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Munc13-1 is a large multifunctional protein essential for synaptic vesicle fusion and neurotransmitter release. Its dysfunction has been linked to many neurological disorders. Evidence suggests that the MUN domain of Munc13-1 collaborates with Munc18-1 to initiate SNARE assembly, thereby priming vesicles for fast calcium-triggered vesicle fusion. The underlying molecular mechanism, however, is poorly understood. Recently, it was found that Munc18-1 catalyzes neuronal SNARE assembly through an obligate template complex intermediate containing Munc18-1 and 2 SNARE proteins—syntaxin 1 and VAMP2. Here, using single-molecule force spectroscopy, we discovered that the MUN domain of Munc13-1 stabilizes the template complex by ∼2.1 kBT. The MUN-bound template complex enhances SNAP-25 binding to the templated SNAREs and subsequent full SNARE assembly. Mutational studies suggest that the MUN-bound template complex is functionally important for SNARE assembly and neurotransmitter release. Taken together, our observations provide a potential molecular mechanism by which Munc13-1 and Munc18-1 cooperatively chaperone SNARE folding and assembly, thereby regulating synaptic vesicle fusion.

Sec1/Munc18-family (SM) proteins are required for SNARE-mediated membrane fusion, but their mechanism(s) of action remain controversial. Using single-molecule force spectroscopy, we found that the SM protein Munc18-1 catalyzes step-wise zippering of three synaptic SNAREs (syntaxin, VAMP2, and SNAP-25) into a four-helix bundle. Catalysis requires formation of an intermediate template complex in which Munc18-1 juxtaposes the N-terminal regions of the SNARE motifs of syntaxin and VAMP2, while keeping their C-terminal regions separated. SNAP-25 binds the templated SNAREs to induce full SNARE zippering. Munc18-1 mutations modulate the stability of the template complex in a manner consistent with their effects on membrane fusion, indicating that chaperoned SNARE assembly is essential for exocytosis. Two other SM proteins, Munc18-3 and Vps33, similarly chaperone SNARE assembly via a template complex, suggesting that SM protein mechanism is conserved.

Synaptic soluble N-ethylmaleimide–sensitive factor attachment protein receptors (SNAREs) couple their stepwise folding to fusion of synaptic vesicles with plasma membranes. In this process, three SNAREs assemble into a stable four-helix bundle. Arguably, the first and rate-limiting step of SNARE assembly is the formation of an activated binary target (t)-SNARE complex on the target plasma membrane, which then zippers with the vesicle (v)-SNARE on the vesicle to drive membrane fusion. However, the t-SNARE complex readily misfolds, and its structure, stability, and dynamics are elusive. Using single-molecule force spectroscopy, we modeled the synaptic t-SNARE complex as a parallel three-helix bundle with a small frayed C terminus. The helical bundle sequentially folded in an N-terminal domain (NTD) and a C-terminal domain (CTD) separated by a central ionic layer, with total unfolding energy of ∼17 kBT, where kB is the Boltzmann constant and T is 300 K. Peptide binding to the CTD activated the t-SNARE complex to initiate NTD zippering with the v-SNARE, a mechanism likely shared by the mammalian uncoordinated-18-1 protein (Munc18-1). The NTD zippering then dramatically stabilized the CTD, facilitating further SNARE zippering. The subtle bidirectional t-SNARE conformational switch was mediated by the ionic layer. Thus, the t-SNARE complex acted as a switch to enable fast and controlled SNARE zippering required for synaptic vesicle fusion and neurotransmission.

