Soluble N-ethylmaleimide-sensitive Factor Attachment Protein Receptor Complex Assembly Research Articles

VAMP2 chaperones α-synuclein in synaptic vesicle co-condensates.

α-Synuclein (α-Syn) aggregation is closely associated with Parkinson's disease neuropathology. Physiologically, α-Syn promotes synaptic vesicle (SV) clustering and soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex assembly. However, the underlying structural and molecular mechanisms are uncertain and it is not known whether this function affects the pathological aggregation of α-Syn. Here we show that the juxtamembrane region of vesicle-associated membrane protein 2 (VAMP2)-a component of the SNARE complex that resides on SVs-directly interacts with the carboxy-terminal region of α-Syn through charged residues to regulate α-Syn's function in clustering SVs and promoting SNARE complex assembly by inducing a multi-component condensed phase of SVs, α-Syn and other components. Moreover, VAMP2 binding protects α-Syn against forming aggregation-prone oligomers and fibrils in these condensates. Our results suggest a molecular mechanism that maintains α-Syn's function and prevents its pathological amyloid aggregation, the failure of which may lead to Parkinson's disease.

Phosphorylation of Doc2 by EphB2 modulates Munc13-mediated SNARE complex assembly and neurotransmitter release.

At the synapse, presynaptic neurotransmitter release is tightly controlled by release machinery, involving the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins and Munc13. The Ca2+ sensor Doc2 cooperates with Munc13 to regulate neurotransmitter release, but the underlying mechanisms remain unclear. In our study, we have characterized the binding mode between Doc2 and Munc13 and found that Doc2 originally occludes Munc13 to inhibit SNARE complex assembly. Moreover, our investigation unveiled that EphB2, a presynaptic adhesion molecule (SAM) with inherent tyrosine kinase functionality, exhibits the capacity to phosphorylate Doc2. This phosphorylation attenuates Doc2 block on Munc13 to promote SNARE complex assembly, which functionally induces spontaneous release and synaptic augmentation. Consistently, application of a Doc2 peptide that interrupts Doc2-Munc13 interplay impairs excitatory synaptic transmission and leads to dysfunction in spatial learning and memory. These data provide evidence that SAMs modulate neurotransmitter release by controlling SNARE complex assembly.

Ykt6 functionally overlaps with vacuolar and exocytic R-SNAREs in the yeast Saccharomyces cerevisiae

The soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex forms a 4-helix coiled-coil bundle consisting of 16 layers of interacting side chains upon membrane fusion. The central layer (layer 0) is highly conserved and comprises three glutamines (Q) and one arginine (R), and thus SNAREs are classified into Qa-, Qb-, Qc-, and R-SNAREs. Homotypic vacuolar fusion in Saccharomyces cerevisiae requires the SNAREs Vam3 (Qa), Vti1 (Qb), Vam7 (Qc), and Nyv1 (R). However, the yeast strain lacking NYV1 (nyv1Δ) shows no vacuole fragmentation, whereas the vam3Δ and vam7Δ strains display fragmented vacuoles. Here, we provide genetic evidence that the R-SNAREs Ykt6 and Nyv1 are functionally redundant in vacuole homotypic fusion in vivo using a newly isolated ykt6 mutant. We observed the ykt6-104 mutant showed no defect in vacuole morphology, but the ykt6-104 nyv1Δ double mutant had highly fragmented vacuoles. Furthermore, we show the defect in homotypic vacuole fusion caused by the vam7-Q284R mutation was compensated by the nyv1-R192Q or ykt6-R165Q mutations, which maintained the 3Q:1R ratio in the layer 0 of the SNARE complex, indicating that Nyv1 is exchangeable with Ykt6 in the vacuole SNARE complex. Unexpectedly, we found Ykt6 assembled with exocytic Q-SNAREs when the intrinsic exocytic R-SNAREs Snc1 and its paralog Snc2 lose their ability to assemble into the exocytic SNARE complex. These results suggest that Ykt6 may serve as a backup when other R-SNAREs become dysfunctional and that this flexible assembly of SNARE complexes may help cells maintain the robustness of the vesicular transport network.

