An Electrostatically Preferred Lateral Orientation of SNARE Complex Suggests Novel Mechanisms for Driving Membrane Fusion

Biological membrane fusion is a basic cellular process catalyzed by SNARE proteins and additional auxiliary factors. Yet, the critical mechanistic details of SNARE-catalyzed membrane fusion are poorly understood, especially during rapid synaptic transmission. Here, we systematically assessed the electrostatic forces between SNARE complex, auxiliary proteins and fusing membranes by the nonlinear Poisson-Boltzmann equation using explicit models of membranes and proteins. We found that a previously unrecognized, structurally preferred and energetically highly favorable lateral orientation exists for the SNARE complex between fusing membranes. This preferred orientation immediately suggests a novel and simple synaptotagmin-dependent mechanistic trigger of membrane fusion. Moreover, electrostatic interactions between membranes, SNARE complex, and auxiliary proteins appear to orchestrate a series of membrane curvature events that set the stage for rapid synaptic vesicle fusion. Together, our electrostatic analyses of SNAREs and their regulatory factors suggest unexpected and potentially novel mechanisms for eukaryotic membrane fusion proteins.     

Synip Arrests Soluble N-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE)-dependent Membrane Fusion as a Selective Target Membrane SNARE-binding Inhibitor

The vesicle fusion reaction in regulated exocytosis requires the concerted action of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) core fusion engine and a group of SNARE-binding regulatory factors. The regulatory mechanisms of vesicle fusion remain poorly understood in most exocytic pathways. Here, we reconstituted the SNARE-dependent vesicle fusion reaction of GLUT4 exocytosis in vitro using purified components. Using this defined fusion system, we discovered that the regulatory factor synip binds to GLUT4 exocytic SNAREs and inhibits the docking, lipid mixing, and content mixing of the fusion reaction. Synip arrests fusion by binding the target membrane SNARE (t-SNARE) complex and preventing the initiation of ternary SNARE complex assembly. Although synip also interacts with the syntaxin-4 monomer, it does not inhibit the pairing of syntaxin-4 with SNAP-23. Interestingly, synip selectively arrests the fusion reactions reconstituted with its cognate SNAREs, suggesting that the defined system recapitulates the biological functions of the vesicle fusion proteins. We further showed that the inhibitory function of synip is dominant over the stimulatory activity of Sec1/Munc18 proteins. Importantly, the inhibitory function of synip is distinct from how other fusion inhibitors arrest SNARE-dependent membrane fusion and therefore likely represents a novel regulatory mechanism of vesicle fusion.     

The Lipid Composition and Physical Properties of the Yeast Vacuole Affect the Hemifusion–Fusion Transition

Yeast vacuole fusion requires the formation of SNARE bundles between membranes. Although the function of vacuolar SNAREs is controlled in part by regulatory lipids, the exact role of the membrane in regulating fusion remains unclear. Because SNAREs are membrane‐anchored and transmit the force required for fusion to the bilayer, we hypothesized that the lipid composition and curvature of the membrane aid in controlling fusion. Here, we examined the effect of altering membrane fluidity and curvature on the functionality of fusion‐incompetent SNARE mutants that are thought to generate insufficient force to trigger the hemifusion–fusion transition. The hemifusion–fusion transition was inhibited by disrupting the 3Q:1R stoichiometry of SNARE bundles with the mutant SNARE Vam7p^Q283R. Similarly, replacing the transmembrane domain of the syntaxin homolog Vam3p with a lipid anchor allowed hemifusion, but not content mixing. Hemifusion‐stalled reactions containing either of the SNARE mutants were stimulated to fuse with chlorpromazine, an amphipathic molecule that alters membrane fluidity and curvature. The activity of mutant SNAREs was also rescued by the overexpression of SNAREs, thus multiplying the force transferred to the membrane. Thus, we conclude that either increasing membrane fluidity, or multiplying SNARE‐generated energy restored the fusogenicity of mutant SNAREs that are stalled at hemifusion. We also found that regulatory lipids differentially modulated the complex formation of wild‐type SNAREs. Together, these data indicate that the physical properties and the lipid composition of the membrane affect the function of SNAREs in promoting the hemifusion–fusion transition.     