Munc18 chaperones assembly of three membrane-anchored soluble N-ethylmaleimidesensitive factor attachment protein receptors (SNAREs) into a four-helix bundle to mediate membrane fusion between vesicles and plasma membranes, leading to neurotransmitter or insulin release, glucose transporter (GLUT4) translocation, or other exocytotic processes. Yet, the molecular mechanism underlying chaperoned SNARE assembly is not well understood. Recent evidence suggests that Munc18-1 and Munc18-3 simultaneously bind their cognate SNAREs to form ternary template complexes – Munc18- 1:Syntaxin-1:VAMP2 for synaptic vesicle fusion and Munc18-3:Syntaxin-4:VAMP2 for GLUT4 translocation and insulin release, which facilitate the binding of SNAP-25 or SNAP-23 to conclude SNARE assembly. Here, we further investigate the structure, dynamics, and function of the template complexes using optical tweezers. Our results suggest that the synaptic template complex transitions to an activated state with a rate of 0.054 s−1 for efficient SNAP-25 binding. The transition depends upon the linker region of syntaxin-1 upstream of its helical bundle-forming SNARE motif. In addition, the template complex is stabilized by a poorly characterized disordered loop region in Munc18-1. While the synaptic template complex efficiently binds both SNAP-25 and SNAP-23, the GLUT4 template complex strongly favors SNAP-23 over SNAP-25, despite the similar stabilities of their assembled SNARE bundles. Together, our data demonstrate that a highly dynamic template complex mediates efficient and specific SNARE assembly.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are evolutionarily conserved machines that couple their folding/assembly to membrane fusion. However, it is unclear how these processes are regulated and function. To determine these mechanisms, we characterized the folding energy and kinetics of four representative SNARE complexes at a single-molecule level using high-resolution optical tweezers. We found that all SNARE complexes assemble by the same step-wise zippering mechanism: slow N-terminal domain (NTD) association, a pause in a force-dependent half-zippered intermediate, and fast C-terminal domain (CTD) zippering. The energy release from CTD zippering differs for yeast (13 kBT) and neuronal SNARE complexes (27 kBT), and is concentrated at the C-terminal part of CTD zippering. Thus, SNARE complexes share a conserved zippering pathway and polarized energy release to efficiently drive membrane fusion, but generate different amounts of zippering energy to regulate fusion kinetics. Many processes in living things need molecules to be transported within, or between, cells. For example, damaged or waste molecules are transported within a cell to structures that can break the molecules down, while nerve impulses are transmitted from one neuron to the next via the release of signaling molecules. Cells—and the compartments within cells—are surrounded by membranes that act as barriers to certain molecules. Vesicles are small, membrane-enclosed packages that are used to transport molecules between different membranes; and in order to release its cargo, a vesicle must fuse with its target membrane. To fuse like this, the forces that act to push membranes away from one another need to be overcome. Proteins called SNARES, which are embedded in both membranes, are the molecular engines that power the fusion process. Once the SNARE proteins from the vesicle and the target membrane bind, they assemble into a more compact complex that pulls the two membranes close together and allows fusion to take place. The final shape of an assembled SNARE complex is essentially the same for all SNARE complexes; however, it is not known whether all of these complexes fold using the same method. Now Zorman et al. have used optical tweezers—an instrument that uses a highly focused laser beam to hold and manipulate microscopic objects—to observe the folding and unfolding of four different types of SNARE complex. All four SNARE complexes followed the same step-by-step process: the leading ends of the SNARE proteins slowly bound to each other; the process paused; then the rest of the proteins rapidly ‘zippered’ together. Zorman et al. revealed that, although the steps in the processes were the same, the energy released in the last step was different when different complexes assembled. This suggests that the energy released by the ‘zippering’ of different SNARE proteins is optimized to match the required speed of different membrane fusion events. Furthermore, Zorman et al. propose that the reason why the majority of energy is released in the later stages of complex assembly is because this is when the repulsion between the two membranes is strongest. The discoveries of Zorman et al. will now aid future efforts aimed at understanding better how the numerous other proteins that interact with SNARE proteins regulate the process of membrane fusion.