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.

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.

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.

Conformational change of Syntaxin-3b in regulating SNARE complex assembly in the ribbon synapses

Neurotransmitter release of synaptic vesicles relies on the assembly of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, consisting of syntaxin and SNAP-25 on the plasma membrane and synaptobrevin on the synaptic vesicle. The formation of the SNARE complex progressively zippers towards the membranes, which drives membrane fusion between the plasma membrane and the synaptic vesicle. However, the underlying molecular mechanism of SNARE complex regulation is unclear. In this study, we investigated the syntaxin-3b isoform found in the retinal ribbon synapses using single-molecule fluorescence resonance energy transfer (smFRET) to monitor the conformational changes of syntaxin-3b that modulate the SNARE complex formation. We found that syntaxin-3b is predominantly in a self-inhibiting closed conformation, inefficiently forming the ternary SNARE complex. Conversely, a phosphomimetic mutation (T14E) at the N-terminal region of syntaxin-3b promoted the open conformation, similar to the constitutively open form of syntaxin LE mutant. When syntaxin-3b is bound to Munc18-1, SNARE complex formation is almost completely blocked. Surprisingly, the T14E mutation of syntaxin-3b partially abolishes Munc18-1 regulation, acting as a conformational switch to trigger SNARE complex assembly. Thus, we suggest a model where the conformational change of syntaxin-3b induced by phosphorylation initiates the release of neurotransmitters in the ribbon synapses.

Conformational dynamics of SNARE recycling mediated by NSF

Neurotransmitter release via synaptic vesicle exocytosis is mediated by the dynamic assembly and disassembly of the neuronal SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment protein receptor) complex, which consists of syntaxin‐1, SNAP‐25, and synaptobrevin‐2. Despite their importance, the molecular mechanism of SNARE complex recycling remains unclear. Individual SNARE proteins are intrinsically disordered and undergo a disorder‐to‐order transition, which assembles into a highly stable four‐helix bundle, providing the energy required to drive membrane fusion between the synaptic vesicle and the plasma membrane. The AAA+ protein NSF later disassembles the SNARE complex to maintain a pool of the individually functional SNARE proteins to be utilized for recurring rounds of synaptic vesicle fusion. Using single‐molecule fluorescence resonance energy transfer (smFRET), we examined the stepwise conformational dynamics of individual SNARE proteins during NSF‐mediated disassembly and reassembly of the SNARE complex. Interestingly, the disorder‐to‐order transition of the SNARE proteins during SNARE complex assembly was reversible by NSF‐mediated disassembly, where the SNARE chaperone Munc18 preserved the intrinsically disordered state of SNAP‐25 and synaptobrevin‐2 by locking syntaxin‐1 in an inhibiting closed conformation. Moreover, we observed a transient “entangled” conformation of SNAP‐25 during the reassembly process of the SNARE complex. Together, NSF acts as a protein quality control mechanism for efficient membrane fusion via proper assembly of the SNARE complex.

SNAP25/syntaxin4/VAMP2/Munc18-1 Complexes in Spinal Dorsal Horn Contributed to Inflammatory Pain

