Membrane-Proximal Tryptophans of Synaptobrevin II Stabilize Priming of Secretory Vesicles

Borisovska, Maria; Schwarz, Yvonne; Dhara, Madhurima; Yarzagaray, Antonio; Hugo, Sandra; Narzi, Daniele; Si, Yain-Whar; Kesavan, Jaideep; Mohrmann, Ralf; Böckmann, Rainer A.; Bruns, Dieter

doi:10.1523/jneurosci.6282-11.2012

Cited by 30 publications

(32 citation statements)

References 67 publications

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“…The most parsimonious explanation for this part of our data is that fusion per se only requires a loose coupling of SNARE-complex assembly to membranes, but that evoked fusion requires a tight coupling of SNARE-complex assembly to membranes because evoked fusion operates on a partly pre-assembled, activated state that is then the substrate of the fusogenic stimulus (Südhof, 1995). The notion of such an activated state involving a tight coupling of SNARE-complex assembly to the membrane is also supported by the dramatic effects of mutations in juxtamembranous residues in synaptobrevin-2 which increase spontaneous fusion but impair evoked fusion (Maximov et al, 2009; Borisovska et al, 2012). …”

Section: Discussionmentioning

confidence: 99%

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

Zhou

Bacaj

Yang

et al. 2013

Neuron

106

107

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SUMMARY Synaptic vesicle fusion during neurotransmitter release is mediated by assembly of SNARE- and SM-protein complexes composed of syntaxin-1, SNAP-25, synaptobrevin-2/VAMP2, and Munc18-1. Current models suggest that SNARE-complex assembly catalyzes membrane fusion by pulling the transmembrane regions (TMRs) of SNARE proteins together, thus allowing their TMRs to form a fusion pore. These models are consistent with the requirement for TMRs in viral fusion proteins. However, the role of the SNARE TMRs in synaptic vesicle fusion has not yet been tested physiologically. Here, we examined whether synaptic SNAREs require TMRs for catalysis of synaptic vesicle fusion, which was monitored electrophysiologically at millisecond time resolution. Surprisingly, we find that both lipid-anchored syntaxin-1 and lipid-anchored synaptobrevin-2 lacking TMRs efficiently promoted spontaneous and Ca2+-triggered membrane fusion. Our data suggest that SNARE proteins function during fusion primarily as force generators, consistent with the notion that forcing lipid membranes close together suffices to induce membrane fusion.

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Section: Discussionmentioning

confidence: 99%

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

Zhou

Bacaj

Yang

et al. 2013

Neuron

106

107

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“…The TMD may contribute to the availability of the SNARE motif and the manner in which the SNARE motif is presented. This could happen if the TMD influenced the conformation of the rest of the protein or if the TMD controlled membrane insertion of the juxtamembrane region (30), which has been shown to influence exocytosis (31). Microlocalization and dimerization are additional possible functional roles for the TMD, although the functional relevance of dimerization has been difficult to establish (32).…”

Section: Discussionmentioning

confidence: 99%

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

Chang¹,

Chiang

Gaffaney

et al. 2016

Journal of Biological Chemistry

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SNARE proteins catalyze many forms of biological membrane fusion, including Ca2؉ -triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca 2؉ -triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca 2؉ -synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-␣, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-␣ may reflect a slower alternative mode of SNARE-mediated membrane fusion.Nearly all biological fusion mediated by SNARE proteins employs at least one t-SNARE and one v-SNARE anchored to the membrane by a transmembrane domain (TMD) 3 (1-3). Ca 2ϩ -triggered exocytosis of neurotransmitter and hormone from many cells requires the vesicle-SNARE synaptobrevin 2 (syb2) and plasma membrane-SNARE syntaxin, both of which have a TMD (4, 5). Mutations in the TMD alter flux through exocytotic fusion pores in a manner consistent with structural models of fusion pores formed by the TMDs of syntaxin (6, 7) and syb2 (8). Yeast t-and v-SNAREs anchored to membranes by geranylgeranyl moieties instead of a TMD support docking but not fusion (9). SNAREs anchored to membranes by lipid instead of a TMD support lipid mixing of liposomes only when the lipid moiety is long enough to span a lipid bilayer or multiple lipid moieties are present (10) or an accessory protein is present (11).Although early work supported a role of SNARE TMDs in membrane fusion, this issue has become controversial. Recent work on fusion of yeast vacuoles suggests that some SNAREs with lipid anchors can support lipid mixing, content mixing, or both, but other SNAREs cannot function when their TMD is replaced by a lipid anchor (12). Lipid-anchored forms of syntaxin and syb2 were reported to rescue neurotransmitter release at synapses as effectively as wild type ...

