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.

Munc18c accelerates SNARE-dependent membrane fusion in the presence of regulatory proteins α-SNAP and NSF.

Intracellular vesicle fusion is driven by the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and their cofactors, including Sec1/Munc18 (SM), α-SNAP, and NSF. α-SNAP and NSF play multiple layers of regulatory roles in the SNARE assembly, disassembling the cis-SNARE complex and the prefusion SNARE complex. How SM proteins coupled with NSF and α-SNAP regulate SNARE-dependent membrane fusion remains incompletely understood. Munc18c, an SM protein involved in the exocytosis of the glucose transporter GLUT4, binds and activates target (t-) SNAREs to accelerate the fusion reaction through a SNARE-like peptide (SLP). Here, using an in vitro reconstituted system, we discovered that α-SNAP blocks the GLUT4 SNAREs-mediated membrane fusion. Munc18c interacts with t-SNAREs to displace α-SNAP, which overcomes the fusion inhibition. Furthermore, Munc18c shields the trans-SNARE complex from NSF/α-SNAP-mediated disassembly and accelerates SNARE-dependent fusion kinetics in the presence of NSF and α-SNAP. The SLP in domain 3a is indispensable in Munc18c-assisted resistance to NSF and α-SNAP. Together, our findings demonstrate that Munc18c protects the prefusion SNARE complex from α-SNAP and NSF, promoting SNARE-dependent membrane fusion through its SLP.

The crustacean DNA virus tegument protein VP26 binds to SNAP29 to inhibit SNARE complex assembly and autophagic degradation.

Autophagy generally functions as a cellular surveillance mechanism to combat invading viruses, but viruses have evolved various strategies to block autophagic degradation and even subvert it to promote viral propagation. White spot syndrome virus (WSSV) is the most highly pathogenic crustacean virus, but little is currently known about whether crustacean viruses such as WSSV can subvert autophagic degradation for escape. Here, we show that even though WSSV proliferation triggers the accumulation of autophagosomes, autophagic degradation is blocked in the crustacean species red claw crayfish. Interestingly, the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex including CqSNAP29, CqVAMP7, and the novel autophagosome SNARE protein CqSyx12 is required for autophagic flux to restrict WSSV replication, as revealed by gene silencing experiments. Simultaneously, the expressed WSSV tegument protein VP26, which likely localizes on autophagic membrane mediated by its transmembrane region, binds the Qb-SNARE domain of CqSNAP29 to competitively inhibit the binding of CqSyx12-Qa-SNARE with CqSNAP29-Qb-SNARE; this in turn disrupts the assembly of the CqSyx12-SNAP29-VAMP7 SNARE complex, which is indispensable for the proposed fusion of autophagosomes and lysosomes. Consequently, the autophagic degradation of WSSV is likely suppressed by the expressed VP26 protein in vivo in crayfish, thus probably protecting WSSV components from degradation via the autophagosome-lysosome pathway, resulting in evasion by WSSV. Collectively, these findings highlight how a DNA virus can subvert autophagic degradation by impairing the assembly of the SNARE complex to achieve evasion, paving the way for understanding host-DNA virus interactions from an evolutionary point of view, from crustaceans to mammals.IMPORTANCEWhite spot syndrome virus (WSSV) is one of the largest animal DNA viruses in terms of its genome size and has caused huge economic losses in the farming of crustaceans such as shrimp and crayfish. Detailed knowledge of WSSV-host interactions is still lacking, particularly regarding viral escape from host immune clearance. Intriguingly, we found that the presence of WSSV-VP26 might inhibit the autophagic degradation of WSSV in vivo in the crustacean species red claw crayfish. Importantly, this study is the first to show that viral protein VP26 functions as a core factor to benefit WSSV escape by disrupting the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, which is necessary for the proposed fusion of autophagosomes with lysosomes for subsequent degradation. These findings highlight a novel mechanism of DNA virus evasion by blocking SNARE complex assembly and identify viral VP26 as a key candidate for anti-WSSV targeting.