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) have been implicated in the trafficking of postsynaptic glutamate receptors, including N-methyl-d-aspartate (NMDA)-subtype glutamate receptors (NMDARs) that are critical for nociceptive plasticity and behavioral sensitization. However, the components of SNAREs complex involved in spinal nociceptive processing remain largely unknown. Here we found that SNAP25, syntaxin4, VAMP2 and Munc18-1 were localized at postsynaptic sites and formed the complex in the superficial lamina of spinal cord dorsal horn of rats. The complex formation between these SNAREs components were accelerated after intraplantar injection of complete Freund’s adjuvant (CFA), pharmacological removal of GABAergic inhibition or activation of NMDAR in intact rats. The increased SNAP25/syntaxin4/VAMP2/Munc18-1 interaction facilitated the surface delivery and synaptic accumulation of NMDAR during inflammatory pain. Disruption of the molecular interaction between SNAP25 with its SNARE partners by using a blocking peptide derived from the C-terminus of SNAP25 effectively repressed the surface and synaptic accumulation of GluN2B-containing NMDARs in CFA-injected rats. This peptide also alleviated inflammatory mechanical allodynia and thermal hypersensitivity. These data suggested that SNAREs complex assembly in spinal cord dorsal horn was involved in the inflammatory pain hypersensitivity through promoting NMDAR synaptic trafficking.

Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores

The opening of a fusion pore during exocytosis creates the first aqueous connection between the lumen of a vesicle and the extracellular space. Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediate the formation of these dynamic structures, and their kinetic transitions are tightly regulated by accessory proteins at the synapse. Here, we utilize two single molecule approaches, nanodisc-based planar bilayer electrophysiology and single-molecule FRET, to address the relationship between SNARE complex assembly and rapid (micro-millisecond) fusion pore transitions, and to define the role of accessory proteins. Synaptotagmin (syt) 1, a major Ca2+-sensor for synaptic vesicle exocytosis, drove the formation of an intermediate: committed trans-SNARE complexes that form large, stable pores. Once open, these pores could only be closed by the action of the ATPase, NSF. Time-resolved measurements revealed that NSF-mediated pore closure occurred via a complex ‘stuttering’ mechanism. This simplified system thus reveals the dynamic formation and dissolution of fusion pores.

K+ Channel-SEC11 Binding Exchange Regulates SNARE Assembly for Secretory Traffic.

Cell expansion requires that ion transport and secretory membrane traffic operate in concert. Evidence from Arabidopsis (Arabidopsis thaliana) indicates that such coordination is mediated by physical interactions between subsets of so-called SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins, which drive the final stages of vesicle fusion, and K+ channels, which facilitate uptake of the cation to maintain cell turgor pressure as the cell expands. However, the sequence of SNARE binding with the K+ channels and its interweaving within the events of SNARE complex assembly for exocytosis remains unclear. We have combined protein-protein interaction and electrophysiological analyses to resolve the binding interactions of the hetero-oligomeric associations. We find that the RYxxWE motif, located within the voltage sensor of the K+ channels, is a nexus for multiple SNARE interactions. Of these, K+ channel binding and its displacement of the regulatory protein SEC11 is critical to prime the Qa-SNARE SYP121. Our results indicate a stabilizing role for the Qbc-SNARE SNAP33 in the Qa-SNARE transition to SNARE complex assembly with the R-SNARE VAMP721. They also suggest that, on its own, the R-SNARE enters an anomalous binding mode with the channels, possibly as a fail-safe measure to ensure a correct binding sequence. Thus, we suggest that SYP121 binding to the K+ channels serves the role of a primary trigger to initiate assembly of the secretory machinery for exocytosis.

Dual Sites for SEC11 on the SNARE SYP121 Implicate a Binding Exchange during Secretory Traffic.

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins facilitate vesicle traffic through their assembly in a heteromeric complex that drives membrane fusion. Much of vesicle traffic at the Arabidopsis (Arabidopsis thaliana) plasma membrane is subject to the Sec1/Munc18 protein SEC11, which, along with plasma membrane K+ channels, selectively binds with the SNARE SYP121 to regulate its assembly in complex. How SEC11 binding is coordinated with the K+ channels is poorly understood, as both SEC11 and the channels are thought to compete for the same SNARE binding site. Here, we identify a second binding motif within the N terminus of SYP121 and demonstrate that this motif affects SEC11 binding independently of the F9xRF motif that is shared with the K+ channels. This second, previously unrecognized motif is centered on residues R20R21 of SYP121 and is essential for SEC11 interaction with SYP121. Mutation of the R20R21 motif blocked vesicle traffic without uncoupling the effects of SYP121 on solute and K+ uptake associated with the F9xRF motif; the mutation also mimicked the effects on traffic block observed on coexpression of the dominant-negative SEC11Δ149 fragment. We conclude that the R20R21 motif represents a secondary site of interaction for the Sec1/Munc18 protein during the transition of SYP121 from the occluded to the open conformation that leads to SNARE complex assembly.

