Synaptotagmin-1 Is an Antagonist for Munc18-1 in SNARE Zippering

Abstract

Background: The molecular mechanisms of the critical necessity of Munc18-1 protein for neurotransmitter release remain unclear. Results: Synaptotagmin-1 competes with Munc18-1 in SNARE zippering and fusion pore opening. Conclusion: Synaptotagmin-1 wins the tug-of-war in gaining control of the SNAREpin at the moment of membrane fusion. Significance: This work clarifies an ambiguity concerning the Munc18-1 function in neuroexocytosis.

SM protein Munc18-2 facilitates transition of Syntaxin 11-mediated lipid mixing to complete fusion for T-lymphocyte cytotoxicity

“…Order-of-addition experiments show that Munc18-2 accelerates the formation cdV8-resistant SNARE complexes, therefore supporting a role of Munc18-2 in proof-reading trans-SNARE complexes (52,(55)(56)(57). Additionally, our data also suggest that Munc18-2 facilitates SNAREpin zippering similarly to other SM proteins (43,54,(58)(59)(60)(61)(62)(63). Thus, although the GPI anchor may be less effective than the transmembrane domain in transducing the energy of SNARE assembly to drive membrane fusion, the increased number of assembled STX11-GPI SNARE complexes generated in the presence of Munc18-2 likely cooperate, giving rise to a higher energetically favorable state that could be sufficient to destabilize the phospholipid bilayers and result in complete membrane fusion.…”

“…These results suggest that the Munc18-2 R65Q mutant stabilizes the assembly of SNARE complexes at an intermediate state before lipid mixing and interferes with complete zippering of the SNARE complex. Therefore, the Munc18-2 R65Q mutant seems to dissociate two distinct functions attributed to SM proteins: the stimulatory effect on membrane fusion observed in ensemble lipid mixing assays (43,53,54) and the inhibitory function recently described in single molecule experiments (55). Order-of-addition experiments show that Munc18-2 accelerates the formation cdV8-resistant SNARE complexes, therefore supporting a role of Munc18-2 in proof-reading trans-SNARE complexes (52,(55)(56)(57).…”

Dramatic effect of levetiracetam in early-onset epileptic encephalopathy due to STXBP1 mutation

“…To our knowledge there are no data available on expression or function of SV2A in humans or rodents with STXBP1 mutations, but we speculate that modulation of SV2A by LEV may act counterbalancing the epileptogenic effects of haploinsufficiency of STXBP1 on neurotransmission. Indeed SV2A regulates synaptotagmin-1, that seems to act as an antagonist for Munc18-1 in SNARE zippering [10].…”

α-Synuclein may cross-bridge v-SNARE and acidic phospholipids to facilitate SNARE-dependent vesicle docking

“…Recombined proteins were expressed and purified as recently described [38,39]. Briefly, all recombinant proteins were expressed in Escherichia coli BL21 (DE3) by growing the cells to OD 600 of 0.6–0.8 at 37°C, and then the cells were induced with 0.5 mM IPTG (isopropyl β-D-1-thiogalactopyranoside) at 18°C for 16 h. The cells were harvested and resuspended in PBS or PBS with 0.2% (w/v) Triton X-100 (PBST) for membrane proteins, supplemented with 1 mM AEBSF [4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride].…”

“…For the t- and v-vesicles with 20% PS or without DOPS, PIP 2 was replaced with an equal amount of POPC, and equivalent amounts of POPC were removed or added to the lipid mixture. For the single-vesicle docking assay, the proteoliposome reconstitution was performed following the procedure described recently [38]. Briefly, the completely dried lipid film was hydrated with dialysis buffer (25 mM HEPES (pH 7.4) and 100 mM KCl).…”