Lipid-anchored Synaptobrevin Provides Little or No Support for Exocytosis or Liposome Fusion

SNARE proteins catalyze many forms of biological membrane fusion, including Ca²⁺-triggered exocytosis. Although fusion mediated by SNAREs generally involves proteins anchored to each fusing membrane by a transmembrane domain (TMD), the role of TMDs remains unclear, and previous studies diverge on whether SNAREs can drive fusion without a TMD. This issue is important because it relates to the question of the structure and composition of the initial fusion pore, as well as the question of whether SNAREs mediate fusion solely by creating close proximity between two membranes versus a more active role in transmitting force to the membrane to deform and reorganize lipid bilayer structure. To test the role of membrane attachment, we generated four variants of the synaptic v-SNARE synaptobrevin-2 (syb2) anchored to the membrane by lipid instead of protein. These constructs were tested for functional efficacy in three different systems as follows: Ca²⁺-triggered dense core vesicle exocytosis, spontaneous synaptic vesicle exocytosis, and Ca²⁺-synaptotagmin-enhanced SNARE-mediated liposome fusion. Lipid-anchoring motifs harboring one or two lipid acylation sites completely failed to support fusion in any of these assays. Only the lipid-anchoring motif from cysteine string protein-α, which harbors many lipid acylation sites, provided support for fusion but at levels well below that achieved with wild type syb2. Thus, lipid-anchored syb2 provides little or no support for exocytosis, and anchoring syb2 to a membrane by a TMD greatly improves its function. The low activity seen with syb2-cysteine string protein-α may reflect a slower alternative mode of SNARE-mediated membrane fusion.     

Docking of liposomes to planar surfaces mediated by trans-SNARE complexes

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) play a key role in membrane fusion in the secretory pathway. In vitro, SNAREs spontaneously assemble into helical SNARE complexes with the transmembrane domains at the C-terminal end. During fusion, SNAREs are thought to bridge the two membranes and assemble in a zipper-like fashion, pulling the membranes together and initiating fusion. However, it is not clear to what extent SNARE assembly contributes to membrane attachment and membrane fusion. Using the neuronal SNAREs synaptobrevin (VAMP), SNAP-25, and syntaxin as examples, we show here that liposomes containing synaptobrevin firmly attach to planar surfaces containing immobilized syntaxin. Attachment requires the formation of SNARE complexes because it is dependent on the presence of SNAP-25. Binding is competed for by soluble SNARE fragments, with noncognate SNAREs such as endobrevin (VAMP8), VAMP4, and VAMP7 (Ti-VAMP) being effective but less potent in some cases. Furthermore, although SNAP-23 is unable to substitute for SNAP-25 in the attachment assay, it forms complexes of comparable stability and is capable of substituting in liposome fusion assays. Vesicle attachment is initiated by SNARE assembly at the N-terminal end of the helix bundle. We conclude that SNAREs can indeed form stable trans-complexes that result in vesicle attachment if progression to fusion is prevented, further supporting the zipper model of SNARE function.     

A Dynamin Homolog Promotes the Transition from Hemifusion to Content Mixing in Intracellular Membrane Fusion

The convergence of the antagonistic reactions of membrane fusion and fission at the hemifusion/hemifission intermediate has generated a captivating enigma of whether Soluble N‐ethylmaleimide sensitive factor Attachment Protein Receptor (SNAREs) and dynamin have unusual counter‐functions in fission and fusion, respectively. SNARE‐mediated fusion and dynamin‐driven fission are fundamental membrane flux reactions known to occur during ubiquitous cellular communication events such as exocytosis, endocytosis and vesicle transport. Here we demonstrate the influence of the dynamin homolog Vps1 (Vacuolar protein sorting 1) on lipid mixing and content mixing properties of yeast vacuoles, and on the incorporation of SNAREs into fusogenic complexes. We propose a novel concept that Vps1, through its oligomerization and SNARE domain binding, promotes the hemifusion‐content mixing transition in yeast vacuole fusion by increasing the number of trans‐SNAREs .      