Focused clamping of a single neuronal SNARE complex by complexin under high mechanical tension

Neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) catalyze synaptic vesicle fusion with presynaptic membranes through the formation of SNARE complexes. Complexin (Cpx) is the only presynaptic protein that tightly binds to SNAREs and regulates membrane fusion, but how it modulates the energy landscape of SNARE complex assembly, especially under mechanical tension on the complex, remains unclear. Here, using magnetic tweezers, we report how Cpx interacts with single SNARE complexes. The effects of Cpx manifest only under high mechanical tensions above 13 pN. Cpx stabilizes the central four-helix bundle of SNARE motifs and, at the same time, prevents the complete zippering of SNAREs by inhibiting linker-domain assembly. These results suggest that Cpx generates a focused clamp for the neuronal SNARE complex in a linker-open conformation. Our results provide a hint as to how Cpx cooperates with neuronal SNAREs to prime synaptic vesicles in preparation for synchronous neurotransmitter release.

SNARE complex assembly and disassembly

A fundamental hallmark of eukaryotic cells is their compartmentalization into functionally distinct organelles, including those of the secretory and endocytic pathways. Transport of cargo between these compartments and to/from the cell surface is mediated by membrane-bound vesicles and tubules. Delivery of cargo is facilitated by SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated membrane fusion of vesicles with their target compartments. Vesicles contain a variety of cargos, including lipids, membrane proteins, signaling molecules, biosynthetic and hydrolytic enzymes, and the trafficking machinery itself. Proper function of membrane trafficking is required for cellular growth, division, movement, and cell-cell communication. Defects in these processes have been implicated in a variety of human diseases, such as cancer, diabetes, neurodegenerative disorders, ciliopathies, and infections. The elucidation of the mechanisms of SNARE assembly and disassembly is key to understanding how membrane fusion is regulated throughout eukaryotes. Here, we introduce the SNARE proteins, their structures and functions in eukaryotic cells, and discuss recent breakthroughs in elucidating the regulation of SNARE assembly and disassembly through the use of high-resolution structural biology and biophysical techniques.

Sec3 promotes the initial binary t-SNARE complex assembly and membrane fusion

The soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (SNAREs) constitute the core machinery for membrane fusion during eukaryotic cell vesicular trafficking. However, how the assembly of the SNARE complex is initiated is unknown. Here we report that Sec3, a component of the exocyst complex that mediates vesicle tethering during exocytosis, directly interacts with the t-SNARE protein Sso2. This interaction promotes the formation of an Sso2-Sec9 ‘binary’ t-SNARE complex, the early rate-limiting step in SNARE complex assembly, and stimulates membrane fusion. The crystal structure of the Sec3-Sso2 complex suggests that Sec3 binding induces conformational changes of Sso2 that are crucial for the relief of its auto-inhibition. Interestingly, specific disruption of the Sec3–Sso2 interaction in cells blocks exocytosis without affecting the function of Sec3 in vesicle tethering. Our study reveals an activation mechanism for SNARE complex assembly, and uncovers a role of the exocyst in promoting membrane fusion in addition to vesicle tethering.

