Conformation of the synaptobrevin transmembrane domain

Bowen, Mark E.; Brunger, Axel T.

doi:10.1073/pnas.0602644103

Cited by 71 publications

(114 citation statements)

References 69 publications

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“…It should be noted that our approach did not allow us to directly assess the conformation of the membrane proximal region of synaptobrevin. A recent study, however, has shown that the transmembrane region of synaptobrevin is tilted at an angle, though it is noteworthy that this study did not propose an interaction of the membrane-proximal region with the membrane (Bowen and Brunger, 2006).…”

Section: Discussionmentioning

confidence: 42%

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: Discussionmentioning

confidence: 42%

Determinants of Synaptobrevin Regulation in Membranes

Siddiqui

Vites

Stein³

et al. 2007

MBoC

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“…It seems likely that these residues are transiently forced into close proximity at the point of membrane fusion (Tong et al, 2009), but our BiFC analysis might not be able to resolve such interactions on the background of fluorescence from individual, non-complexed VAMP2 molecules interacting via their TMDs. The predicted TMD of VAMP2 is unusually large, and is embedded in the membrane with a tilt with respect to the bilayer normal (Bowen and Brunger, 2006). Thus, the TMD-mediated interaction of VAMP2 molecules identified here by BiFC probably involves a conformation whereby glycine residues oriented toward the helix-helix interface contribute to the packing of two tilted helices; however, additional transient conformations close to the point of membrane fusion might also exist.…”

Section: Discussionmentioning

confidence: 86%

Transmembrane-domain determinants for SNARE-mediated membrane fusion

Fdez

Martínez-Salvador

Beard

et al. 2010

Journal of Cell Science

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SummaryNeurosecretion involves fusion of vesicles with the plasma membrane. Such membrane fusion is mediated by the SNARE complex, which is composed of the vesicle-associated protein synaptobrevin (VAMP2), and the plasma membrane proteins syntaxin-1A and SNAP-25. Although clearly important at the point of membrane fusion, the precise structural and functional requirements for the transmembrane domains (TMDs) of SNAREs in bringing about neurosecretion remain largely unknown. Here, we used a bimolecular fluorescence complementation (BiFC) approach to study SNARE protein interactions involving TMDs in vivo. VAMP2 molecules were found to dimerise through their TMDs in intact cells. Dimerisation was abolished when replacing a glycine residue in the centre of the TMD with residues of increasing molecular volume. However, such mutations still were fully competent in bringing about membrane-fusion events, suggesting that dimerisation of the VAMP2 TMDs does not have an important functional role. By contrast, a series of deletion or insertion mutants in the C-terminal half of the TMD were largely deficient in supporting neurosecretion, whereas mutations in the N-terminal half did not display severe secretory deficits. Thus, structural length requirements, largely confined to the C-terminal half of the VAMP2 TMD, seem to be essential for SNARE-mediated membrane-fusion events in cells.

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“…But beyond simple localization, tryptophan residues have been hypothesized to play a direct role in facilitating membrane fusion through anchoring, lipid mixing (Hu et al 2002;Jahn and Sudhof 1999;Sutton et al 1998), and imparting an angle to the transmembrane domain (Bowen and Brunger 2006). Interestingly, viral fusion proteins, which must overcome the same fundamental biophysical challenges as SNARE proteins, often have conserved tryptophan residues localized at the membrane interface.…”

Section: Discussionmentioning

confidence: 99%