Determinants of Synaptobrevin Regulation in Membranes

Abstract

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...

A Role for Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Dimerization during Neurosecretion

“…Although detailed electrophysiological experiments will be needed to determine the precise role of SNARE complex dimers in membrane fusion events, one can speculate from their solution structure how they might contribute toward formation of an oligomeric complex around a fusion pore, consistent with the energies required to bring about membrane fusion (Li et al, 2007). It is interesting to note that the syb2(W89A,W90A) mutant does not seem to be defective in mediating liposomal fusion events (Siddiqui et al, 2007), whereas it profoundly inhibits Ca 2ϩ -dependent secretion. Thus, secretory defects using the syb2 dimerization mutants only seem to be evident in cell systems requiring cooperativity between SNARE complexes.…”

Investigation of the juxtamembrane region of neuronal-Synaptobrevin in synaptic transmission at theDrosophilaneuromuscular junction

“…This is not the case, as n-syb AA is identical to n-syb WT during the first 10 stimuli and actually demonstrates greater depression than n-syb WT during the first 90 s. Therefore it is safe to conclude that the synaptic deficits observed in n-syb AA result from a mechanistic alteration of vesicle fusion rather than simply not enough n-Syb. (Kweon et al 2003) or no effect (Siddiqui et al 2007) using VAMP2 with W89S/ W90S mutations. These results are contrasted by an observed decrease in the exocytotic burst phase from chromaffin cells expressing W89S/W90S mutations (Borisovska et al 2012).…”

“…In an NMR study, we observed reduced membrane anchoring with mutation of tryptophan to alanine in Drosophila neuronal-Synaptobrevin (n-Syb) (Al-Abdul-Wahid et al 2012). Prior studies of vesicle fusion with Syb mutant residues have produced mixed results, such as decreased secretion from PC-12 cells (Quetglas et al 2002), increased liposome fusion (Kweon et al 2003), or no effect on liposome fusion (Siddiqui et al 2007). However, neural models of Ca 2ϩ -evoked transmitter release have demonstrated reducedamplitude evoked potentials and increased miniature frequency in cultured mouse pyramidal neurons (Maximov et al 2009) and reduced vesicle priming inhibiting the exocytotic burst phase in cultured chromaffin cells (Borisovska et al 2012) when tryptophan residues are modified.…”

A Fast Mode of Membrane Fusion Dependent on Tight SNARE Zippering

“…On zippering of the complex, the membrane-proximal region of SNAREs could thus bend or destabilize the membrane. This may account for the severe reduction of membrane fusion that results from mutations or deletion of the C-terminal hydrophobic layers of SNAREs (Sorensen et al, 2006;Siddiqui et al, 2007). Insertion of a hydrophilic linker in Syb2 could perturb the insertion of the membrane-proximal region into the phospholipid bilayer and thus force-transmission to the membrane (Baaden, personal communication).…”