Potential role of the cell-penetrating peptide-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor motif of vesicle-associated membrane protein 2-patterned peptide in novel cosmeceutical skin product development.

AIM: This study aimed to investigate and verify the effect of cell-penetrating peptide (CPP)-conjugated soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) motif of vesicle-associated membrane protein 2 (VAMP2)-patterned peptide (INCI name: Acetyl sh-Oligopeptide-26 sh-Oligopeptide-27 SP, trade name: M.Biome-BT) on improving skin function in vitro. METHODS: The cytotoxicity of CPP-conjugated SNARE motif of VAMP2-patterned peptide (CVP) was investigated using the 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT) assay against B16-F10 cells and human dermal fibroblasts (HDFs) and a reconstructed skin irritation test. The anti-wrinkle activity of M.Biome-BT was determined by assessing the release of norepinephrine and dopamine in PC-12 cells via ELISA. The skin-whitening effects of CVP were assessed in B16-F10 cells by measuring the intra- and extracellular melanin contents and expression levels of melanin production-related genes, such as microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein-1 (TRP-1), and TRP-2. RESULTS: CVP is not cytotoxic to B16-F10 cells and HDFs, and no skin irritation was observed. CVP treatment considerably diminished K+ -induced norepinephrine and dopamine secretion compared with the non-treated control group (62% and 40%, respectively). Additionally, the inhibition ability of CVP on norepinephrine and dopamine release was comparable to that of botulinum neurotoxin type A (BoNT/A). CVP also increased intracellular melanin content in a dose-dependent manner, whereas extracellular melanin content decreased (76%-85%). However, CVP treatment did not affect the mRNA expression of MITF, TYR, TRP-1, and TRP-2. These results suggest that CVP does not inhibit melanin production; however, it may induce a whitening effect by inhibiting melanin transport. CONCLUSIONS: Taken together, our findings indicate that CVP could be used as an active and safe cosmeceutical ingredient for antiaging applications.

Analysis of NAPA Gene Expression in Brain Structures of Wistar Rats during the Formation of Long-Term Spatial Memory and Physical Activity under Stress Situation.

In the cerebellum, hippocampus, and prefrontal cortex of mature male Wistar rats with trained spatial navigational skill in the Morris water maze, the transcriptional activity the NAPA gene that regulates the transport and secretion of synaptic vesicles, release of neurotransmitters, and protein degradation was determined by real-time PCR. Animals subjected to forced swimming in a time-matched regime (active control) and naïve rats were used as the comparison groups. Suppression of NAPA gene activity was found in the hippocampus and cerebellum of the active control group, while navigation skill training led to a significant increase in gene expression in all brain structures under study. The findings suggest the existence of specific mechanisms regulating NAPA gene activity during the formation of spatial memory and adaptive behavior under stress conditions.

Loss-of-function of an α-SNAP gene confers resistance to soybean cyst nematode.

Plant-parasitic nematodes are one of the most economically impactful pests in agriculture resulting in billions of dollars in realized annual losses worldwide. Soybean cyst nematode (SCN) is the number one biotic constraint on soybean production making it a priority for the discovery, validation and functional characterization of native plant resistance genes and genetic modes of action that can be deployed to improve soybean yield across the globe. Here, we present the discovery and functional characterization of a soybean resistance gene, GmSNAP02. We use unique bi-parental populations to fine-map the precise genomic location, and a combination of whole genome resequencing and gene fragment PCR amplifications to identify and confirm causal haplotypes. Lastly, we validate our candidate gene using CRISPR-Cas9 genome editing and observe a gain of resistance in edited plants. This demonstrates that the GmSNAP02 gene confers a unique mode of resistance to SCN through loss-of-function mutations that implicate GmSNAP02 as a nematode virulence target. We highlight the immediate impact of utilizing GmSNAP02 as a genome-editing-amenable target to diversify nematode resistance in commercially available cultivars.

MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion.