Syntaxin 6: the promiscuous behaviour of a SNARE protein

Vesicle flow within the cell is responsible for the dynamic maintenance of and communication between intracellular compartments. In addition, vesicular transport is crucial for communication between the cell and its surrounding environment. The ability of a vesicle to recognise and fuse with an appropriate compartment or vesicle is determined by its protein and lipid composition as well as by proteins in the cytosol. SNARE proteins present on both vesicle as well as target organelle membranes provide one component necessary for the process of membrane fusion. While in mammalian cells the main focus of interest about SNARE function has centred on those involved in exocytosis, recent data on SNAREs involved in intracellular membrane-trafficking steps have provided a deeper insight into the properties of these proteins. We take, as an example, the promiscuous SNARE syntaxin 6, a SNARE involved in multiple membrane fusion events. The properties of syntaxin 6 reveal similarities but also differences in the behaviour of intracellular SNAREs and the highly specialised exocytotic SNARE molecules.     

SNARE interactions in membrane trafficking: a perspective from mammalian central synapses

SNAREs (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) are a large family of proteins that are present on all organelles involved in intracellular vesicle trafficking and secretion. The interaction of complementary SNAREs found on opposing membranes presents an attractive lock-and-key mechanism, which may underlie the specificity of vesicle trafficking. Moreover, formation of the tight complex between a vesicle membrane SNARE and corresponding target membrane SNAREs could drive membrane fusion. In synapses, this tight complex, also referred to as the synaptic core complex, is essential for neurotransmitter release. However, recent observations in knockout mice lacking major synaptic SNAREs challenge the prevailing notion on the executive role of these proteins in fusion and open up several questions about their exact role(s) in neurotransmitter release. Persistence of a form of regulated neurotransmitter release in these mutant mice also raises the possibility that other cognate or non-cognate SNAREs may partially compensate for the loss of a particular SNARE. Future analysis of SNARE function in central synapses will also have implications for the role of these molecules in other vesicle trafficking events such as endocytosis and vesicle replenishment. Such analysis can provide a molecular basis for synaptic processes including certain forms of short-term synaptic plasticity.     

Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane

The influence of the lipid environment on docking and fusion of synaptobrevin 2 (Syb2) vesicles with target SNARE complex membranes was examined in a planar supported membrane fusion assay with high time-resolution. Previously, we showed that approximately eight SNARE complexes are required to fuse phosphatidylcholine (PC) and cholesterol model membranes in ∼20 ms. Here we present experiments, in which phosphatidylserine (PS) and phosphatidylethanolamine (PE) were added to mixtures of PC/cholesterol in different proportions in the Syb2 vesicle membranes only or in both the supported bilayers and the Syb2 vesicles. We found that PS and PE both reduce the probability of fusion and that this reduction is fully accounted for by the lipid composition in the vesicle membrane. However, the docking efficiency increases when the PE content in the vesicle (and target membrane) is increased from 0 to 30%. The fraction of fast-activating SNARE complexes decreases with increasing PE content. As few as three SNARE complexes are sufficient to support membrane fusion when at least 5% PS and 10% PE are present in both membranes or 5% and 30% PE are present in the vesicle membrane only. Despite the smaller number of required SNAREs, the SNARE activation and fusion rates are almost as fast as previously reported in reconstituted PC/cholesterol bilayers, i.e., of 10 and ∼20 ms, respectively.     

The Arabidopsis R-SNARE VAMP721 Interacts with KAT1 and KC1 K+ Channels to Moderate K+ Current at the Plasma Membrane

SNARE (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) proteins drive vesicle traffic, delivering membrane and cargo to target sites within the cell and at its surface. They contribute to cell homeostasis, morphogenesis, and pathogen defense. A subset of SNAREs, including the Arabidopsis thaliana SNARE SYP121, are known also to coordinate solute uptake via physical interactions with K⁺ channels and to moderate their gating at the plasma membrane. Here, we identify a second subset of SNAREs that interact to control these K⁺ channels, but with opposing actions on gating. We show that VAMPs (vesicle-associated membrane proteins), which target vesicles to the plasma membrane, also interact with and suppress the activities of the inward-rectifying K⁺ channels KAT1 and KC1. Interactions were evident in yeast split-ubiquitin assays, they were recovered in vivo by ratiometric bimolecular fluorescence complementation, and they were sensitive to mutation of a single residue, Tyr-57, within the longin domain of VAMP721. Interaction was also recovered on exchange of the residue at this site in the homolog VAMP723, which normally localizes to the endoplasmic reticulum and otherwise did not interact. Functional analysis showed reduced channel activity and alterations in voltage sensitivity that are best explained by a physical interaction with the channel gates. These actions complement those of SYP121, a cognate SNARE partner of VAMP721, and lead us to propose that the channel interactions reflect a “hand-off” in channel control between the two SNARE proteins that is woven together with vesicle fusion.     