Synaptic-soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins couple their stage-wise folding/assembly to rapid exocytosis of neurotransmitters in a Munc18-1-dependent manner. The functions of the different assembly stages in exocytosis and the role of Munc18-1 in SNARE assembly are not well understood. Using optical tweezers, we observed four distinct stages of assembly in SNARE N-terminal, middle, C-terminal, and linker domains (or NTD, MD, CTD, and LD, respectively). We found that SNARE layer mutations differentially affect SNARE assembly. Comparison of their effects on SNARE assembly and on exocytosis reveals that NTD and CTD are responsible for vesicle docking and fusion, respectively, whereas MD regulates SNARE assembly and fusion. Munc18-1 initiates SNARE assembly and structures t-SNARE C-terminus independent of syntaxin N-terminal regulatory domain (NRD) and stabilizes the half-zippered SNARE complex dependent upon the NRD. Our observations demonstrate distinct functions of SNARE domains whose assembly is intimately chaperoned by Munc18-1. Plants, animals and other eukaryotes transport many large molecules within their cells inside membrane-bound packages called vesicles. These vesicles can fuse with the membrane of a target compartment in the cell to deliver their contents inside, or fuse with the cell’s membrane to release the contents outside of the cell. Membrane fusion is carried out by a group of proteins called SNAREs. These proteins are embedded on the membranes of both the vesicle and its target, and they bind to each other to form a tight complex. This complex docks the vesicle to the target and then acts like a “zipper” to pull the two membranes close enough to fuse. The best-studied SNARE proteins act in nerve cells and fuse vesicles to the cell’s membrane in order to release molecules called neurotransmitters. This process is essential for communication between nerve cells, and relies on a protein called Munc18-1. However, it is not well understood how SNARE proteins assemble into the complex and how Munc18-1 regulates this process. Ma et al. have now used a tool called “optical tweezers” to pull an assembled SNARE complex apart in the laboratory and then observe how it folds and assembles in a step-by-step process. These experiments showed that the complex assembled in four stages and not three as has been reported in previous work. SNARE proteins are made up of four parts called domains, and Ma et al. observed that the N-terminal domains were the first to bind to each other. Next, the binding progressed to the middle domain, then to the C-terminal domain and finally to the linker domain. An intermediate, half-zippered form was also observed. Ma et al. next analysed each domain in more detail and found that the N-terminal and C-terminal domains drive the docking of vesicles to the target membrane, the middle domain is crucial for assembling the SNARE complex correctly, and all three domains regulate the fusing of the membranes. Further experiments showed that Munc18-1 promoted the assembly of new SNARE complexes and stabilized the half-zippered form, rather than stabilizing the complex after it had fully assembled. This study will provide a new tool to examine many other proteins that regulate SNARE assembly, and a basis to understand the role of SNARE proteins in brain activity.

α-Synuclein (α-Syn) is a presynaptic protein implicated in the regulation of synaptic vesicle (SV) organization, soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly, and neurotransmitter release through reversible interactions with curved lipid membranes. Increasing evidence indicates that α-Syn functions as a curvature-sensitive membrane adaptor whose N-terminal amphipathic helices selectively recognize the nanoscale architecture of SVs, thereby promoting vesicle tethering, clustering, and membrane fusion dynamics essential for synaptic transmission. Recent structural and biophysical studies demonstrate that SVs act as catalytic platforms in which membrane curvature, lipid packing defects, molecular crowding, and local physicochemical conditions determine whether α-Syn remains membrane-bound or transitions into pathogenic assemblies. Post-translational modifications, including phosphorylation, nitration, acetylation, and ubiquitination, dynamically regulate α-Syn conformation, membrane engagement, proteostatic turnover, aggregation propensity, and intracellular localization by modulating its structural plasticity and interactions with synaptic membranes. These modifications can alter electrostatic interactions, destabilize amphipathic helices, and shift α-Syn from functional membrane-associated states toward soluble oligomeric and amyloidogenic species. Aging further exacerbates these transitions through alterations in SV lipid composition, membrane fluidity, oxidative membrane damage, impaired proteostasis, and defective vesicle trafficking, thereby destabilizing α-Syn-membrane interactions. The present review discusses how membrane remodeling, vesicle lipid composition, post-translational modifications, protein conformational dynamics, and aging collectively regulate α-Syn phase behavior, membrane binding, and pathological aggregation at the presynaptic terminal. Integrating structural biology, lipidomics, proteomics, and live-cell imaging approaches will identify mechanistic transitions linking physiological membrane engagement to neurodegenerative synucleinopathies and reveal therapeutic opportunities for preventing synaptic dysfunction, toxic condensate formation, and neurodegeneration in Parkinson's disease and related disorders associated with pathological α-Syn aggregation.

