SNARE Assembly Research Articles

Intermediate steps in the formation of neuronal SNARE complexes

Neuronal exocytosis requires the assembly of three SNARE proteins, syntaxin and SNAP25 on the plasma membrane and synaptobrevin on the vesicle membrane. However, the precise steps in this process and the points at which assembly and fusion are controlled by regulatory proteins are unclear. In the present work, we examine the kinetics and intermediate states during SNARE assembly in vitro using a combination of time resolved fluorescence and EPR spectroscopy. We show that syntaxin rapidly forms a dimer prior to forming the kinetically stable 2:1 syntaxin:SNAP25 complex, and that the 2:1 complex is not diminished by the presence of excess SNAP25. Moreover, the 2:1 complex is temperature dependent with a reduced concentration at 37°C. The two segments of SNAP25 behave differently. The N-terminal SN1 segment of SNAP25 exhibits a pronounced increase in backbone ordering from the N- to the C-terminus that is not seen in the C-terminal SNAP25 segment SN2. Both the SN1 and SN2 segments of SNAP25 will assemble with syntaxin; however, while the association of the SN1 segment with syntaxin produces a stable 2:2 (SN1:syntaxin) complex, the complex formed between SN2 and syntaxin is largely disordered. Synaptobrevin fails to bind syntaxin alone, but will associate with syntaxin in the presence of either the SN1 or SN2 segments; however, the synaptobrevin:syntaxin:SN2 complex remains disordered. Taken together, these data suggest that synaptobrevin and syntaxin do not assemble in the absence of SNAP25, and that the SN2 segment of SNAP25 is the last to enter the SNARE complex.

Identification of Key Genes and Imbalanced SNAREs Assembly in the Comorbidity of Polycystic Ovary Syndrome and Depression.

Women with polycystic ovary syndrome (PCOS) have increased odds of concurrent depression, indicating that the relationship between PCOS and depression is more likely to be comorbid. However, the underlying mechanism remains unclear. Here, we aimed to use bioinformatic analysis to screen for the genetic elements shared between PCOS and depression. Differentially expressed genes (DEGs) were screened out through GEO2R using the PCOS and depression datasets in NCBI. Protein-protein interaction (PPI) network analysis and enrichment analysis were performed to identify the potential hub genes. After verification using other PCOS and depression datasets, the associations between key gene polymorphism and comorbidity were further studied using data from the UK biobank (UKB) database. In this study, three key genes, namely, SNAP23, VTI1A, and PRKAR1A, and their related SNARE interactions in the vesicular transport pathway were identified in the comorbidity of PCOS and depression. The rs112568544 at SNAP23, rs11077579 and rs4458066 at PRKAR1A, and rs10885349 at VTI1A might be the genetic basis of this comorbidity. Our study suggests that the SNAP23, PRKAR1A, and VTI1A genes can directly or indirectly participate in the imbalanced assembly of SNAREs in the pathogenesis of the comorbidity of PCOS and depression. These findings may provide new strategies in diagnosis and therapy for this comorbidity.

Sec18 binds the tethering/SM complex HOPS to engage the Qc-SNARE for membrane fusion.

Membrane fusion is regulated by Rab GTPases, their tethering effectors such as HOPS, SNARE proteins on each fusion partner, SM proteins to catalyze SNARE assembly, Sec17 (SNAP), and Sec18 (NSF). Though concentrated HOPS can support fusion without Sec18, we now report that fusion falls off sharply at lower HOPS levels, where direct Sec18 binding to HOPS restores fusion. This Sec18-dependent fusion needs adenine nucleotide but neither ATP hydrolysis nor Sec17. Sec18 enhances HOPS recognition of the Qc-SNARE. With high levels of HOPS, Qc has a Km for fusion of a few nM. Either lower HOPS levels, or substitution of a synthetic tether for HOPS, strikingly increases the Km for Qc to several hundred nM. With dilute HOPS, Sec18 returns the Km for Qc to low nM. In contrast, HOPS concentration and Sec18 have no effect on Qb-SNARE recognition. Just as Qc is required for fusion but not for the initial assembly of SNAREs in trans, impaired Qc recognition by limiting HOPS without Sec18 still allows substantial trans-SNARE assembly. Thus, in addition to the known Sec18 functions of disassembling SNARE complexes, oligomerizing Sec17 for membrane association, and allowing Sec17 to drive fusion without complete SNARE zippering, we report a fourth Sec18 function, the Sec17-independent binding of Sec18 to HOPS to enhance functional Qc-SNARE engagement.

