab action. HOPS initially associates with vacuole SNAREs in "cis" and, after release by priming, hops to Ypt7p, activating this Ypt/Rab switch to initiate docking.     

A new role for a SNARE protein as a regulator of the Ypt7/Rab-dependent stage of docking

The homotypic fusion of yeast vacuoles occurs in an ordered cascade of priming, docking, and fusion. The linkage between these steps has so far remained unclear. We now report that Vam7p (the vacuolar SNAP-23/25 homolog) signals from the cis-SNARE complex to Ypt7p (the vacuolar Rab/Ypt) to initiate the docking process. After Vam7p has been released from the cis-SNARE complex by Sec18p-mediated priming, it is still required for Ypt7p-dependent docking and it needs Ypt7p to remain on the vacuole. Thus, after priming, Vam7p is released from the vacuole altogether if Ypt7p has been extracted by Gdi1p or inactivated by antibody but is not released if docking is blocked simply by vacuole dilution; it is therefore Ypt7p function, and not docking per se, that retains Vam7p. In accord with this finding, cells deleted for the gene encoding Ypt7 have normal amounts of Vam7p but have little Vam7p on their isolated vacuoles. Interaction of Vam7p and Ypt7p is further indicated by two-hybrid analysis [Uetz, P., Giot, L., Cagney, G., Mansfield, T. A., Judson, R. S., Knight, J. R., Lockshon, D., Narayan, V., Srinivasan, M., Pochart, P., et al. (2000) Nature (London) 403, 623-627] and by the effect of Vam7p on the association of the Rab/Ypt-effector HOPS complex (homotypic fusion and vacuole protein sorting; Vam2p and Vam6p plus four vacuole protein sorting class C proteins) with Ypt7p. Vam7p provides a functional link between the priming step, which releases it from the cis-SNARE complex, and docking.     

Three-dimensional structure of the amino-terminal domain of syntaxin 6, a SNAP-25 C homolog

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins are required for intracellular membrane fusion, and are differentially localized throughout the cell. SNAREs on vesicle and target membranes contain "SNARE motifs" which interact to form a four-helix bundle that contributes to the fusion of two membranes. SNARE motif sequences fall into four classes, homologous to the neuronal proteins syntaxin 1a, VAMP 2, and the N- and C-terminal SNARE motifs of SNAP-25 (S25N and S25C), and it is thought that one member from each class interacts to form a SNARE complex. Many SNAREs also feature N-terminal domains believed to function in regulating SNARE complex assembly or other aspects of vesicle transport. Syntaxin 6 is a SNARE found primarily in endosomal transport vesicles and whose SNARE motif shows significant homology to both syntaxin 1a and S25C. The crystal structure of the syntaxin 6 N-terminal domain reveals strong structural similarity with the N-terminal domains of syntaxin family members syntaxin 1a, Sso1p, and Vam3p, despite a very low level of sequence similarity. The syntaxin 6 SNARE motif can substitute for S25C in in vitro binding experiments, supporting the classification of syntaxin 6 as an S25C family member. Secondary structure prediction of SNARE proteins shows that the N-terminal domains of many syntaxin, S25N, and S25C family members are likely to be similar to one another, but are distinct from those of VAMP family members, indicating that syntaxin, S25N, and S25C SNAREs may have shared a common ancestor.     

Excess vacuolar SNAREs drive lysis and Rab bypass fusion

Although concentrated soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) drive liposome fusion and lysis, the fusion of intracellular membranes also requires Rab GTPases, Rab effectors, SM proteins, and specific regulatory lipids and is accompanied by little or no lysis. To rationalize these findings, we generated yeast strains that overexpress all four vacuolar SNAREs (4SNARE(++)). Although vacuoles with physiological levels of Rab, Rab effector/SM complex, and SNAREs support rapid fusion without Rab- and SNARE-dependent lysis, vacuoles from 4SNARE(++) strains show extensive lysis and a reduced need for the Rab Ypt7p or regulatory lipids for fusion. SNARE overexpression and the addition of pure homotypic fusion and vacuole protein sorting complex (HOPS), which bears the vacuolar SM protein, enables ypt7Delta vacuoles to fuse, allowing direct comparison of Rab-dependent and Rab-independent fusion. Because 3- to 40-fold more of each of the five components that form the SNARE/HOPS fusion complex are required for vacuoles from ypt7Delta strains to fuse at the same rate as vacuoles from wild-type strains, the apparent forward rate constant of 4SNARE/HOPS complex assembly is enhanced many thousand-fold by Ypt7p. Rabs function in normal membrane fusion by concentrating SNAREs, other proteins (e.g., SM), and key lipids at a fusion site and activating them for fusion without lysis.     