SNAREs contribute to the specificity of membrane fusion

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses a large number of SNARE proteins that are localized to particular membrane compartments, suggesting that the fidelity of vesicle trafficking might in part be determined by specific SNARE pairing. However, the promiscuity of SNARE pairing in vitro suggests that the information for membrane compartment organization is not encoded in the inherent ability of SNAREs to form complexes. Here, we show that exocytosis of norepinephrine from PC12 cells is only inhibited or rescued by specific SNAREs. The data suggest that SNARE pairing does underlie vesicle trafficking fidelity, and that specific SNARE interactions with other proteins may facilitate the correct pairing.     

Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion

Exocytosis in yeast requires the assembly of the secretory vesicle soluble N-ethylmaleimide-sensitive factor attachment protein receptor (v-SNARE) Sncp and the plasma membrane t-SNAREs Ssop and Sec9p into a SNARE complex. High-level expression of mutant Snc1 or Sso2 proteins that have a COOH-terminal geranylgeranylation signal instead of a transmembrane domain inhibits exocytosis at a stage after vesicle docking. The mutant SNARE proteins are membrane associated, correctly targeted, assemble into SNARE complexes, and do not interfere with the incorporation of wild-type SNARE proteins into complexes. Mutant SNARE complexes recruit GFP-Sec1p to sites of exocytosis and can be disassembled by the Sec18p ATPase. Heterotrimeric SNARE complexes assembled from both wild-type and mutant SNAREs are present in heterogeneous higher-order complexes containing Sec1p that sediment at greater than 20S. Based on a structural analogy between geranylgeranylated SNAREs and the GPI-HA mutant influenza virus fusion protein, we propose that the mutant SNAREs are fusion proteins unable to catalyze fusion of the distal leaflets of the secretory vesicle and plasma membrane. In support of this model, the inverted cone-shaped lipid lysophosphatidylcholine rescues secretion from SNARE mutant cells.     

Mechanism of neurotransmitter release coming into focus

Research for three decades and major recent advances have provided crucial insights into how neurotransmitters are released by Ca²⁺‐triggered synaptic vesicle exocytosis, leading to reconstitution of basic steps that underlie Ca²⁺‐dependent membrane fusion and yielding a model that assigns defined functions for central components of the release machinery. The soluble N‐ethyl maleimide sensitive factor attachment protein receptors (SNAREs) syntaxin‐1, SNAP‐25, and synaptobrevin‐2 form a tight SNARE complex that brings the vesicle and plasma membranes together and is key for membrane fusion. N‐ethyl maleimide sensitive factor (NSF) and soluble NSF attachment proteins (SNAPs) disassemble the SNARE complex to recycle the SNAREs for another round of fusion. Munc18‐1 and Munc13‐1 orchestrate SNARE complex formation in an NSF‐SNAP‐resistant manner by a mechanism whereby Munc18‐1 binds to synaptobrevin and to a self‐inhibited “closed” conformation of syntaxin‐1, thus forming a template to assemble the SNARE complex, and Munc13‐1 facilitates assembly by bridging the vesicle and plasma membranes and catalyzing opening of syntaxin‐1. Synaptotagmin‐1 functions as the major Ca²⁺ sensor that triggers release by binding to membrane phospholipids and to the SNAREs, in a tight interplay with complexins that accelerates membrane fusion. Many of these proteins act as both inhibitors and activators of exocytosis, which is critical for the exquisite regulation of neurotransmitter release. It is still unclear how the actions of these various proteins and multiple other components that control release are integrated and, in particular, how they induce membrane fusion, but it can be expected that these fundamental questions can be answered in the near future, building on the extensive knowledge already available.     

Synaptobrevin N-terminally bound to syntaxin–SNAP-25 defines the primed vesicle state in regulated exocytosis

Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)–dependent exocytosis pathway at an intermediate “cocked” state, from which fusion can be triggered by Ca²⁺. It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin–SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca²⁺-triggered C-terminal assembly and membrane fusion.     