Yeast vacuolar HOPS tethers membranes, catalyzes trans-SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted-vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage. The MED fusion blockade is overcome by either salt extraction, inactivation with the MED ligand calmodulin, or addition of Sec17/Sec18. During incubation with MED, SNAREs assemble stable complexes in trans and fusion becomes resistant to antibody to the Qa SNARE. When Q-SNAREs are preassembled, a synthetic tether can replace HOPS for fusion. With a synthetic tether, fusion needs both complete SNARE zippering and Sec17/Sec18 to overcome a MED block. In contrast, when SNARE domains are only two-third zippered, only HOPS will support Sec17/Sec18 driven fusion without needing complete zippering. HOPS thus remains engaged with SNAREs during zippering. MED facilitates the study of distinct fusion stages: tethering, initial trans-SNARE assembly and its sensitivity to Sec17, SNARE zippering, Sec17/Sec18 engagement, and lipid and lumenal mixing.

Structural Mechanism of Soluble N-ethylmaleimide-Sensitive Factor Attachment Protein Receptor Complex Assembly in Lipid Bilayers Revealed by Solid-State NMR.

Synaptic vesicle fusion is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins, including synaptobrevin-2 (Syb-2), syntaxin-1 (Syx-1), and SNAP-25. However, it remains controversial whether the formation of thoroughly contacted α-helical bundle from the SNARE motifs to the end of the transmembrane domains (TMDs) is necessary for SNARE-mediated membrane fusion. In this study, we characterized the conformation of Syb-2 in different assembly states using a combination of dipolar- and scalar-based solid-state NMR experiments in lipid bilayers. Our spectral analysis revealed a highly dynamic nature of the Syb-2 TMD with considerable α-helical contents. Chemical shift perturbation and mutational analysis indicated that the coupling between Syb-2 and Syx-1 TMDs mediated by residue Gly-100 of Syb-2 together with high mobility of the C-terminal segment of Syb-2 TMD are required for inner membrane merger. Our results provide new insights into the role of the Syb-2 TMD in driving membrane fusion, which improves the current understanding of the structural mechanism of SNARE complex assembly. This study highlights the significance of membrane environments in elucidating the mechanism of membrane proteins.

Sensory deficit screen identifies nsf mutation that differentially affects SNARE recycling and quality control.

The AAA+ NSF complex is responsible for SNARE complex disassembly both before and after membrane fusion. Loss of NSF function results in pronounced developmental and degenerative defects. In a genetic screen for sensory deficits in zebrafish, we identified a mutation in nsf, I209N, that impairs hearing and balance in a dosage-dependent manner without accompanying defects in motility, myelination, and innervation. In vitro experiments demonstrate that while the I209N NSF protein recognizes SNARE complexes, the effects on disassembly are dependent upon the type of SNARE complex and I209N concentration. Higher levels of I209N protein produce a modest decrease in binary (syntaxin-SNAP-25) SNARE complex disassembly and residual ternary (syntaxin-1A-SNAP-25-synaptobrevin-2) disassembly, whereas at lower concentrations binary disassembly activity is strongly reduced and ternary disassembly activity is absent. Our study suggests that the differential effect on disassembly of SNARE complexes leads to selective effects on NSF-mediated membrane trafficking and auditory/vestibular function.

Hepatocyte CD36 modulates UBQLN1-mediated proteasomal degradation of autophagic SNARE proteins contributing to septic liver injury.

