Reconstitution of Ca
            <sup>2+</sup>
            -Regulated Membrane Fusion by Synaptotagmin and SNAREs

Tucker, Ward C.; Weber, Thomas; Chapman, Edwin R.

doi:10.1126/science.1097196

Cited by 354 publications

(438 citation statements)

References 22 publications

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“…However, recent experiments on fusion of synaptobrevin-containing liposomes are difficult to reconcile with the view of an intrinsic inactivation of the protein. Although fusion of synaptobrevin-containing liposomes with liposomes containing SNAP-25 and syntaxin is slow (Weber et al, 1998;Schuette et al, 2004;Tucker et al, 2004), the fusion rate is accelerated dramatically when the syntaxin/SNAP-25 binding site for synaptobrevin is stabilized (Pobbati et al, 2006). Thus it appears that the availability of the acceptor site rather than the intrinsic activity of membrane-anchored synaptobrevin is rate-limiting.…”

Section: Introductionmentioning

confidence: 91%

“…Although it is conceivable that differences in the protein and vesicle purification protocols may account for some of the differences (for instance, Hu et al used proteins purified by preparative denaturing SDS-PAGE for reconstitution), we have no obvious explanation for these discrepancies. Indeed, several laboratories reported that synaptobrevin-containing liposomes readily fuse with liposomes containing SNAP-25 and syntaxin, a reaction that clearly requires active synaptobrevin (Weber et al, 1998;Schuette et al, 2004;Tucker et al, 2004). Furthermore, clostridial neurotoxins readily cleave membrane-bound synaptobrevin both in liposomes and in synaptic vesicles, with toxin action requiring access to most of the cytoplasmic domain of synaptobrevin (Montecucco et al, 2005).…”

Section: Discussionmentioning

confidence: 99%

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Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion. INTRODUCTIONCommunication between neurons is mediated by neurotransmitters that are released from presynaptic nerve endings by Ca 2ϩ -dependent exocytosis of synaptic vesicles. Exocytotic fusion of the vesicle with the synaptic plasma membrane is mediated by the proteins synaptobrevin 2/VAMP2, SNAP-25, and syntaxin 1 (Jahn and Scheller, 2006;Rizo et al., 2006). These proteins are members of the SNARE protein family that are involved in all fusion events of the secretory pathway. SNAREs are characterized by stretches of 60-70 amino acids arranged in heptad repeats, termed SNARE motifs (Weimbs et al., 1997;Fasshauer et al., 1998b;Bock et al., 2001;Day et al., 2006). Syntaxin and synaptobrevin each contain a single SNARE motif that is located adjacent to a C-terminal transmembrane domain. In contrast, SNAP-25 contains two SNARE motifs connected by a palmitoylated linker region that serves as membrane anchor.Synaptobrevin resides in synaptic vesicles, whereas SNAP-25 and syntaxin 1 reside in the plasma membrane. The SNARE motifs of syntaxin, SNAP-25, and synaptobrevin readily assemble into quarternary bundles of ␣-helices (Fasshauer et al., 1997;Sutton et al., 1998). Assembly would thus lead to a tight connection between the membranes. According to this view, assembly is nucleated at the Nterminal ends of the SNARE-motifs and proceeds toward the C-terminal membrane anchor ("zippering"), resulting in a strained "trans"-complex . During membrane merger, the trans-complex would relax into a "cis"-complex in which the transmembrane domains are aligned in parallel. To regenerate the SNAREs for another round of fusion, SNARE complexes need to be disassembled by the AAAϩ-ATPase NEM-sensitive factor (NSF) in conjunction with cofactors termed soluble NSF attachment proteins (SNAPs; Sollner et al., 1993).Although the "zippering" hypothesis of SNARE function has received a lot of experimental support, it is still unclear...

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Section: Introductionmentioning

confidence: 91%

Section: Discussionmentioning

confidence: 99%

Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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“…10a) may enhance flippase activity resulting in an unfavorable local lipid environment to support fusion. CAPS, rabphilin, and Syt1 support exocytosis via interaction with PIP [53,65,79]. CAPS, a component of the synaptic vesicle priming machinery, maintains a fusion-competent vesicle pool for transmitter release [47].…”

Section: Roles For Polyphosphoinositides In Regulated Releasementioning

confidence: 99%

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Rogasevskaia

Coorssen

2011

J Chem Biol

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Studies using isolated sea urchin cortical vesicles have proven invaluable in dissecting mechanisms of Ca 2+ -triggered membrane fusion. However, only acute molecular manipulations are possible in vitro. Here, using selective pharmacological manipulations of sea urchin eggs ex vivo, we test the hypothesis that specific lipidic components of the membrane matrix selectively affect defined late stages of exocytosis, particularly the Ca 2+ -triggered steps of fast membrane fusion. Egg treatments with cholesterol-lowering drugs resulted in the inhibition of vesicle fusion. Exogenous cholesterol recovered fusion extent and efficiency in cholesterol-depleted membranes; α-tocopherol, a structurally dissimilar curvature analogue, selectively restored fusion extent. Inhibition of phospholipase C reduced vesicle phosphatidylethanolamine and suppressed both the extent and kinetics of fusion. Although phosphatidylinositol-3-kinase inhibition altered levels of polyphosphoinositide species and reduced all fusion parameters, sequestering polyphosphoinositides selectively inhibited fusion kinetics. Thus, cholesterol and phosphatidylethanolamine play direct roles in the fusion pathway, contributing negative curvature. Cholesterol also organizes the physiological fusion site, defining fusion efficiency. A selective influence of phosphatidylethanolamine on fusion kinetics sheds light on the local microdomain structure at the site of docking/fusion. Polyphosphoinositides have modulatory upstream roles in priming: alterations in specific polyphosphoinositides likely represent the terminal priming steps defining fully docked, release-ready vesicles. Thus, this pharmacological approach has the potential to be a robust high-throughput platform to identify molecular components of the physiological fusion machine critical to docking, priming, and triggered fusion.