Vam10p defines a Sec18p-independent step of priming that allows yeast vacuole tethering

YOR068c, termed VAM10 (altered vacuole morphology), lies within the VPS5 gene on the opposite DNA strand. VAM10 deletion causes vacuole fragmentation in vivo. The in vitro fusion of purified yeast vacuoles is stimulated by recombinant Vam10p and blocked by antibody to Vam10p. Vam10p acts early in the priming stage of fusion, independent of Sec18p. After priming, recombinant Vam10p will not stimulate fusion and anti-Vam10p antibodies will not inhibit; Vam10p provides a functional marker for this Sec18p-independent priming step. Pure Vam10p restores normal, Ypt7p-dependent tethering to vacuoles from a vam10Delta strain.     

Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18

Sec17 [soluble N-ethylmaleimide–sensitive factor (NSF) attachment protein; α-SNAP] and Sec18 (NSF) perform ATP-dependent disassembly of cis-SNARE complexes, liberating SNAREs for subsequent assembly of trans-complexes for fusion. A mutant of Sec17, with limited ability to stimulate Sec18, still strongly enhanced fusion when ample Sec18 was supplied, suggesting that Sec17 has additional functions. We used fusion reactions where the four SNAREs were initially separate, thus requiring no disassembly by Sec18. With proteoliposomes bearing asymmetrically disposed SNAREs, tethering and trans-SNARE pairing allowed slow fusion. Addition of Sec17 did not affect the levels of trans-SNARE complex but triggered sudden fusion of trans-SNARE paired proteoliposomes. Sec18 did not substitute for Sec17 in triggering fusion, but ADP- or ATPγS-bound Sec18 enhanced this Sec17 function. The extent of the Sec17 effect varied with the lipid headgroup and fatty acyl composition of the proteoliposomes. Two mutants further distinguished the two Sec17 functions: Sec17^L291A,L292A did not stimulate Sec18 to disassemble cis-SNARE complex but triggered the fusion of trans-SNARE paired membranes. Sec17^F21S,M22S, with diminished apolar character to its hydrophobic loop, fully supported Sec18-mediated SNARE complex disassembly but had lost the capacity to stimulate the fusion of trans-SNARE paired membranes. To model the interactions of SNARE-bound Sec17 with membranes, we show that Sec17, but not Sec17^F21S,M22S, interacted synergistically with the soluble SNARE domains to enable their stable association with liposomes. We propose a model in which Sec17 binds to trans-SNARE complexes, oligomerizes, and inserts apolar loops into the apposed membranes, locally disturbing the lipid bilayer and thereby lowering the energy barrier for fusion.Intracellular vesicular traffic between organelles is the basis of cell growth, hormone secretion, and neurotransmission. At each step of exocytic and endocytic trafficking, membranes dock and fuse, mixing their lipids and luminal contents while keeping them separate from the cytosol. Families of proteins, conserved from yeast to humans, mediate docking and fusion. Fusion requires Rab family GTPases and “effector” proteins that bind to a Rab in its active, GTP-bound state (1). Among the effectors are large, organelle-specific tethering complexes. Fusion requires SNARE proteins and their chaperones. SNAREs (2) are proteins that can “snare” (bind to) each other, in cis (when anchored to the same membrane) or in trans (when anchored to apposed, tethered membranes). SNAREs have a conserved “SNARE domain” with a characteristic heptad repeat. SNAREs are categorized as R-SNAREs if they have a central arginyl residue, or Qa-, Qb-, or Qc-SNAREs with a central glutamyl residue (3). SNAREs form RQaQbQc quaternary cis- or trans-SNARE complexes, which bind SNARE chaperones, including the Sec1/Munc18 family of SNARE binding proteins, and Sec18/NSF (N-ethylmaleimide–sensitive factor), an AAA family ATPase that drives SNARE complex disassembly (4). Sec17/α-SNAP (soluble NSF attachment protein) is a cochaperone to Sec18 that enhances its rate of SNARE complex disassembly (5).We study fusion with yeast vacuoles. The homotypic fusion of vacuoles has been studied extensively through genetic identification of vacuole morphology (vam) mutants (6) and vacuole protein sorting (vps) mutants (7), through a colorimetric assay of the fusion of isolated vacuoles (8), and more recently through the fusion of proteoliposomes reconstituted with defined, purified proteins and lipids (9–11). Sec17, Sec18, and ATP catalyze the first stage of vacuole fusion, in which cis-SNARE complexes are disassembled (12). Tethering is then supported by the Rab Ypt7 and the large, multisubunit tethering complex termed HOPS (13). Vps33, one of the HOPS subunits, is the vacuolar SM (Sec1/mUNC-18 family) protein. HOPS has direct affinity for vacuolar SNAREs (14–16), and helps to catalyze SNARE complex assembly and the subsequent fusion (17).During in vitro fusion incubations, most Sec17 is released from vacuoles during cis-SNARE complex disassembly (12). However, a few percent of the vacuolar SNAREs form trans-SNARE complexes (18), and Sec17 is a major constituent of these complexes (19). Furthermore, although Sec17 and Sec18 can disassemble trans-SNARE complexes (19) and will block fusion events in which tethering is supplied by an unphysiological agent such as polyethylene glycol (13), Sec17 and Sec18 work synergistically with HOPS to promote fusion (9, 20). This synergy is even seen when the SNAREs are initially disposed with the R-SNARE on one set of proteoliposomes and the three Q-SNAREs on the others (9), a condition that per se does not require cis-SNARE complex disassembly by Sec18. Finally, added Sec17 restores fusion to vacuoles where fusion is blocked by a defined C-terminal truncation in the SNARE domain of the Qc-SNARE Vam7, in the apparent absence of ATP or Sec18 activity (21). These observations prompted us to reevaluate the roles of Sec17 and Sec18 in the fusion pathway.We now exploit proteoliposomes bearing purified vacuolar Rab and SNAREs to reinvestigate the roles of Sec17 and Sec18. Generous amounts of Sec18 alone can disassemble cis-SNARE complexes, allowing proteoliposomes bearing all four vacuolar SNAREs to fuse at a moderate rate. The rate of fusion can be stimulated by wild-type Sec17, as expected, but also by a Sec17 mutant that has greatly diminished capacity to stimulate Sec18. This suggested that Sec17 could act in ways other than through Sec18 stimulation. We therefore examined fusion incubations where the SNAREs are disposed on complementary proteoliposomes such that Sec18 is not required at all. We find that Sec17 can trigger rapid fusion of proteoliposomes that are already joined by trans-SNARE associations.     

Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation

SNARE proteins play a critical role in intracellular membrane fusion by forming tight complexes that bring two membranes together and involve sequences called SNARE motifs. These motifs have a high tendency to form amphipathic coiled-coils that assemble into four-helix bundles, and often precede transmembrane regions. NMR studies in dodecylphosphocholine (DPC) micelles suggested that the N-terminal half of the SNARE motif from the neuronal SNARE synaptobrevin binds to membranes, which appeared to contradict previous biophysical studies of synaptobrevin in liposomes. NMR analyses of synaptobrevin reconstituted into nanodiscs and into liposomes now show that most of its SNARE motif, except for the basic C terminus, is highly flexible, exhibiting cross-peak patterns and transverse relaxation rates that are very similar to those observed in solution. Considering the proximity to the bilayer imposed by membrane anchoring, our data show that most of the synaptobrevin SNARE motif has a remarkable reluctance to bind membranes. This conclusion is further supported by NMR experiments showing that the soluble synaptobrevin SNARE motif does not bind to liposomes, even though it does bind to DPC micelles. These results show that nanodiscs provide a much better membrane model than DPC micelles in this system, and that most of the SNARE motif of membrane-anchored synaptobrevin is accessible for SNARE complex formation. We propose that the charge and hydrophobicity of SNARE motifs is optimized to enable formation of highly stable SNARE complexes while at the same time avoiding membrane binding, which could hinder SNARE complex assembly.     