Tomosyns attenuate SNARE assembly and synaptic depression by binding to VAMP2-containing template complexes

Tomosyns are widely thought to attenuate membrane fusion by competing with synaptobrevin-2/VAMP2 for SNARE-complex assembly. Here, we present evidence against this scenario. In a novel mouse model, tomosyn-1/2 deficiency lowered the fusion barrier and enhanced the probability that synaptic vesicles fuse, resulting in stronger synapses with faster depression and slower recovery. While wild-type tomosyn-1m rescued these phenotypes, substitution of its SNARE motif with that of synaptobrevin-2/VAMP2 did not. Single-molecule force measurements indeed revealed that tomosyn’s SNARE motif cannot substitute synaptobrevin-2/VAMP2 to form template complexes with Munc18-1 and syntaxin-1, an essential intermediate for SNARE assembly. Instead, tomosyns extensively bind synaptobrevin-2/VAMP2-containing template complexes and prevent SNAP-25 association. Structure-function analyses indicate that the C-terminal polybasic region contributes to tomosyn’s inhibitory function. These results reveal that tomosyns regulate synaptic transmission by cooperating with synaptobrevin-2/VAMP2 to prevent SNAP-25 binding during SNARE assembly, thereby limiting initial synaptic strength and equalizing it during repetitive stimulation.

Annexin A1 may contribute to the morphological changes in podocytes by mediating endocytic vesicle fusion and transport via promotion of SNARE assembly in idiopathic membranous nephropathy.

Annexin A1 is a membrane-associated calcium-binding protein that participates in the progression of many diseases by facilitating vesicle aggregation. It has been documented that reducing vesicle formation alleviates podocyte injury and albuminuria in idiopathic membranous nephropathy (IMN). However, the role of Annexin A1 (ANXA1) in IMN is unknown. Electron microscopy was used to observe the numbers of vesicles in podocytes. The expression of ANXA1 in IMN was investigated by bioinformatics analysis. We validated the hub genes with the Nephroseq V5 online tool and microarray data from the GEO. Immunohistochemical staining and qPCR were performed to measure gene and protein expression. The numbers of vesicles in IMN podocytes were significantly increased. Bioinformatics analysis showed that ANXA1, one of the differentially expressed genes, was upregulated in glomeruli from IMN patients. In the validation database and dataset, we confirmed that ANXA1 expression was upregulated in the glomeruli of IMN patients. We revealed that the increased expression of ANXA1 was negatively correlated with the glomerular filtration rate (GFR) and proteinuria. Moreover, ANXA1 was enriched in the biological process of vesicle fusion, in which the expression of SNAREs and the SNARE complex was increased. Finally, the expression of ANXA1 and genes related to SNAREs and the SNARE complex was upregulated in glomeruli from IMN patients according to immunohistochemical staining and qPCR. We conclude that ANXA1 may mediate endocytic vesicle fusion and transport by promoting SNARE assembly, contributing to the morphological changes in podocytes and massive proteinuria in IMN.

Roles for diacylglycerol in synaptic vesicle priming and release revealed by complete reconstitution of core protein machinery

Here, we introduce the full functional reconstitution of genetically validated core protein machinery (SNAREs, Munc13, Munc18, Synaptotagmin, and Complexin) for synaptic vesicle priming and release in a geometry that enables detailed characterization of the fate of docked vesicles both before and after release is triggered with Ca2+. Using this setup, we identify new roles for diacylglycerol (DAG) in regulating vesicle priming and Ca2+-triggered release involving the SNARE assembly chaperone Munc13. We find that low concentrations of DAG profoundly accelerate the rate of Ca2+-dependent release, and high concentrations reduce clamping and permit extensive spontaneous release. As expected, DAG also increases the number of docked, release-ready vesicles. Dynamic single-molecule imaging of Complexin binding to release-ready vesicles directly establishes that DAG accelerates the rate of SNAREpin assembly mediated by chaperones, Munc13 and Munc18. The selective effects of physiologically validated mutations confirmed that the Munc18-Syntaxin-VAMP2 "template" complex is a functional intermediate in the production of primed, release-ready vesicles, which requires the coordinated action of Munc13 and Munc18.

Efficient fusion requires a membrane anchor on the vacuolar Qa-SNARE.

