Transmembrane-domain determinants for SNARE-mediated membrane fusion

Fdez, Elena; Martínez-Salvador, M.; Beard, Matthew; Woodman, Philip; Hilfiker, Sabine

doi:10.1242/jcs.061325

Cited by 46 publications

(55 citation statements)

References 63 publications

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“…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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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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“…Similarly, replacement of the Vam3 TMD by a prenyl anchor reduced fusion activity of vacuoles (16). Dimerization of the TMDs of the R-SNARE Vamp2 was observed in intact cells, but it was shown not to have an important functional role (26). C-terminal extension of the Vamp2 TMD inhibited exocytosis, leading to the proposal that the TMD contributes to the resolution of the hemifusion intermediate by being pulled into the inner membrane leaflet (51).…”

Section: Discussionmentioning

confidence: 99%

“…Dimerization of R-SNAREs through their TMDs was observed in intact cells, but it was shown not to have an important functional role (26,27). Additionally, mutations in the transmembrane helix of the SNARE Vam3 were shown to reduce the ability of the full-length protein to induce content mixing to different extents.…”

mentioning

confidence: 99%

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

Pieren

Desfougères

Michaillat

et al. 2015

Journal of Biological Chemistry

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Background: Contribution of SNARE transmembrane domains (TMDs) to membrane fusion is unclear. Results: Exchange of yeast vacuolar SNARE TMDs against unrelated TMDs yielded normal fusion activities; lipid anchors do not support fusion pore opening. Conclusion: SNARE TMDs serve as nonspecific membrane anchors in vacuole fusion but they need to be proteinaceous. Significance: SNARE TMDs participate in fusion pore opening.

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“…31,48 The dominant-negative approach obtained through expressing a soluble form of VAMP2 corroborated that this isoform was implicated in Ig secretion. These truncated mutants are known to act as selective inhibitors of exocytosis, presumably by competing with endogenous proteins in the formation of SNARE complexes.…”

Section: Discussionmentioning

confidence: 94%

“…8 To further support the involvement of VAMP2 in the antibody secretion process and to alleviate the possible protein redundancy effect after siRNA or TeNT-LC approaches, we decided to perform a dominant-negative assay. 31,32 Thus, we constructed a wild-type VAMP2 (wtVAMP2) and VAMP2 protein lacking the TMD (VAMP2-ΔTMD), bearing both monomer red fluorescent protein, Ruby, fused at their carboxyl-terminus. Ruby-only-expressing vector and wtVAMP2-Ruby and VAMP2-ΔTMD-Ruby-fused proteinexpressing vectors were transfected into U266 cells.…”

Section: Co-localized In Vesicles Even In Different Cell Lines Produmentioning

confidence: 99%

VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

et al. 2016

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The production and secretion of antibodies by human plasma cells (PCs) are two essential processes of humoral immunity. The secretion process relies on a group of proteins known as soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), which are located in the plasma membrane (t-SNAREs) and in the antibody-carrying vesicle membrane (v-SNARE), and mediate the fusion of both membranes. We have previously shown that SNAP23 and STX4 are the t-SNAREs responsible for antibody secretion. Here, using human PCs and antibody-secreting cell lines, we studied and characterized the expression and subcellular distribution of vesicle associated membrane protein (VAMP) isoforms, demonstrating that all isoforms (with the exception of VAMP1) are expressed by the referenced cells. Furthermore, the functional role in antibody secretion of each expressed VAMP isoform was tested using siRNA. Our results show that VAMP2 may be the v-SNARE involved in vesicular antibody release. To further support this conclusion, we used tetanus toxin light chain to cleave VAMP2, conducted experiments to verify co-localization of VAMP2 in antibody-carrying vesicles, and demonstrated the coimmunoprecipitation of VAMP2 with STX4 and SNAP23 and the in situ interaction of VAMP2 with STX4. Taken together, these findings implicate VAMP2 as the main VAMP isoform functionally involved in antibody secretion.

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Transmembrane-domain determinants for SNARE-mediated membrane fusion

Cited by 46 publications

References 63 publications

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

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

Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing

VAMP2 is implicated in the secretion of antibodies by human plasma cells and can be replaced by other synaptobrevins

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