A new catch in the SNARE

Vesicle traffic underpins cell homeostasis, growth and development in plants. Traffic is facilitated by a superfamily of proteins known as SNAREs ( soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors) that interact to draw vesicle and target membrane surfaces together for fusion of the bilayers. Several recent findings now indicate that plant SNAREs might not be limited to the conventional 'housekeeping' activities commonly attributed to vesicle trafficking. In the past five years, six different SNAREs have been implicated in stomatal movements, gravisensing and pathogen resistance. These proteins almost certainly do contribute to specific membrane fusion events but they are also essential for signal transduction and response. Some SNAREs can modulate the activity of non-SNARE proteins, notably ion channels. Other examples might reflect SNARE interactions with different scaffolding and structural components of the cell.     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统,通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控,如Coat、SM、Tether、SNARE和Rab蛋白等,其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白,分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE,两类SNARE结合形成SNARE复合体,促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥（Arabidopsis thaliana）SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

SNARE and regulatory proteins induce local membrane protrusions to prime docked vesicles for fast calcium‐triggered fusion

Synaptic vesicles fuse with the plasma membrane in response to Ca²⁺ influx, thereby releasing neurotransmitters into the synaptic cleft. The protein machinery that mediates this process, consisting of soluble N‐ethylmaleimide‐sensitive factor attachment protein receptors (SNAREs) and regulatory proteins, is well known, but the mechanisms by which these proteins prime synaptic membranes for fusion are debated. In this study, we applied large‐scale, automated cryo‐electron tomography to image an in vitro system that reconstitutes synaptic fusion. Our findings suggest that upon docking and priming of vesicles for fast Ca²⁺‐triggered fusion, SNARE proteins act in concert with regulatory proteins to induce a local protrusion in the plasma membrane, directed towards the primed vesicle. The SNAREs and regulatory proteins thereby stabilize the membrane in a high‐energy state from which the activation energy for fusion is profoundly reduced, allowing synchronous and instantaneous fusion upon release of the complexin clamp.     

拟南芥SNARE因子在膜泡运输中的功能

高等植物细胞含有复杂的内膜系统, 通过其特有的膜泡运输机制来完成细胞内和细胞间的物质交流。膜泡运输主要包括运输囊泡的出芽、定向移动、拴留和膜融合4个过程。这4个过程受到许多因子的调控, 如Coat、SM、Tether、SNARE和Rab蛋白等, 其中SNARE因子在膜融合过程中发挥重要功能。SNARE因子是小分子跨膜蛋白, 分为定位于运输囊泡上的v-SNARE和定位于靶位膜上的t-SNARE, 两类SNARE结合形成SNARE复合体, 促进膜融合的发生。SNARE蛋白在调控植物体生长发育以及对外界环境响应等生理过程中起重要作用。该文对模式植物拟南芥(Arabidopsis thaliana)SNARE因子的最新细胞内定位和功能分析等研究进展进行了概述。     

Interaction of the Complexin Accessory Helix with the C-Terminus of the SNARE Complex: Molecular-Dynamics Model of the Fusion Clamp

SNARE complexes form between the synaptic vesicle protein synaptobrevin and the plasma membrane proteins syntaxin and SNAP25 to drive membrane fusion. A cytosolic protein, complexin (Cpx), binds to the SNARE bundle, and its accessory helix (AH) functions to clamp synaptic vesicle fusion. We performed molecular-dynamics simulations of the SNARE/Cpx complex and discovered that at equilibrium the Cpx AH forms tight links with both synaptobrevin and SNAP25. To simulate the effect of electrostatic repulsion between vesicle and membrane on the SNARE complex, we calculated the electrostatic force and performed simulations with an external force applied to synaptobrevin. We found that the partially unzipped state of the SNARE bundle can be stabilized by interactions with the Cpx AH, suggesting a simple mechanistic explanation for the role of Cpx in fusion clamping. To test this model, we performed experimental and computational characterizations of the syx^3-69Drosophila mutant, which has a point mutation in syntaxin that causes increased spontaneous fusion. We found that this mutation disrupts the interaction of the Cpx AH with synaptobrevin, partially imitating the cpx null phenotype. Our results support a model in which the Cpx AH clamps fusion by binding to the synaptobrevin C-terminus, thus preventing full SNARE zippering.