As a prelude to fusion, the R-SNARE on one membrane zippers with Qa-, Qb-, and Qc-SNAREs from its apposed fusion partner, forming a four-helical bundle that draws the two membranes together. Because Qa- and Qb-SNAREs are anchored to the same membrane and are adjacent in the 4-SNARE bundle, their two anchors might be redundant. Using the recombinant pure protein catalysts of yeast vacuole fusion, we now report that the specific distribution of transmembrane (TM) anchors on the Q-SNAREs is critical for efficient fusion. A TM anchor on the Qa-SNARE supports rapid fusion even when the other two Q-SNAREs are unanchored, while a TM anchor on the Qb-SNARE is dispensable and is insufficient for rapid fusion as the sole Q-SNARE anchor. This does not depend on which specific TM domain is attached to the Qa-SNARE but rather is due to the Qa-SNARE being anchored per se. The need for Qa-SNARE anchoring is even seen when the homotypic fusion and vacuole protein sorting protein (HOPS), the physiological catalyst of tethering and SNARE assembly, is replaced by an artificial tether. The need for a Qa TM anchor is thus a fundamental property of vacuolar SNARE zippering-induced fusion and may reflect the need for the Qa juxtamembrane (JxQa) region to be anchored between its SNARE and TM domains. This requirement for Qa-SNARE anchoring and correct JxQa position is bypassed by Sec17/Sec18, exploiting a platform of partially zippered SNAREs. Because Qa is the only synaptic Q-SNARE with a TM anchor, the need for Qa-specific anchoring may reflect a general requirement for SNARE-mediated fusion.

An Hspa8 variant is a shocking modifier of spinal muscular atrophy in mice

In this issue of Neuron, Kim etal.1 show that an Hspa8 variant modifies disease phenotypes in a mouse model of spinal muscular atrophy. Hspa8 facilitates the correct folding of proteins, enhances SNARE assembly, and influences SMN2 splicing.

SNARE Proteins in Synaptic Vesicle Fusion.

Neurotransmitters are stored in small membrane-bound vesicles at synapses; a subset of synaptic vesicles is docked at release sites. Fusion of docked vesicles with the plasma membrane releases neurotransmitters. Membrane fusion at synapses, as well as all trafficking steps of the secretory pathway, is mediated by SNARE proteins. The SNAREs are the minimal fusion machinery. They zipper from N-termini to membrane-anchored C-termini to form a 4-helix bundle that forces the apposed membranes to fuse. At synapses, the SNAREs comprise a single helix from syntaxin and synaptobrevin; SNAP-25 contributes the other two helices to complete the bundle. Unc13 mediates synaptic vesicle docking and converts syntaxin into the permissive "open" configuration. The SM protein, Unc18, is required to initiate and proofread SNARE assembly. The SNAREs are then held in a half-zippered state by synaptotagmin and complexin. Calcium removes the synaptotagmin and complexin block, and the SNAREs drive vesicle fusion. After fusion, NSF and alpha-SNAP unwind the SNAREs and thereby recharge the system for further rounds of fusion. In this chapter, we will describe the discovery of the SNAREs, their relevant structural features, models for their function, and the central role of Unc18. In addition, we will touch upon the regulation of SNARE complex formation by Unc13, complexin, and synaptotagmin.

SNARE assembly enlightened by cryo-EM structures of a synaptobrevin-Munc18-1-syntaxin-1 complex.

Munc18-1 forms a template to organize assembly of the neuronal SNARE complex that triggers neurotransmitter release, binding first to a closed conformation of syntaxin-1 where its amino-terminal region interacts with the SNARE motif, and later binding to synaptobrevin. However, the mechanism of SNARE complex assembly remains unclear. Here, we report two cryo-EM structures of Munc18-1 bound to cross-linked syntaxin-1 and synaptobrevin. The structures allow visualization of how syntaxin-1 opens and reveal how part of the syntaxin-1 amino-terminal region can help nucleate interactions between the amino termini of the syntaxin-1 and synaptobrevin SNARE motifs, while their carboxyl termini bind to distal sites of Munc18-1. These observations, together with mutagenesis, SNARE complex assembly experiments, and fusion assays with reconstituted proteoliposomes, support a model whereby these interactions are critical to initiate SNARE complex assembly and multiple energy barriers enable diverse mechanisms for exquisite regulation of neurotransmitter release.

Molecular determinants of complexin clamping and activation function.