In vitro assay using engineered yeast vacuoles for neuronal SNARE-mediated membrane fusion

Intracellular membrane fusion requires not only SNARE proteins but also other regulatory proteins such as the Rab and Sec1/Munc18 (SM) family proteins. Although neuronal SNARE proteins alone can drive the fusion between synthetic liposomes, it remains unclear whether they are also sufficient to induce the fusion of biological membranes. Here, through the use of engineered yeast vacuoles bearing neuronal SNARE proteins, we show that neuronal SNAREs can induce membrane fusion between yeast vacuoles and that this fusion does not require the function of the Rab protein Ypt7p or the SM family protein Vps33p, both of which are essential for normal yeast vacuole fusion. Although excess vacuolar SNARE proteins were also shown to mediate Rab-bypass fusion, this fusion required homotypic fusion and vacuole protein sorting complex, which bears Vps33p and was accompanied by extensive membrane lysis. We also show that this neuronal SNARE-driven vacuole fusion can be stimulated by the neuronal SM protein Munc18 and blocked by botulinum neurotoxin serotype E, a well-known inhibitor of synaptic vesicle fusion. Taken together, our results suggest that neuronal SNARE proteins are sufficient to induce biological membrane fusion, and that this new assay can be used as a simple and complementary method for investigating synaptic vesicle fusion mechanisms.Membrane fusion mediates a variety of biological processes, such as fertilization and cell growth, hormone secretion, neurotransmission, nutrient uptake, and viral infection (1). Vesicle trafficking between organelles, a major tool for intracellular transport of materials, is also regulated by membrane fusion: Membrane fusion between transport vesicles and target compartments releases the cargo stored in the vesicles into the lumen of the compartments. To maintain the unique chemical environment of each organelle, biological membrane fusion occurs with spatiotemporal precision but without leakage of the luminal contents. This nature of biological membrane fusion may be achieved through the cooperation of various proteins, such as Rab GTPases and their effectors, SNARE [soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor] proteins, and SNARE chaperones (2, 3). SNARE proteins bring two membrane bilayers into close proximity, which promotes the fusion of the apposed membranes (4, 5). The molecular mechanism by which SNARE proteins mediate membrane fusion has been intensively studied in synaptic vesicle fusion, which mediates neurotransmission at the synapse, whereby neurotransmitters released by presynaptic neurons are recognized by their receptors on postsynaptic neurons (6, 7). Depolarization of presynaptic nerve terminals by an action potential opens Ca²⁺ channels in the presynaptic membrane. Ca²⁺ influx into the presynaptic cell then triggers membrane fusion between the presynaptic plasma membrane and synaptic vesicles, leading to the release of neurotransmitter. Synaptic vesicle fusion is mediated by three neuronal SNARE proteins: syntaxin, SNAP25, and synaptobrevin (also referred to as VAMP). Synaptobrevin and syntaxin-1 each contain one SNARE motif, whereas SNAP25 contains two. One SNARE motif from synaptobrevin (v-SNARE) on a synaptic vesicle and three SNARE motifs provided by syntaxin-1 and SNAP25 (t-SNAREs) from the plasma membrane assemble into a tight trans-SNARE complex that brings the two membranes into close apposition. This close apposition, in turn, induces lipid bilayer merging, thus releasing neurotransmitter into the synaptic cleft.In addition to the neuronal SNARE proteins, many other regulatory proteins, such as Munc18 and synaptotagmin, are required for synaptic vesicle fusion in vivo (8, 9). Munc18, a member of the Sec1/Munc18 (SM) protein family, seems to play a variety of roles in synaptic vesicle fusion. First, Munc18 binds to free syntaxin molecules and keeps them in a closed, inactive state, helping to prevent the formation of premature SNARE complexes (10, 11). Additionally, Munc18 interacts with the syntaxin molecule within assembled t-SNARE complexes, guiding them in a manner conducive to productive trans-SNARE complex formation, which triggers membrane fusion (12). Neuronal synaptotagmin, anchored to synaptic vesicles, functions as a calcium sensor for synaptic vesicle fusion (9, 13, 14). Although its mode of action is not yet fully defined, calcium binding to synaptotagmin triggers synaptic vesicle fusion. Despite the importance of these and many other proteins for synaptic vesicle fusion, the three neuronal SNARE proteins are thought to constitute the minimal components sufficient to drive membrane fusion: On their own, they can induce fusion between proteoliposomes carrying both syntaxin and SNAP25 and those reconstituted with synaptobrevin (15). Although these reconstitution experiments strongly support the concept that neuronal SNARE proteins suffice to induce membrane fusion, it is still unclear whether they can also induce fusion between biological membranes for the following reasons: (i) Liposome fusion, unlike biological membrane fusion, is intrinsically promiscuous: Even protein-free liposomes can fuse under certain conditions (16). (ii) Liposome fusion assays often rely on detection of lipid mixing, which can occur without content mixing. Liposome rupture (17) or clustering without fusion (18) can generate false-positive signals. (iii) Finally, because detergent is used to reconstitute SNARE proteins into liposomes, the potential presence of residual detergent, which affects the integrity of liposomes and their lipid mixing, cannot be completely excluded.Homotypic yeast vacuole fusion has been used to study membrane fusion mechanisms (19–21). In vitro assays using isolated yeast vacuoles have been established that measure the mixing of vacuole luminal compartments (22, 23). The fusion of isolated yeast vacuoles in vitro is mediated by evolutionarily conserved membrane fusion machinery, which involves not only SNARE proteins but also Rab and SM proteins. In this study, to address whether neuronal SNARE proteins are sufficient to induce biological membrane fusion, we engineered the budding yeast Saccharomyces cerevisiae to express neuronal SNARE proteins in vacuoles. Using these yeast vacuoles, we then show that neuronal SNARE proteins can induce vacuole fusion in a Rab- and SM protein-independent manner.     

