Neuronal SNARE proteins mediate neurotransmitter release at the synapse by facilitating the fusion of vesicles to the presynaptic plasma membrane. Cognate v-SNAREs and t-SNAREs from the vesicle and the plasma membrane, respectively, zip up and bring about the apposition of two membranes attached at the Cterminal ends. Here, we demonstrate that SNARE zippering can be modulated in the midways by wedging with small hydrophobic molecules. Myricetin, which intercalated into the hydrophobic inner core near the middle of the SNARE complex, stopped SNARE zippering in motion and accumulated the trans-complex, where the N-terminal region of v-SNARE VAMP2 is in the coiled coil with the frayed C-terminal region. Delphinidin and cyanidin inhibited N-terminal nucleation of SNARE zippering. Neuronal SNARE complex in PC12 cells showed the same pattern of vulnerability to small hydrophobic molecules. We propose that the half-zipped trans-SNARE complex is a crucial intermediate waiting for a calcium trigger that leads to fusion pore opening.polyphenol | hemifusion | neurotransmission | neuron N eurotransmitter release at the synapse, which serves as the brain's major form of cell-cell communication, requires the fusion of synaptic vesicles with the presynaptic plasma membrane. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins mediate this synaptic fusion event (1-5), and the formation of a four-helical bundle (6-8) is believed to generate the force required for fusion. A zipper model has been proposed for SNARE complex formation, initiating assembly at the N-terminal region and zipping toward the C-terminal membrane-proximal region (6-9). To account for fast neuroexocytosis, the SNAREs in primed readily releasable vesicles have been proposed as being partially zipped in the trans-configuration bridging the two membranes.Although the structure of the fully assembled cis-SNARE complex, which is believed to represent the postfusion state, has been determined (10), the structure of the trans-complex is poorly understood and is purely imaginary, most likely because of its inherently transient nature. Precisely linking the degrees of SNARE zippering to specific stages of membrane fusion seems to be prerequisite for determining the structure of the trans-complex and for providing answers to the questions of how fast fusion is controlled in neurons and how the trans-complexes set up the readily releasable vesicles with other regulatory proteins.Here, we show that certain small hydrophobic molecules (SHM) enable layer-by-layer control of SNARE zippering by wedging into various points of the SNARE zipper. SNAREmediated membrane fusion is dissected via this wedge-like action of SHMs. Analysis of the captured replication fork-like structure allowed us to understand the basic architecture of the putative trans-complex. Results SNARE-Driven Membrane Fusion Can Be Controlled by SHMs withDifferent Modes of Action. As an initial step to examine the feasibility of whether SHM works as a wedge for the SNARE zipp...

SNAREpins must be formed between two membranes to allow vesicle fusion, a required process for neurotransmitter release. Although its post-fusion structure has been well characterized pre-fusion conformations have been elusive. We used single molecule FRET and EPR to investigate the SNAREpin assembled between two nanodisc membranes. The SNAREpin shows at least three distinct dynamic states, which might represent pre-fusion intermediates. While the N-terminal half above the conserved ionic layer maintains a robust helical bundle structure the membrane-proximal C-terminal half shows either high FRET representing a helical bundle (45%), low FRET reflecting a frayed conformation (39%), or mid FRET revealing an yet unidentified structure (16%). It is generally thought that SNAREpins are trapped at a partially zipped conformation in the pre-fusion state, and complete SNARE assembly happens concomitantly with membrane fusion. However, our results show that the complete SNARE complex can be formed without membrane fusion, which suggests that the complete SNAREpin formation could precede membrane fusion, providing an ideal access to the fusion regulators such as complexins and synaptotagmin 1.

Membrane fusion is mediated by the SNARE complex which is formed through a zippering process. Here, we developed a chemical controller for the progress of membrane fusion. A hemifusion state was arrested by a polyphenol myricetin which binds to the SNARE complex. The arrest of membrane fusion was rescued by an enzyme laccase that removes myricetin from the SNARE complex. The rescued hemifusion state was metastable and long-lived with a decay constant of 39 min. This membrane fusion controller was applied to delineate how Ca2+ stimulates fusion-pore formation in a millisecond timescale. We found, using a single-vesicle fusion assay, that such myricetin-primed vesicles with synaptotagmin 1 respond synchronously to physiological concentrations of Ca2+. When 10 µM Ca2+ was added to the hemifused vesicles, the majority of vesicles rapidly advanced to fusion pores with a time constant of 16.2 ms. Thus, the results demonstrate that a minimal exocytotic membrane fusion machinery composed of SNAREs and synaptotagmin 1 is capable of driving membrane fusion in a millisecond time scale when a proper vesicle priming is established. The chemical controller of SNARE-driven membrane fusion should serve as a versatile tool for investigating the differential roles of various synaptic proteins in discrete fusion steps.

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.

In neuronal exocytosis, SNARE assembly into a stable four-helix bundle drives membrane fusion. Previous studies have revealed that the SM protein Munc18-1 plays a critical role for precise SNARE assembly with the help of Munc13-1, but the underlying mechanism remains unclear. Here, we used single-molecule FRET assays with a nanodisc membrane reconstitution system to investigate the conformational dynamics of SNARE/Munc18-1 complexes in multiple intermediate steps towards the SNARE complex. We found that single Munc18-1 proteins induce the closed conformation of syntaxin-1 not only in the free syntaxin-1 but also in the t-SNARE (syntaxin-1/SNAP-25) complex. These results implicate that Munc18-1 may act as a gatekeeper for both binary and ternary SNARE complex formation by locking the syntaxin-1 in a cleft of Munc18-1. Furthermore, the kinetic analysis of the opening/closing transition reveals that the closed syntaxin-1 in the syntaxin-1/SNAP-25/ Munc18-1 complex is less stable than that in the closed syntaxin-1/Munc18-1 complex, which is manifested by the infrequent closing transition, indicating that the conformational equilibrium of the ternary complex is biased toward the open conformation of syntaxin-1 compared with the binary complex. Neuronal exocytosis for neurotransmitter release is driven by the assembly of the three soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, which are syntaxin-1 and SNAP-25 in the presynaptic plasma membrane and synaptobrevin in the synaptic vesicle membrane 1,2. The assembly of these proteins generates a stable four-helix bundle between the vesicle and target membranes, thus promoting membrane fusion 3,4. Although the membrane fusion can be induced by the SNAREs alone in vitro, a number of auxiliary proteins are required for membrane fusion with high speed and high fidelity in vivo 5,6. Among those, the Sec1/Munc18 (SM) family proteins Munc18-1 and Munc13-1 are known to be essential for SNARE-mediated membrane fusion 7-9. Extensive studies on roles of the Munc18-1 and Munc13-1 in the membrane fusion have established that these proteins are critically involved in the regulation of SNARE complex formation. Initially, Munc18-1 induces the closed conformation of syntaxin-1, locking the syntaxin-1 protein in a cleft of Munc18-1, that inhibits the spontaneous binding of SNAP-25 to syntaxin-1 10,11. Recent reports have revealed that the MUN domain of Munc13-1 promotes the transition from the syntaxin-1/Munc18-1 complex to the ternary SNARE complex in the presence of SNAP-25 and synaptobrevin, suggesting that Munc13-1 plays a role in opening syntaxin-1 for the subsequent SNARE assembly 12-15. On the other hand, it is also known that Munc18-1 stimulates membrane fusion when it binds to a fully assembled SNARE complex 16-18. Despite such major advances, many important questions concerning the mechanisms underlying the precise regulation of SNARE complex formation by the Munc18-1 and Munc13-1 still remain unanswered. For instance,