Previously we reported that Synaptotagmin-1 and Complexin synergistically clamp the SNARE assembly process to generate and maintain a pool of docked vesicles that fuse rapidly and synchronously upon Ca2+ influx (Ramakrishnan et al., 2020). Here, using the same in vitro single-vesicle fusion assay, we determine the molecular details of the Complexin-mediated fusion clamp and its role in Ca2+-activation. We find that a delay in fusion kinetics, likely imparted by Synaptotagmin-1, is needed for Complexin to block fusion. Systematic truncation/mutational analyses reveal that continuous alpha-helical accessory-central domains of Complexin are essential for its inhibitory function and specific interaction of the accessory helix with the SNAREpins enhances this functionality. The C-terminal domain promotes clamping by locally elevating Complexin concentration through interactions with the membrane. Independent of their clamping functions, the accessory-central helical domains of Complexin also contribute to rapid Ca2+-synchronized vesicle release by increasing the probability of fusion from the clamped state.

The small GTPase RABA2a recruits SNARE proteins to regulate the secretory pathway in parallel with the exocyst complex in Arabidopsis

Delivery of proteins to the plasma membrane occurs via secretion, which requires tethering, docking, priming, and fusion of vesicles. In yeast and mammalian cells, an evolutionarily conserved RAB GTPase activation cascade functions together with the exocyst and SNARE proteins to coordinate vesicle transport with fusion at the plasma membrane. However, it is unclear whether this is the case in plants. In this study, we show that the small GTPase RABA2a recruits and interacts with the VAMP721/722-SYP121-SNAP33 SNARE ternary complex for membrane fusion. Through immunoprecipitation coupled with mass spectrometry analysis followed by the validatation with a series of biochemical assays, we identified the SNARE proteins VAMP721 and SYP121 as the interactors and downstream effectors of RABA2a. Further expreiments showed that RABA2a interacts with all members of the SNARE complex in its GTP-bound form and modulates the assembly of the VAMP721/722-SYP121-SNAP33 SNARE ternary complex. Intriguingly, we did not observe the interaction of the exocyst subunits with either RABA2a or theSNARE proteins in several different experiments. Neither RABA2a inactivation affects the subcellular localization or assembly of the exocystnor the exocyst subunit mutant exo84b shows the disrupted RABA2a-SNARE association or SNARE assembly, suggesting that the RABA2a-SNARE- and exocyst-mediated secretory pathways are largely independent. Consistently, our live imaging experiments reveal that the two sets of proteins follow non-overlapping trafficking routes, and genetic and cell biologyanalyses indicate that the two pathways select different cargos. Finally, we demonstrate that the plant-specific RABA2a-SNARE pathway is essential for the maintenance of potassium homeostasis in Arabisopsis seedlings. Collectively, our findings imply that higher plants might have generated different endomembrane sorting pathways during evolution and may enable the highly conserved endomembrane proteins to participate in plant-specific trafficking mechanisms for adaptation to the changing environment.

Fusion of tethered membranes can be driven by Sec18/NSF and Sec17/αSNAP without HOPS.

Yeast vacuolar membrane fusion has been reconstituted with R, Qa, Qb, and Qc-family SNAREs, Sec17/αSNAP, Sec18/NSF, and the hexameric HOPS complex. HOPS tethers membranes and catalyzes SNARE assembly into RQaQbQc trans-complexes which zipper through their SNARE domains to promote fusion. Previously, we demonstrated that Sec17 and Sec18 can bypass the requirement of complete zippering for fusion (Song et al., 2021), but it has been unclear whether this activity of Sec17 and Sec18 is directly coupled to HOPS. HOPS can be replaced for fusion by a synthetic tether when the three Q-SNAREs are pre-assembled. We now report that fusion intermediates with arrested SNARE zippering, formed with a synthetic tether but without HOPS, support Sec17/Sec18-triggered fusion. This zippering-bypass fusion is thus a direct result of Sec17 and Sec18 interactions: with each other, with the platform of partially zippered SNAREs, and with the apposed tethered membranes. As these fusion elements are shared among all exocytic and endocytic traffic, Sec17 and Sec18 may have a general role in directly promoting fusion.

Room for Two: The Synaptophysin/Synaptobrevin Complex.