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“…These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impressive specificity for cognate SNARE pairs (9) and can be directly promoted by other fusion-regulatory factors such as synaptotagmin and calcium (10) or Sec1p (11) under conditions that minimize lysis (12).…”

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confidence: 99%

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Fratti¹,

Collins²,

Hickey

et al. 2007

Journal of Biological Chemistry

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SNARE proteins form bundles of four ␣-helical SNARE domains with conserved polar amino acids, 3Q and 1R, at the "0-layer" of the bundle. Previous studies have confirmed the importance of 3Q⅐1R for fusion but have not shown whether it regulates SNARE complex assembly or the downstream functions of assembled SNAREs. Yeast vacuole fusion requires regulatory lipids (ergosterol, phosphoinositides, and diacylglycerol), the Rab Ypt7p, the Rab-effector complex HOPS, and 4 SNAREs: the Q-SNAREs Vti1p, Vam3p, and Vam7p and the R-SNARE Nyv1p. We now report that alterations in the 0-layer Gln or Arg residues of Vam7p or Nyv1p, respectively, strongly inhibit fusion. Vacuoles with wild-type Nyv1p show exquisite discrimination for the wild-type Vam7p over Vam7 Q283R , yet Vam7 Q283R is preferred by vacuoles with Nyv1 R191Q . Rotation of the position of the arginine in the 0-layer increases the K m for Vam7p but does not affect the maximal rate of fusion. Vam7 Q283R forms stable 2Q⅐2R complexes that do not promote fusion. However, fusion is restored by the lipophilic amphiphile chlorpromazine or by the phospholipase C inhibitor U73122, perturbants of the lipid phase of the membrane. Thus, SNARE function as regulated by the 0-layer is intimately coupled to the lipids, which must rearrange for fusion. SNARE 4 proteins (1) are vital for membrane fusion. Initially discovered in neuronal tissues, SNAREs are found on all organelles of the exocytic and endocytic pathways, from yeast to humans. Their characteristic feature is a heptad-repeat "SNARE domain," flanked by varied N-domains and by C-terminal membrane anchors, either a single membrane-spanning apolar polypeptide or an acyl anchor. SNAREs form 4-helical complexes through their SNARE domains (2). Although the residues in each SNARE that face the others in a 4-helical complex are generally apolar, 3 glutamine (Q) and 1 arginine (R) near the center of the 4-complexed SNARE domains form a conserved and polar "0-layer" (3). Recombinant neuronal SNAREs spontaneously assemble into stable bundles, yet SNARE complex assembly in vivo requires SNARE-binding proteins of the Sec1-Munc18 "SM" family. SNARE complexes are disassembled by two chaperones: Sec17p/␣-SNAP, which binds directly to SNARE complexes, and Sec18p/NSF, which binds to Sec17p/␣-SNAP and couples the energy of ATP binding and hydrolysis to SNARE complex disassembly (4). SNARE complexes form in cis, with each SNARE anchored to the same membrane, or in trans, with SNAREs anchored to apposed, "tethered" membranes.SNARE function has been studied in vivo, on isolated organelles, and through the reconstitution of recombinant SNAREs into proteoliposomes. When several SNAREs, which are found in vivo on a "target" membrane, are co-reconstituted into one population of liposomes, they form a t-SNARE complex. These liposomes can selectively interact with other liposomes bearing a reconstituted "vesicle," or v-, SNARE to allow selective lipid mixing (5), vesicle content mixing (6), or lysis (7,8). This reconstituted reaction shows impress...

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Reconstitution of Ca ²⁺ -Regulated Membrane Fusion by Synaptotagmin and SNAREs

Cited by 354 publications

References 22 publications

Determinants of Synaptobrevin Regulation in Membranes

Determinants of Synaptobrevin Regulation in Membranes

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

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Reconstitution of Ca 2+ -Regulated Membrane Fusion by Synaptotagmin and SNAREs

Cited by 354 publications

References 22 publications

Determinants of Synaptobrevin Regulation in Membranes

Determinants of Synaptobrevin Regulation in Membranes

A new approach to the molecular analysis of docking, priming, and regulated membrane fusion

Stringent 3Q·1R Composition of the SNARE 0-Layer Can Be Bypassed for Fusion by Compensatory SNARE Mutation or by Lipid Bilayer Modification

Contact Info

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Reconstitution of Ca ²⁺ -Regulated Membrane Fusion by Synaptotagmin and SNAREs