CFTR chloride channels are regulated by a SNAP-23/syntaxin 1A complex

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate membrane fusion reactions in eukaryotic cells by assembling into complexes that link vesicle-associated SNAREs with SNAREs on target membranes (t-SNAREs). Many SNARE complexes contain two t-SNAREs that form a heterodimer, a putative intermediate in SNARE assembly. Individual t-SNAREs (e.g., syntaxin 1A) also regulate synaptic calcium channels and cystic fibrosis transmembrane conductance regulator (CFTR), the epithelial chloride channel that is defective in cystic fibrosis. Whether the regulation of ion channels by individual t-SNAREs is related to SNARE complex assembly and membrane fusion is unknown. Here we show that CFTR channels are coordinately regulated by two cognate t-SNAREs, SNAP-23 (synaptosome-associated protein of 23 kDa) and syntaxin 1A. SNAP-23 physically associates with CFTR by binding to its amino-terminal tail, a region that modulates channel gating. CFTR-mediated chloride currents are inhibited by introducing excess SNAP-23 into HT29-Cl.19A epithelial cells. Conversely, CFTR activity is stimulated by a SNAP-23 antibody that blocks the binding of this t-SNARE to the CFTR amino-terminal tail. The physical and functional interactions between SNAP-23 and CFTR depend on syntaxin 1A, which binds to both proteins. We conclude that CFTR channels are regulated by a t-SNARE complex that may tune CFTR activity to rates of membrane traffic in epithelial cells.     

A SNARE complex containing SGR3/AtVAM3 and ZIG/VTI11 in gravity-sensing cells is important for Arabidopsis shoot gravitropism

Plants can sense the direction of gravity and change the growth orientation of their organs. The molecular mechanisms of gravity sensing and signal transduction during gravitropism are not well known. We have isolated several shoot gravitropism (sgr) mutants of Arabidopsis. The sgr3-1 mutant exhibits a reduced gravitropic response in the inflorescence stems. In the inflorescence stems of Arabidopsis, gravity is sensed in endodermal cells that contain sedimentable amyloplasts. In sgr3-1, some amyloplasts in the endodermis failed to sediment in the direction of gravity. SGR3 encodes a syntaxin, AtVAM3, which had previously been cloned as a homologue of yeast Vam3p. AtVAM3 is localized to the prevacuolar compartment and vacuole and is suggested to function in vesicle transport to the vacuole. We have also cloned another soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE), ZIG/AtVTI11, a mutation that causes abnormal gravitropism. This mutant displayed an abnormal distribution of amyloplasts in the endodermal cells similar to that in sgr3-1. Endodermis-specific expression of SGR3 and ZIG by using the SCR promoter could complement the abnormal shoot gravitropism of each mutant. Protein-protein interaction between AtVAM3 and AtVTI11 in the endodermal cells was detected immunologically. The sgr3-1 mutation appeared to reduce the affinity of AtVAM3 for AtVTI11 or SYP5. These results suggest that vesicle transport to the prevacuolar compartment/vacuole in the endodermal cells, mediated by a specific SNARE complex containing AtVAM3 and AtVTI11, plays an important role in shoot gravitropism.     

Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate

The assembly of the three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure, little is known about the prefusion state of individual membrane-bound SNAREs and how they change conformation from the unzippered prefusion to the zippered postfusion state in a membrane environment. We have solved the solution NMR structure of micelle-bound syntaxin-1A in its prefusion conformation. In addition to the transmembrane helix, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the “0-layer” residue Gln226. This unexpected structural order of the N- and C-terminal halves of the uncomplexed SNARE motif suggests the formation of partially zippered SNARE complex intermediates, with the 0-layer serving as a proofreading site for correct SNARE assembly. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices of syntaxin interact with lipid bilayers and that association with the other target-membrane SNARE SNAP-25 lifts the SNARE motif off the membrane as a critical prerequisite for SNARE complex assembly and membrane fusion.Neurotransmitter release in eukaryotic cells requires the fusion of synaptic vesicles with the presynaptic plasma membrane. This fast and precise process is mediated by many proteins (1–3). The minimal neuronal exocytotic fusion machinery includes three soluble N-ethylmaleimide-sensitive (NSF) factor attachment protein receptor (SNARE) proteins: synaptobrevin-2 (syb) in the vesicle membrane and syntaxin-1A (syx) and SNAP-25 (SN25) in the plasma membrane (4). These proteins possess either one or two conserved SNARE motifs of 60–70 amino acids, which on SNARE assembly are capable of forming a four-stranded coiled-coil helical bundle (5). Folding, assumed to occur by N- to C-terminal zippering of these helices, is thought to be sufficient to merge the two opposing membranes. However, little is known about how the individual SNARE proteins change their conformations from the unzippered prefusion to the zippered postfusion states in membranes. Whereas SN25 and syb mostly consist of the SNARE motifs and, in the case of syb, a transmembrane (TM) domain, syx possesses two regulatory domains, a three-helix bundle (Habc) domain (6, 7) and a short N-terminal peptide (N-peptide) (8), in addition to its SNARE motif (H3 domain) and TM domain. Although neither of these additional domains are required in vitro for fast fusion per se (9, 10), the interaction between these domains and the SNARE motif in the presence of Munc18-1, Munc13, and other regulatory proteins is required upstream to “precondition” the SNAREs for efficient assembly and subsequent fusion. The Habc and H3 domains of syx are in dynamic equilibrium between “closed” and “open” conformations, which is regulated by Munc18-1 and Munc13, and which thereby controls synaptic vesicle fusion (11–14). Only the open, not the closed, conformation promotes formation of the SNARE complex, and thus membrane fusion.Although the three-helix Habc domain is well-ordered, the H3 domain is flexible in a soluble syx construct without its TM domain (15). Two different modes of binding of Munc18-1 to soluble syx have been observed. In a crystal structure of the syx-Munc18 complex, Munc18 locks syx in a “closed” conformation. The N-terminal half of the H3 domain forms an interrupted helix that is stabilized by interaction with Habc and Munc18 (16). Alternatively, Munc18 can just bind to the N-terminal peptide of syx (13, 17). Interestingly, the Munc18-regulated open/closed equilibrium of syx is shifted toward being more open in the membrane-bound and more closed in the soluble form of syx (18). In the soluble and the detergent-bound postfusion SNARE complex, the SNARE motifs form uninterrupted helices (19, 20). Moreover, the SNARE helices continue through the juxtamembrane linker regions to the TM domains in the latter structure (20). Because some structures and conformational equilibria are different for membrane-bound than for soluble SNAREs (18, 21), and as all previously reported high-resolution structures of prefusion syx have been obtained for their soluble portions only (6, 15, 16), it is of great interest to elucidate the structure of prefusion syx in a membrane-mimetic environment and to demonstrate its relevance in lipid bilayers. Here, we used solution NMR to study the structure and dynamics of syx in lipid micelles and fluorescence interference contrast (FLIC) microscopy to validate its conformation and activity in lipid bilayers.     