Synaptic vesicle release is regulated by upwards of 30 proteins at the fusion complex alone, but disruptions in any one of these components can have devastating consequences for neuronal communication. Aberrant molecular responses to calcium signaling at the pre-synaptic terminal dramatically affect vesicle trafficking, docking, fusion, and release. At the organismal level, this is reflected in disorders such as epilepsy, depression, and neurodegeneration. Among the myriad pre-synaptic proteins, perhaps the most functionally mysterious is synaptophysin (SYP). On its own, this vesicular transmembrane protein has been proposed to function as a calcium sensor, a cholesterol-binding protein, and to form ion channels across the phospholipid bilayer. The downstream effects of these functions are largely unknown. The physiological relevance of SYP is readily apparent in its interaction with synaptobrevin (VAMP2), an integral element of the neuronal SNARE complex. SNAREs, soluble NSF attachment protein receptors, comprise a family of proteins essential for vesicle fusion. The complex formed by SYP and VAMP2 is thought to be involved in both trafficking to the pre-synaptic membrane as well as regulation of SNARE complex formation. Recent structural observations specifically implicate the SYP/VAMP2 complex in anchoring the SNARE assembly at the pre-synaptic membrane prior to vesicle fusion. Thus, the SYP/VAMP2 complex appears vital to the form and function of neuronal exocytotic machinery.

Phosphatidylinositol and phosphatidylinositol-3-phosphate activate HOPS to catalyze SNARE assembly, allowing small headgroup lipids to support the terminal steps of membrane fusion.

Intracellular membrane fusion requires Rab GTPases, tethers, SNAREs of the R, Qa, Qb, and Qc families, and SNARE chaperones of the Sec17 (SNAP), Sec18 (NSF), and SM (Sec1/Munc18) families. The vacuolar HOPS complex combines the functions of membrane tethering and SM catalysis of SNARE assembly. HOPS is activated for this catalysis by binding to the vacuolar lipids and Rab. Of the eight major vacuolar lipids, we now report that phosphatidylinositol and phosphatidylinositol-3-phosphate are required to activate HOPS for SNARE complex assembly. These lipids plus ergosterol also allow full trans-SNARE complex assembly, yet do not support fusion, which is reliant on either phosphatidylethanolamine (PE) or on phosphatidic acid (PA), phosphatidylserine (PS), and diacylglycerol (DAG). Fusion with a synthetic tether and without HOPS, or even without SNAREs, still relies on either PE or on PS, PA, and DAG. These lipids are thus required for the terminal bilayer rearrangement step of fusion, distinct from the lipid requirements for the earlier step of activating HOPS for trans-SNARE assembly.

128-OR: DOC2b Boosts Insulin Secretion via a Yes Kinase-Dependent Mechanism

Exocytosis regulatory protein DOC2b is a crucial regulator of SNARE assembly in β-cells and enrichment of DOC2b enhances glucose-stimulated insulin secretion (GSIS). However, the underlying mechanism of how DOC2b boosts GSIS remains to be explored. We hypothesize that tyrosine-phosphorylation of DOC2b’s functionally indispensable C2AB domain is required for DOC2b’s beneficial function on GSIS. Biochemical analysis revealed that DOC2b is tyrosine-phosphorylated specifically by glucose within 1-2 min of stimulation in MIN6 β-cells and human islets. Pervanadate (pV) treatment, which inhibits tyrosine-specific phosphatases, validated the phosphorylation event to be tyrosine-specific. Further, the Src family kinases (SFKs) were implicated in this phosphorylation event, based upon the ability of SFK inhibitor SU-6656 to markedly attenuate the level of tyrosine-phosphorylated DOC2b under pV treated conditions. Phosphorylated/activated SFKY416 co-immunoprecipitated with DOC2b from pV treated β-cells; SU-6656 abrogated the interaction. YES kinase, an SFK, was also co-immunoprecipitated with DOC2b following glucose stimulation or pV treatment, suggesting YES kinase to be the specific SFK that phosphorylates DOC2b in β-cells. All-atom molecular dynamics simulations identified a specific tyrosine residue present in DOC2b’s C2B domain, Y301, as solvent-exposed and available for modification. Our results using phosphodeficient and phosphomimetic point mutants validated the involvement of the Y301 phosphorylation in for the boosting effect of DOC2b on GSIS. Taken together, these results resolve the first glucose-induced post-translational modification of DOC2b in β-cells, pinpointing the kinase and the site of action. These findings support a new model for DOC2b in GSIS: 1) glucose stimulation of the β-cell rapidly phosphorylates DOC2b on residue Y301 via YES kinase at the plasma membrane, yielding 2) an activated form of DOC2b that regulates insulin release. Disclosure D. Chatterjee bhowmick: None. A. Aslamy: None. S. Bhattacharya: None. E. Oh: None. M. Ahn: None. D. C. Thurmond: Consultant; Self; Immutics. Funding National Institute of Diabetes and Digestive and Kidney Diseases (DK067912, DK112917, DK102233 to D.C.T.); JDRF (3-PDF-2020-934-A-N to D.C.B.)