VAMP721 Conformations Unmask an Extended Motif for K+ Channel Binding and Gating Control.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins play a major role in membrane fusion and contribute to cell expansion, signaling, and polar growth in plants. The SNARE SYP121 of Arabidopsis thaliana that facilitates vesicle fusion at the plasma membrane also binds with, and regulates, K+ channels already present at the plasma membrane to affect K+ uptake and K+-dependent growth. Here, we report that its cognate partner VAMP721, which assembles with SYP121 to drive membrane fusion, binds to the KAT1 K+ channel via two sites on the protein, only one of which contributes to channel-gating control. Binding to the VAMP721 SNARE domain suppressed channel gating. By contrast, interaction with the amino-terminal longin domain conferred specificity on VAMP721 binding without influencing gating. Channel binding was defined by a linear motif within the longin domain. The SNARE domain is thought to wrap around this structure when not assembled with SYP121 in the SNARE complex. Fluorescence lifetime analysis showed that mutations within this motif, which suppressed channel binding and its effects on gating, also altered the conformational displacement between the VAMP721 SNARE and longin domains. The presence of these two channel-binding sites on VAMP721, one also required for SNARE complex assembly, implies a well-defined sequence of events coordinating K+ uptake and the final stages of vesicle traffic. It suggests that binding begins with VAMP721, and subsequently with SYP121, thereby coordinating K+ channel gating during SNARE assembly and vesicle fusion. Thus, our findings also are consistent with the idea that the K+ channels are nucleation points for SNARE complex assembly.

Distinct Functions of Syntaxin-1 in Neuronal Maintenance, Synaptic Vesicle Docking, and Fusion in Mouse Neurons.

Neurotransmitter release requires the formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes by SNARE proteins syntaxin-1 (Stx1), synaptosomal-associated protein 25 (SNAP-25), and synaptobrevin-2 (Syb2). In mammalian systems, loss of SNAP-25 or Syb2 severely impairs neurotransmitter release; however, complete loss of function studies for Stx1 have been elusive due to the functional redundancy between Stx1 isoforms Stx1A and Stx1B and the embryonic lethality of Stx1A/1B double knock-out (DKO) mice. Here, we studied the roles of Stx1 in neuronal maintenance and neurotransmitter release in mice with constitutive or conditional deletion of Stx1B on an Stx1A-null background. Both constitutive and postnatal loss of Stx1 severely compromised neuronal viability in vivo and in vitro, indicating an obligatory role of Stx1 for maintenance of developing and mature neurons. Loss of Munc18-1, a high-affinity binding partner of Stx1, also showed severely impaired neuronal viability, but with a slower time course compared with Stx1A/1B DKO neurons, and exogenous Stx1A or Stx1B expression significantly delayed Munc18-1-dependent lethality. In addition, loss of Stx1 completely abolished fusion-competent vesicles and severely impaired vesicle docking, demonstrating its essential roles in neurotransmission. Putative partial SNARE complex assembly with the SNARE motif mutant Stx1A(AV) (A240V, V244A) was not sufficient to rescue neurotransmission despite full recovery of vesicle docking and neuronal survival. Together, these data suggest that Stx1 has independent functions in neuronal maintenance and neurotransmitter release and complete SNARE complex formation is required for vesicle fusion and priming, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Syntaxin-1 (Stx1) is a component of the synaptic vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex and is essential for neurotransmission. We present the first detailed loss-of-function characterization of the two Stx1 isoforms in central mammalian neurons. We show that Stx1 is fundamental for maintenance of developing and mature neurons and also for vesicle docking and neurotransmission. We also demonstrate that neuronal maintenance and neurotransmitter release are regulated by Stx1 through independent functions. Furthermore, we show that SNARE complex formation is required for vesicle fusion, whereas partial SNARE complex formation is sufficient for vesicle docking and neuronal maintenance. Therefore, our work provides insights into differential functions of Stx1 in neuronal maintenance and neurotransmission, with the latter explored further into its functions in vesicle docking and fusion.

Amyloid-β Oligomers May Impair SNARE-Mediated Exocytosis by Direct Binding to Syntaxin 1a.