Role of the synaptobrevin C terminus in fusion pore formation

Neurotransmitter release is mediated by the SNARE proteins synaptobrevin II (sybII, also known as VAMP2), syntaxin, and SNAP-25, generating a force transfer to the membranes and inducing fusion pore formation. However, the molecular mechanism by which this force leads to opening of a fusion pore remains elusive. Here we show that the ability of sybII to support exocytosis is inhibited by addition of one or two residues to the sybII C terminus depending on their energy of transfer from water to the membrane interface, following a Boltzmann distribution. These results suggest that following stimulation, the SNARE complex pulls the C terminus of sybII deeper into the vesicle membrane. We propose that this movement disrupts the vesicular membrane continuity leading to fusion pore formation. In contrast to current models, the experiments suggest that fusion pore formation begins with molecular rearrangements at the intravesicular membrane leaflet and not between the apposed cytoplasmic leaflets.     

A yeast assay probes the interaction between botulinum neurotoxin serotype B and its SNARE substrate

The seven functionally distinct serotypes (A-G) of botulinum neurotoxin (BoNT) are dichains consisting of light chain (LC) with zinc-dependent endoprotease activity connected by one disulfide bond to heavy chain with neuronal-cell translocation and receptor-binding domains. LC-mediated proteolysis of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and consequent inhibition of synaptic vesicle fusion to the presynaptic membrane of human motor neurons are responsible for flaccid paralysis associated with botulism. LC endoproteolysis is complex, requiring highly extended SNARE sequences at the surface of intracellular membranes and prompting our development of a genetically amenable assay to monitor the interaction between BoNT/LC and its SNARE substrate. Using BoNT serotype B as a model, the assay employs a chimeric SNARE protein where a portion of neuronal synaptobrevin (Sb) is fused to Snc2p, a Sb ortholog required for protein secretion from yeast cells. Regulated expression of serotype B-LC in yeast leads to cleavage of the chimera and a conditional growth defect. To assess utility of this assay for monitoring SNARE protein cleavage, we growth-selected chimeric SNARE mutations that inhibited proteolysis. When these mutations were introduced into Sb and examined for cleavage, substrate residues located near and distal to the cleavage site were important, including residues positioned near the Sb transmembrane domain, an unexplored aspect of BoNT cell intoxication. Additional mutations were positioned in a nine-residue SNARE motif, supporting a previously assigned role for this motif in LC recognition and providing proof of principle for the application of yeast-based technology to study intracellular BoNT/LC endoproteases.     

Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation

The synaptic vesicle protein synaptobrevin engages with syntaxin and SNAP-25 to form the SNARE complex, which drives membrane fusion in neuronal exocytosis. In the SNARE complex, the SNARE motif of synaptobrevin forms a 55-residue helix, but it has been assumed to be mostly unstructured in its prefusion form. NMR data for full-length synaptobrevin in dodecylphosphocholine micelles reveals two transient helical segments flanked by natively disordered regions and a third more stable helix. Transient helix I comprises the most N-terminal part of the SNARE motif, transient helix II extends the SNARE motif into the juxtamembrane region, and the more stable helix III is the transmembrane domain. These helices may have important consequences for SNARE complex folding and fusion: helix I likely forms a nucleation site, the C-terminal disordered SNARE motif may act as a folding arrest signal, and helix II likely couples SNARE complex folding and fusion.     

Specific SNARE complex binding mode of the Sec1/Munc-18 protein, Sec1p

The Sec1/Munc-18 (SM) family of proteins is required for vesicle fusion in eukaryotic cells and has been linked to the membrane-fusion proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs). SM proteins may activate the target-membrane SNARE, syntaxin, for assembly into the fusogenic SNARE complex. In support of an activation role, SM proteins bind directly to their cognate syntaxins. An exception is the yeast Sec1p, which does not bind the yeast plasma-membrane syntaxin, Sso1p. This exception could be explained if the SM interaction motif were blocked by the highly stable closed conformation of Sso1p. We tested the possibility of a latent binding motif using sso1 mutants in yeast and reconstituted the Sec1p binding specificity observed in vivo with purified proteins in vitro. Our results indicate there is no latent binding motif in Sso1p. Instead, Sec1p binds specifically to the ternary SNARE complex, with no detectable binding to the binary t-SNARE complex or any of the three individual SNAREs in their uncomplexed forms. We propose that vesicle fusion requires a specific interaction between the SM protein and the ternary SNARE complex.