Macroautophagy/autophagy plays a protective role in sepsis-induced liver injury. As a member of class B scavenger receptors, CD36 plays important roles in various disorders, such as atherosclerosis and fatty liver disease. Here we found that the expression of CD36 in hepatocytes was increased in patients and a mouse model with sepsis, accompanied by impaired autophagy flux. Furthermore, hepatocyte cd36 knockout (cd36-HKO) markedly improved liver injury and the impairment of autophagosome-lysosome fusion in lipopolysaccharide (LPS)-induced septic mice. Ubqln1 (ubiquilin 1) overexpression (OE) in hepatocyte blocked the protective effect of cd36-HKO on LPS-induced liver injury in mice. Mechanistically, with LPS stimulation, CD36 on the plasma membrane was depalmitoylated and distributed to the lysosome, where CD36 acted as a bridge molecule linking UBQLN1 to soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and hence promoting the proteasomal degradation of SNARE proteins, resulting in fusion impairment. Overall, our data reveal that CD36 is essential for modulating the proteasomal degradation of autophagic SNARE proteins in a UBQLN1-dependent manner. Targeting CD36 in hepatocytes is effective for improving autophagic flux in sepsis and therefore represents a promising therapeutic strategy for clinical treatment of septic liver injury.Abbreviations: AAV8: adeno-associated virus 8; AOSC: acute obstructive suppurative cholangitis; ATP1A1: ATPase, Na+/K+ transporting, alpha 1 polypeptide; CASP3: caspase 3; CASP8: caspase 8; CCL2: chemokine (C-C motif) ligand 2; cd36-HKO: hepatocyte-specific cd36 knockout; Co-IP: co-immunoprecipitation; CQ: chloroquine; Cys: cysteine; GOT1: glutamic-oxaloacetic transaminase 1, soluble; GPT: glutamic-pyruvic transaminase, soluble; IL1B: interleukin 1 beta; IL6: interleukin 6; KO: knockout; LAMP1: lysosomal associated membrane protein 1; LDH, lactate dehydrogenase; LPS: lipopolysaccharide; LYPLA1: lysophospholipase 1; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; OE: overexpression; qPCR: quantitative polymerase chain reaction; SNAP29: synaptosome associated protein 29; SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SQSTM1/p62: sequestosome 1; STX17: syntaxin 17; TNF: tumor necrosis factor; TRIM: tripartite motif-containing; UBA: ubiquitin-associated; UBL: ubiquitin-like; UBQLN: ubiquilin; VAMP8: vesicle associated membrane protein 8; WT: wild-type.

Perfluorooctanoic acid induces tight junction injury of Sertoli cells by blocking autophagic flux.

Perfluorooctanoic acid (PFOA), a man-made chemical widely used in consumers, could cause male reproductive toxicity by disrupting blood-testis barrier (BTB) integrity. Autophagy in Sertoli cells is essential for regulation of spermatogenesis and BTB. However, it remains a mystery that whether PFOA-induced BTB injury is associated with autophagy in Sertoli cells. In this study, we found that PFOA dose-dependently disrupted tight junction (TJ) function in Sertoli cells in vivo and in vitro. Furthermore, the results from transmission electron microscopy, Western blot and immunofluorescence analysis revealed that PFOA induced the accumulation of autophagosome in testicular Sertoli cells as well as TM4 cells. Further study confirmed that autophagosome accumulation resulted from the blockage of autophagic degradation because of disruption of autophagosome and lysosome fusion via downregulation of the expression of α-SNAP. In parallel, the overexpressed MMP9 was also observed in vivo and in vitro. Conversely, overexpression of α-SNAP inhibited the expression of MMP9 in TM4 cells. In conclusion, PFOA blocks autophagic flux through downregulating the expression levels of α-SNAP in Sertoli cells, and then induces the accumulation of MMP9 leading to disruption of TJ function. This finding will provide clues for effective prevention and treatment of PFOA-induced male reproductive toxicity.

Unveiling the Nanoscale Dynamics of the Exocytic Machinery in Chromaffin Cells with Single-Molecule Imaging.

Neuronal and hormonal communication relies on the exocytic fusion of vesicles containing neurotransmitters and hormones with the plasma membrane. This process is tightly regulated by key protein-protein and protein-lipid interactions and culminates in the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex formation and zippering that promotes vesicular fusion. Located on both sides of the vesicle and the plasma membrane, the zippering of the SNARE complex acts to overcome the energy barrier afforded by the repulsive electrostatic force stemming from apposing two negatively charged phospholipid membranes. Another component opposing the timely organization of the fusion machinery is thermal Brownian energy that tends to homogenize all cellular molecules by constantly switching their motions and directions through short-lived molecular interactions. Much less is known of the mechanisms counteracting these chaotic forces, allowing seamless cellular functions such as exocytic fusion. Super-resolution microscopy techniques such as single-molecule imaging have proven useful to start uncovering these nanoscale mechanisms. Here, we used single-particle tracking photoactivatable localization microscopy (sptPALM) to track syntaxin-1-mEos, a SNARE protein located on the plasma membrane of cultured bovine chromaffin cells. We demonstrate that syntaxin-1-mEos undergoes dramatic change in its mobility in response to secretagogue stimulation leading to increased nanoclustering. These nanoclusters are transient in nature and likely to provide docked vesicles with a molecular environment conducive to exocytic fusion.

