Exocytotic pore in a SNARE

In 2013, the Nobel prize was awarded for discoveries related to the regulation of the cellular transport system (https://www.nobelprize.org/nobel_prizes/medicine/ laureates/2013/). In addition to studies by Südhof’s lab of signals that tell secretory vesicles when to release their cargo and the work of Schekman’s lab describing a set of genes required for vesicle transport, the Rothman’s lab determined the proteins termed SNAREs (soluble NSFattachment protein receptors), that allow vesicles to fuse with their targets and thus transfer materials. Although the three interacting SNARE proteins (vesicle-associated membrane protein [VAMP]/synaptobrevin, synaptosomeassociated protein of 25 kDa [SNAP-25] and syntaxin) had previously been identified by several scientists, and were localized to the presynaptic region, their function was largely unknown. VAMP/synaptobrevin was found to reside on the vesicle, whereas SNAP-25 and syntaxin were found at the plasmalemma. This led to the SNARE hypothesis, which stipulated that target and vesicle SNAREs were critical for vesicle fusion through a set of sequential steps of synaptic docking, activation and fusion of vesicle with the plasmalemma. This interpretation was influenced by bulk biochemical studies, which revealed that the ternary SNARE complex is a thermally stable structure [1], meaning that once the ternary SNARE complex is formed, its disassembly may take a long time, unless special enzymes are in action [2]. Moreover, these studies also implied that SNARE complex formation is associated with the vesicle membrane merger with the plasmalemma, enabling the vesicle cargo to be released in an all-or-none fashion, as originally considered by B. Katz. However, studies using atomic force spectroscopy of single molecule interactions revealed the disassembly properties of the ternary SNARE complex are occurring in the time domain of 0.2-2 s [3]. Therefore, assembly/ disassembly of the ternary SNARE complex, influenced by SNARE accessory proteins, may take place not only at vesicle docking, facilitating the vesicle membrane merger with the plasmalemma, but also at other exocytotic stage intermediates (Figure 1), such as transient fusion pore widening and fusion pore dwell-time regulation, leading to full-fusion, a complete integration of vesicle membrane into the plasmalemma [4]. When the function of the accessory SNARE protein Munc18-1 was studied at the level of a single vesicle by the high-resolution capacitance technique, an ideal approach to study the membrane merger between a single vesicle and the plasmalemma [5], it was revealed that there may be multiple sites where the SNARE complex may play a role along the exocytotic intermediates [6]. In this study, Munc18-1 mutants were transfected into secretory cells to affect the interaction of Munc18-1 with syntaxin1 (Synt1) (R39C), Rab3A (E466K), and Mint proteins (P242S). In comparison with wild-type Munc18-1, mutant Munc18-1E466K increased the frequency of the unitary fusion events, consistent with the view that Rab3A protein facilitates vesicle docking at the plasmalemma. While the other Munc18-1 mutants (R39C and P242S) increased the fusion pore dwell-time, all the mutants stabilized a narrow fusion pore geometry, preventing transitions into a more widely or fully open one. Single-molecule atomic force microscopy experiments revealed that wild-type Munc181, but not Munc18-1R39C, abrogates the interaction between synaptobrevin2 (Syb2) and Synt1 binary trans complexes [6]. Importantly, neither form of Munc181 affected the interaction of Syb2 with the preformed binary cis-Synt1-SNAP25 complexes, revealing that Munc18-1 performs a proofing function by inhibiting tethering of Syb2-containing vesicles solely to Synt1 at the plasmalemma and promoting vesicular tethering via Syb2 to the preformed binary cis complex of Synt1-SNAP25 (Figure 1A). The last transition prior to full-fusion is inhibited by the dominant-negative domain of synaptobrevin 2 protein peptide (dnSNARE, Figure 1B). This peptide Editorial