Chaperoning SNARE Folding and Assembly.

SNARE proteins and Sec1/Munc18 (SM) proteins constitute the core molecular engine that drives nearly all intracellular membrane fusion and exocytosis. While SNAREs are known to couple their folding and assembly to membrane fusion, the physiological pathways of SNARE assembly and the mechanistic roles of SM proteins have long been enigmatic. Here, we review recent advances in understanding the SNARE-SM fusion machinery with an emphasis on biochemical and biophysical studies of proteins that mediate synaptic vesicle fusion. We begin by discussing the energetics, pathways, and kinetics of SNARE folding and assembly in vitro. Then, we describe diverse interactions between SM and SNARE proteins and their potential impact on SNARE assembly in vivo. Recent work provides strong support for the idea that SM proteins function as chaperones, their essential role being to enable fast, accurate SNARE assembly. Finally, we review the evidence that SM proteins collaborate with other SNARE chaperones, especially Munc13-1, and briefly discuss some roles of SNARE and SM protein deficiencies in human disease.

The Accessory Helix of Complexin Stabilizes a Partially Unzippered State of the SNARE Complex and Mediates the Complexin Clamping Function In Vivo.

Spontaneous synaptic transmission is regulated by the protein complexin (Cpx). Cpx binds the SNARE complex, a coil-coiled four-helical bundle that mediates the attachment of a synaptic vesicle (SV) to the presynaptic membrane (PM). Cpx is thought to clamp spontaneous fusion events by stabilizing a partially unraveled state of the SNARE bundle; however, the molecular detail of this mechanism is still debated. We combined electrophysiology, molecular modeling, and site-directed mutagenesis in Drosophila to develop and validate the atomic model of the Cpx-mediated clamped state of the SNARE complex. We took advantage of botulinum neurotoxins (BoNTs) B and G, which cleave the SNARE protein synaptobrevin (Syb) at different sites. Monitoring synaptic depression on BoNT loading revealed that the clamped state of the SNARE complex has two or three unraveled helical turns of Syb. Site-directed mutagenesis showed that the Cpx clamping function is predominantly maintained by its accessory helix (AH), while molecular modeling suggested that the Cpx AH interacts with the unraveled C terminus of Syb and the SV lipid bilayer. The developed molecular model was employed to design new Cpx poor-clamp and super-clamp mutations and to tested the predictions in silico employing molecular dynamics simulations. Subsequently, we generated Drosophila lines harboring these mutations and confirmed the poor-clamp and super-clamp phenotypes in vivo. Altogether, these results validate the atomic model of the Cpx-mediated fusion clamp, wherein the Cpx AH inserts between the SNARE bundle and the SV lipid bilayer, simultaneously binding the unraveled C terminus of Syb and preventing full SNARE assembly.

Munc13 binds and recruits SNAP25 to chaperone SNARE complex assembly.

Synaptic vesicle fusion is mediated by SNARE proteins-VAMP2 on the vesicle and Syntaxin-1/SNAP25 on the presynaptic membrane. Chaperones Munc18-1 and Munc13-1 cooperatively catalyze SNARE assembly via an intermediate 'template' complex containing Syntaxin-1 and VAMP2. How SNAP25 enters this reaction remains a mystery. Here, we report that Munc13-1 recruits SNAP25 to initiate the ternary SNARE complex assembly by direct binding, as judged by bulk FRET spectroscopy and single-molecule optical tweezer studies. Detailed structure-function analyses show that the binding is mediated by the Munc13-1 MUN domain and is specific for the SNAP25 'linker' region that connects the two SNARE motifs. Consequently, freely diffusing SNAP25 molecules on phospholipid bilayers are concentrated and bound in~1:1 stoichiometry by the self-assembled Munc13-1 nanoclusters.

SCGN-regulated Stage-wise SNARE Assembly: Novel Insight into Synaptic Exocytosis.

SCGN-regulated Stage-wise SNARE Assembly: Novel Insight into Synaptic Exocytosis.