VAMP724 and VAMP726 are involved in autophagosome formation in Arabidopsis thaliana.

Macroautophagy/autophagy, an evolutionarily conserved degradative process essential for cell homeostasis and development in eukaryotes, involves autophagosome formation and fusion with a lysosome/vacuole. The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play important roles in regulating autophagy in mammals and yeast, but relatively little is known about SNARE function in plant autophagy. Here we identified and characterized two Arabidopsis SNAREs, AT4G15780/VAMP724 and AT1G04760/VAMP726, involved in plant autophagy. Phenotypic analysis showed that mutants of VAMP724 and VAMP726 are sensitive to nutrient-starved conditions. Live-cell imaging on mutants of VAMP724 and VAMP726 expressing YFP-ATG8e showed the formation of abnormal autophagic structures outside the vacuoles and compromised autophagic flux. Further immunogold transmission electron microscopy and electron tomography (ET) analysis demonstrated a direct connection between the tubular autophagic structures and the endoplasmic reticulum (ER) in vamp724-1 vamp726-1 double mutants. Further transient co-expression, co-immunoprecipitation and double immunogold TEM analysis showed that ATG9 (autophagy related 9) interacts and colocalizes with VAMP724 and VAMP726 in ATG9-positive vesicles during autophagosome formation. Taken together, VAMP724 and VAMP726 regulate autophagosome formation likely working together with ATG9 in Arabidopsis.Abbreviations: ATG, autophagy related; BTH, benzo-(1,2,3)-thiadiazole-7-carbothioic acid S-methyl ester; Conc A, concanamycin A; EM, electron microscopy; ER, endoplasmic reticulum; FRET, Förster/fluorescence resonance energy transfer; MS, Murashige and Skoog; MVB, multivesicular body; PAS, phagophore assembly site; PM, plasma membrane; PVC, prevacuolar compartment; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; TEM, transmission electron microscopy; TGN, trans-Golgi network; WT, wild-type.

SNARE Modulators and SNARE Mimetic Peptides.

The soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein (SNAP) receptor (SNARE) proteins play a central role in most forms of intracellular membrane trafficking, a key process that allows for membrane and biocargo shuffling between multiple compartments within the cell and extracellular environment. The structural organization of SNARE proteins is relatively simple, with several intrinsically disordered and folded elements (e.g., SNARE motif, N-terminal domain, transmembrane region) that interact with other SNAREs, SNARE-regulating proteins and biological membranes. In this review, we discuss recent advances in the development of functional peptides that can modify SNARE-binding interfaces and modulate SNARE function. The ability of the relatively short SNARE motif to assemble spontaneously into stable coiled coil tetrahelical bundles has inspired the development of reduced SNARE-mimetic systems that use peptides for biological membrane fusion and for making large supramolecular protein complexes. We evaluate two such systems, based on peptide-nucleic acids (PNAs) and coiled coil peptides. We also review how the self-assembly of SNARE motifs can be exploited to drive on-demand assembly of complex re-engineered polypeptides.

Diesel exhaust particles induce human umbilical vein endothelial cells apoptosis by accumulation of autophagosomes and caspase-8 activation.