Alzheimer's disease (AD) is closely associated with synaptic dysfunction, and thus current treatments often aim to stimulate neurotransmission to improve cognitive impairment. Whereas the formation of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic transmission, the correlation between SNAREs and AD neuropathology is unknown. Here, we report that intracellular amyloid-β (Aβ) oligomers directly inhibit SNARE-mediated exocytosis by impairing SNARE complex formation. We observe abnormal reduction of SNARE complex levels in the brains of APP/PS1 transgenic (TG) mice compared to age-matched wild-types. We demonstrate that Aβ oligomers block SNARE complex assembly through the direct interaction with a target membrane (t)-SNARE syntaxin 1a invitro. Furthermore, the results of the invitro single-vesicle content-mixing assay reveal that Aβ oligomers inhibit SNARE-mediated fusion pores. Thus, our study identifies apotential molecular mechanism by which intracellular Aβ oligomers hamper SNARE-mediated exocytosis, likely leading to AD-associated synaptic dysfunctions.

Examination of Sec22 Homodimer Formation and Role in SNARE-dependent Membrane Fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) protein complexes play essential roles in catalyzing intracellular membrane fusion events although the assembly pathway and molecular arrangement of SNARE complexes in membrane fusion reactions are not well understood. Here we monitored interactions of the R-SNARE protein Sec22 through a cysteine scanning approach and detected efficient formation of cross-linked Sec22 homodimers in cellular membranes when cysteine residues were positioned in the SNARE motif or C terminus of the transmembrane domain. When specific Sec22 cysteine derivatives are present on both donor COPII vesicles and acceptor Golgi membranes, the formation of disulfide cross-links provide clear readouts on trans- and cis-SNARE arrangements during this fusion event. The Sec22 transmembrane domain was required for efficient homodimer formation and for membrane fusion suggesting a functional role for Sec22 homodimers. We propose that Sec22 homodimers promote assembly of higher-order SNARE complexes to catalyze membrane fusion. Sec22 is also reported to function in macroautophagy and in formation of endoplasmic reticulum-plasma membrane contact sites therefore homodimer assembly may regulate Sec22 activity across a range of cellular processes.

Prefusion Structures of Lipid-Bound SNARE Proteins Suggest Folding Pathways of Trans-SNARE Complex

The assembly of three neuronal soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) proteins synaptobrevin 2, syntaxin-1A, and SNAP-25 is the key step that leads to exocytotic fusion of synaptic vesicles. In the fully assembled SNARE complex, these three proteins form a coiled-coil four-helix bundle structure by interaction of their respective SNARE motifs. Although biochemical and mutational analyses strongly suggest that the heptad-repeat SNARE motifs zipper into the final structure in the N- to C-terminal direction, little is known about the prefusion state of individual membrane-bound SNAREs and whether they change conformation from the unzippered prefusion to the zippered postfusion state in a continuous or step-wise fashion in membrane environments. We have solved the solution NMR structures of micelle-bound synaptobrevin and syntaxin-1A in their prefusion conformations. In addition to their respective transmembrane helices, the SNARE motifs of both proteins have considerable degrees of helical content. For synaptobrevin, only the N-terminal half (residues 36-54) of the SNARE motif forms a transient helix, and the fraction of helical content and interfacial association decreases as the protein is moved from micelle to bicelle to bilayer environments, suggesting that membrane curvature affects the folding of synaptobrevin. For syntaxin, the SNARE motif consists of two well-ordered, membrane-bound helices separated by the “0-layer” residue. These unexpected structural orders of the N- and C-terminal halves of the prefusion SNARE motifs suggest the formation of partially zippered SNARE complex intermediates. Interferometric fluorescence measurements in lipid bilayers confirm that the open SNARE motif helices interact with lipid bilayers and that the assembly of SNARE complexes involves the segmented movements of N- and C-terminal halves of SNARE motifs in relation to the membrane surface.