Neurotransmitter release by Ca2+‐triggered synaptic vesicle exocytosis is essential for information transmission in the nervous system. The soluble N‐ethylmaleimide sensitive factor attachment protein receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin‐2 form the SNARE complex to bring synaptic vesicles and the plasma membranes together and to catalyze membrane fusion. Munc18‐1 and Munc13‐1 regulate synaptic vesicle priming via orchestrating neuronal SNARE complex assembly. In this review, we summarize recent advances toward the functions and molecular mechanisms of Munc18‐1 and Munc13‐1 in guiding neuronal SNARE complex assembly, and discuss the functional similarities and differences between Munc18‐1 and Munc13‐1 in neurons and their homologs in other intracellular membrane trafficking systems. Munc18‐1 and Munc13‐1 regulate synaptic vesicle exocytosis via orchestrating neuronal SNARE complex (syntaxin‐1, SNAP‐25, and synaptobrevin‐2) assembly. In this review, we summarized recent advances towards the molecular mechanisms of Munc18‐1 and Munc13‐1 in guiding neuronal SNARE complex assembly and discussed the similarities and differences between Munc18‐1 and Munc13‐1 in neurons and their homologs in other intracellular membrane trafficking systems.

Sec1/Munc18 (SM) proteins are central to intracellular transport and neurotransmitter release but their exact role is still elusive. Several SM proteins, like the neuronal N‐Sec1 and the yeast Sly1 protein, bind their cognate t‐SNAREs with high affinity. This has been thought to be critical for their function. Here, we show that various mutant forms of Sly1p and the Golgi‐localized syntaxin Sed5p, which abolish their high‐affinity interaction, are fully functional in vivo , indicating that the tight interaction of the two molecules per se is not relevant for proper function. Mutant Sly1p unable to bind Sed5p is excluded from core SNARE complexes, also demonstrating that Sly1p function is not directly coupled to assembled SNARE complexes thought to execute membrane fusion. We also find that wild‐type Sly1p and mutant Sly1p unable to bind Sed5p directly interact with selected ER‐to‐Golgi and intra‐Golgi nonsyntaxin SNAREs. The newly identified, direct interactions of the SM protein with nonsytaxin SNAREs might provide a molecular mechanism by which SNAREs can be activated to engage in pairing and assemble into fusogenic SNARE complexes.

Formation of the trans -SNARE complex is believed to generate a force transfer to the membranes to promote membrane fusion, but the underlying mechanism remains elusive. In this study, we show that helix-breaking and/or length-increasing insertions in the juxtamembrane linker region of synaptobrevin-2 exert diverse effects on liposome fusion, in a manner dependent on the insertion position relative to the two conserved tryptophan residues (W 89 /W 90 ). Helical extension of synaptobrevin-2 to W 89 /W 90 is a prerequisite for initiating membrane merger. The transmembrane region of synaptobrevin-2 enables proper localization of W 89 /W 90 at the membrane interface to gate force transfer. Besides, our data indicate that the SNARE regulatory components Munc18-1 and Munc13-1 impose liposome fusion strong demand on tight coupling between the SNARE motif and the transmembrane region of synaptobrevin-2.