Diesel exhaust particles (DEP) are risk factors for endothelial cells (ECs) dysfunction. However, the mechanism by which DEP induce ECs apoptosis remains unclear. Here, we investigated how DEP induce death of human umbilical vein ECs (HUVECs), with a focus on the autophagy-mediated apoptotic pathway. DEP induced dose-dependent HUVECs death and exposure to the IC50 concentration of DEP (70 µg/ml) led to apoptosis. DEP phosphorylated Beclin-1 (Ser93) and increased protein levels of p62 and LC3BII and the number of LC3B puncta, indicating autophagy initiation. DEP increased expression of pro- and mature forms of cathepsin D, which increases lysosomal activity. However, DEP suppressed expression of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor proteins (STX17, VAMP8, SNAP29, YKT6, and STX7) to inhibit autolysosome formation, resulting in accumulation of autophagosomes. LC3B, p62, and caspase-8 form a tertiary complex in accumulated autophagosomes, which is known to serve as a platform for caspase-8 activation. Indeed, DEP activates caspase-8 and pretreatment with a caspase-8 inhibitor suppressed DEP-induced apoptosis. Furthermore, depletion of p62 decreased caspase-8 and caspase-3 activation and inhibited the DEP-induced apoptosis. Taken together, these findings demonstrated that DEP induced HUVECs apoptosis by inhibiting autophagosome maturation and identified caspase-8 as a novel mediator of DEP-induced ECs apoptosis.

Rab3 and synaptotagmin proteins in the regulation of vesicle fusion and neurotransmitter release.

Ca2+-triggered neurotransmitter release involves complex regulatory mechanisms, including a series of protein-protein interactions. Three proteins, synaptobrevin (VAMP), synaptosomal-associated protein of 25kDa (SNAP-25) and syntaxin, constitute the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core complex that plays key roles in controlling vesicle fusion and exocytosis. Many other proteins participate in the regulation of the processes via direct and/or indirect interaction with the SNARE complex. Although much effort has been made, the regulatory mechanism for exocytosis is still not completely clear. Accumulated evidence indicates that the small GTPase Rab3 and synaptotagmin proteins play important regulatory roles during vesicle fusion and neurotransmitter release. This review outlines our present understanding of the two regulatory proteins, with the focus on the interaction of Rab3 with synaptotagmin in the regulatory process.

Rabphilin 3A binds the N-peptide of SNAP-25 to promote SNARE complex assembly in exocytosis.

Exocytosis of secretory vesicles requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and small GTPase Rabs. As a Rab3/Rab27 effector protein on secretory vesicles, Rabphilin 3A was implicated to interact with SNAP-25 to regulate vesicle exocytosis in neurons and neuroendocrine cells, yet the underlying mechanism remains unclear. In this study, we have characterized the physiologically relevant binding sites between Rabphilin 3A and SNAP-25. We found that an intramolecular interplay between the N-terminal Rab-binding domain and C-terminal C2AB domain enables Rabphilin 3A to strongly bind the SNAP-25 N-peptide region via its C2B bottom α-helix. Disruption of this interaction significantly impaired docking and fusion of vesicles with the plasma membrane in rat PC12 cells. In addition, we found that this interaction allows Rabphilin 3A to accelerate SNARE complex assembly. Furthermore, we revealed that this interaction accelerates SNARE complex assembly via inducing a conformational switch from random coils to α-helical structure in the SNAP-25 SNARE motif. Altogether, our data suggest that the promotion of SNARE complex assembly by binding the C2B bottom α-helix of Rabphilin 3A to the N-peptide of SNAP-25 underlies a pre-fusion function of Rabphilin 3A in vesicle exocytosis.

Sec18 supports membrane fusion by promoting Sec17 membrane association.