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“…These results are consistent with the hypothesis that fusion is facilitated by a movement of the syb2 TM domain. In another study using chromaffin cells, a marked reduction in rapid release following stimulation was also observed, but an increase in spontaneous events was not detected (4). This apparent discrepancy is still to be resolved.…”

Section: The Role Of Snare Protein Tm Domains In Fusionmentioning

confidence: 93%

How Could SNARE Proteins Open a Fusion Pore?

Fang

Lindau

2014

Physiology

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The SNARE (Soluble NSF Attachment protein REceptor) complex, which in mammalian neurosecretory cells is composed of the proteins synaptobrevin 2 (also called VAMP2), syntaxin, and SNAP-25, plays a key role in vesicle fusion.In this review, we discuss the hypothesis that, in neurosecretory cells, fusion pore formation is directly accomplished by a conformational change in the SNARE complex via movement of the transmembrane domains.Qinghua Fang 1,2 and Manfred Lindau (49) has three components. The v-SNARE synaptobrevin 2 (syb2 also called VAMP2) is a 116-amino acid protein anchored in the vesicle membrane by a single transmembrane (TM) domain. Syntaxin (stx) is correspondingly anchored in the plasma membrane via a single TM helix. The third component, SNAP-25, has lipid anchors in the plasma membrane. SNAP-25 and stx are called t-SNAREs, being in the target membrane for fusion of secretory vesicles. The key importance of these proteins in the fusion mechanism has been demonstrated by the finding that proteolytic cleavage of the SNARE proteins by specific neurotoxins results in strong inhibition of transmitter release in neurons as well as in chromaffin cells (42,44). One example for the medical relevance of the SNARE complex is the BoTox treatment, which exerts its function through inhibition of transmitter release by specific cleavage of the SNARE protein SNAP-25. The cytosolic so-called SNARE domains of these three proteins form a coiled coil, which incorporates one helix each from syb2 and stx and two helices (named SN1 and SN2) from SNAP-25. The coiled coil structure of the SNARE domains has been solved by X-ray crystallography several years ago (57). Based on this structure, the configuration shown in FIGURE 1A was proposed for a trans configuration, in which a SNARE complex links the vesicle membrane and plasma membrane. The arrows indicate the cleavage sites for the neurotoxins mentioned above. More recently, a crystal structure of the SNARE complex, including the syb2 and stx TM helices, was solved (56), which shows helical extension from the SNARE domains through the linkers into the TM domains (FIGURE 1B). This structure is thought to resemble a post-fusion state in which all components of the SNARE complex are in a cis configuration, located in the same fused membrane.Synaptic vesicles contain ϳ70 copies of syb2 (59), but it is unknown and controversial how many of these copies are acting together in the formation of a fusion pore (43). Interestingly, neurosecretory vesicles in PC 12 cells recruit t-SNARE clusters containing a similar number of stx and SNAP-25 molecules (26). It has been shown that in reconstituted systems a single SNARE complex is sufficient to promote lipid vesicle fusion (50, 62). However, at least three SNARE complexes appear to be required for fast membrane fusion kinetics (8,17,41,50). In cultured hippocampal neurons, two copies of syb2 were found to be sufficient for fusion, but the experiments were performed with low time resolution (52). The Triggering MechanismTransmitter rel...

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Membrane-Proximal Tryptophans of Synaptobrevin II Stabilize Priming of Secretory Vesicles

Cited by 30 publications

References 67 publications

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

Lipid-Anchored SNAREs Lacking Transmembrane Regions Fully Support Membrane Fusion during Neurotransmitter Release

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

How Could SNARE Proteins Open a Fusion Pore?

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