Membrane fusion is driven by Sec17, Sec18, and SNARE zippering. Sec17 bound to SNAREs promotes fusion through its membrane-proximal N-terminal apolar loop domain. At its membrane-distal end, Sec17 serves as a high-affinity receptor for Sec18. At that distance from the fusion site, it has been unclear how Sec18 can aid Sec17 to promote fusion. We now report that Sec18, with ATPγS, lowers the Km of Sec17 for fusion. A C-terminal and membrane-distal Sec17 mutation, L291A,L292A, diminishes Sec17 affinity for Sec18. High levels of wild-type Sec17 or Sec17-L291AL292A show equivalent fusion without Sec18, but Sec18 causes far less fusion enhancement with low levels of Sec17-L291AL292A than with wild-type Sec17. Another mutant, Sec17-F21SM22S, has reduced N-loop apolarity. Only very high levels of this mutant protein support fusion, but Sec18 still lowers the apparent fusion Km for Sec17-F21SM22S. Thus Sec18 stimulates fusion through Sec17 and acts at the well-described interface between Sec18 and Sec17. ATP acts as a ligand to activate Sec18 for Sec17-dependent fusion, but ATP hydrolysis is not required. Even without SNAREs, Sec18 and Sec17 exhibit interdependent stable association with lipids, with several Sec17 bound for each Sec18 hexamer, explaining how Sec18 stabilization of surface-concentrated clusters of Sec17 lowers the Sec17 Km for assembly with SNAREs. Each of the associations, between SNARE complex, Sec18, Sec17, and lipid, helps assemble the fusion machinery.

Complexin-1 and synaptotagmin-1 compete for binding sites on membranes containing PtdInsP2.

Complexin-1 is an essential protein for neuronal exocytosis that acts to depress spontaneous fusion events while enhancing evoked neurotransmitter release. In addition to binding soluble N-ethylmaleimide-sensitive factor attachment protein receptors, it is well established that complexin associates with membranes in a manner that depends upon membrane curvature. In the present work, we examine the membrane binding of complexin using electron paramagnetic resonance spectroscopy, fluorescence anisotropy, and total internal reflection fluorescence microscopy. The apparent membrane affinity of complexin is found to strongly depend upon the concentration of protein used in the binding assay, and this is a result of a limited number of binding sites for complexin on the membrane interface. Although both the N- and C-terminal regions of complexin associate with the membrane interface, membrane affinity is driven by its C-terminus. Complexin prefers to bind liquid-disordered membrane phases and shows an enhanced affinity toward membranes containing phosphatidylinositol 4-5-bisphosphate (PI(4,5)P2). In the presence of PI(4,5)P2, complexin is displaced from the membrane surface by proteins that bind to or sequester PI(4,5)P2. In particular, the neuronal calcium sensor synaptotagmin-1 displaces complexin from the membrane but only when PI(4,5)P2 is present. Complexin and synaptotagmin compete on the membrane interface in the presence of PI(4,5)P2, and this interaction may play a role in calcium-triggered exocytosis by displacing complexin from its fusion-inhibiting state.

Munc18 - Munc13-dependent pathway of SNARE complex assembly is resistant to NSF and α-SNAP.

Synaptic exocytosis requires efficient SNARE complex assembly that is precisely regulated by multiple regulatory proteins. Increasing evidence suggests that Munc18-1 and Munc13-1 protect SNARE complex assembly in a manner resistant to NSF and α-SNAP. However, the protective mechanisms of Munc18-1 and Munc13-1 are not fully understood. Here, by analyzing two pathways of SNARE complex assembly (i.e., the Munc18 - Munc13-dependent pathway and the Munc18 - Munc13-independent pathway), we found that the Munc18 - Munc13-dependent pathway of SNARE complex assembly is resistant to NSF - α-SNAP. In this pathway, Munc18-1 and Munc13-1 each, independently, have protective effects. The protective effect of Munc18-1 relies on the interaction with the C-terminal part of Syb2 during the transition from the Munc18-1/Syx1 complex to the SNARE complex. Moreover, the protective effect of Munc13-1 is most likely attributed to its ability in templating the assembling SNAREs. In addition, we found that the Munc18 - Munc13-dependent pathway opposes the association of α-SNAP with the SNARE bundle, thus explaining how this pathway is resistant to NSF - α-SNAP disassembly. Although the above results were derived from the studies on SNARE complex in solution or in cis-configurations, instead of trans-configurations residing on the opposite membrane, our data could still help to understand the protective mechanism of Munc18-1 and Munc13-1 in SNARE-mediated synaptic exocytosis.