SNARE Complex Zipping as a Driving Force in the Dilation of Proteinaceous Fusion Pores

The assembly of SNARE proteins into a tight complex has been hypothesized to drive membrane fusion. A model of the initial fusion pore as a proteinaceous channel formed by SNARE proteins places their membrane anchors in separate membranes. This leaves the possibility of a final assembly step that brings the membrane anchors together and drives fusion pore expansion. The present study develops a model for expansion in which the final SNARE complex zipping step drives a transition from a proteinaceous fusion pore to a lipidic fusion pore. An estimate of the energy released upon merger of the helical segments of the SNARE motifs with the helical segments of the membrane anchors indicates that completing the assembly of a few SNARE complexes can overcome the elastic energy that opposes lipid bilayer deformation into a narrow fusion pore. The angle between the helical axes of the membrane anchor and SNARE motif serves as a useful reaction coordinate for this transition. Energy was calculated as a function of this angle, incorporating contributions from membrane bending, SNARE complex assembly, membrane anchor flexing and hydrophobic interactions. The rate of this transition was evaluated as a process of diffusion over the barrier imposed by these combined energies, and the rates estimated were consistent with experimental measurements.     

Plasma membrane targeting of exocytic SNARE proteins

SNARE proteins play a central role in the process of intracellular membrane fusion. Indeed, the interaction of SNAREs present on two opposing membranes is generally believed to provide the driving force to initiate membrane fusion. Eukaryotic cells express a large number of SNARE isoforms, and the function of individual SNAREs is required for specific intracellular fusion events. Exocytosis, the fusion of secretory vesicles with the plasma membrane, employs the proteins syntaxin and SNAP-25 as plasma membrane SNAREs. As a result, exocytosis is dependent upon the targeting of these proteins to the plasma membrane; however, the mechanisms that underlie trafficking of exocytic syntaxin and SNAP-25 proteins to the cell surface are poorly understood. The intracellular trafficking itinerary of these proteins is particularly intriguing as syntaxins are tail-anchored (or Type IV) membrane proteins, whereas SNAP-25 is anchored to membranes via a central palmitoylated domain-there is no common consensus for the trafficking of such proteins within the cell. In this review, we discuss the plasma membrane targeting of these essential exocytic SNARE proteins.     

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.     

What is the role of SNARE proteins in membrane fusion?

Soluble N-ethylmaleimide-sensitive factor activating protein receptor (SNARE) proteins have been at the fore-front of research on biological membrane fusion for some time. The subcellular localization of SNAREs and their ability to form the so-called SNARE complex may be integral to determining the specificity of intracellular fusion (the SNARE hypothesis) and/or serving as the minimal fusion machinery. Both the SNARE hypothesis and the idea of the minimal fusion machinery have been challenged by a number of experimental observations in various model systems, suggesting that SNAREs may have other functions. Considering recent advances in the SNARE literature, it appears that SNAREs may actually function as part of a complex fusion "machine." Their role in the machinery could be any one or a combination of roles, including establishing tight membrane contact, formation of a scaffolding on which to build the machine, binding of lipid surfaces, and many others. It is also possible that complexations other than the classic SNARE complex participate in membrane fusion.     

α-SNAP Interferes with the Zippering of the SNARE Protein Membrane Fusion Machinery

Neuronal exocytosis is mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins. Before fusion, SNARE proteins form complexes bridging the membrane followed by assembly toward the C-terminal membrane anchors, thus initiating membrane fusion. After fusion, the SNARE complex is disassembled by the AAA-ATPase N-ethylmaleimide-sensitive factor that requires the cofactor α-SNAP to first bind to the assembled SNARE complex. Using chromaffin granules and liposomes we now show that α-SNAP on its own interferes with the zippering of membrane-anchored SNARE complexes midway through the zippering reaction, arresting SNAREs in a partially assembled trans-complex and preventing fusion. Intriguingly, the interference does not result in an inhibitory effect on synaptic vesicles, suggesting that membrane properties also influence the final outcome of α-SNAP interference with SNARE zippering. We suggest that binding of α-SNAP to the SNARE complex affects the ability of the SNARE complex to harness energy or transmit force to the membrane.     

Probing domain swapping for the neuronal SNARE complex with electron paramagnetic resonance

Highly conserved soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins control membrane fusion at synapses. The target plasma membrane-associated SNARE proteins and the vesicle-associated SNARE protein assemble into a parallel four-helix bundle. Using a novel EPR approach, it is found that the SNARE four-helix bundles are interconnected via domain swapping that is achieved by substituting one of the two SNAP-25 helices with the identical helix from the second four-helical bundle. Domain swapping is likely to play a role in the multimerization of the SNARE complex that is required for successful membrane fusion. The new EPR application employed here should be useful to study other polymerizing proteins.     

Sec1/Munc18 protein stabilizes fusion-competent syntaxin for membrane fusion in Arabidopsis cytokinesis

Intracellular membrane fusion requires complexes of syntaxins with other SNARE proteins and regulatory Sec1/Munc18 (SM) proteins. In membrane fusion mediating, e.g., neurotransmitter release or glucose-stimulated insulin secretion in mammals, SM proteins preferentially interact with the inactive closed, rather than the active open, conformation of syntaxin or with the assembled SNARE complex. Other membrane fusion processes such as vacuolar fusion in yeast involve like membranes carrying cis-SNARE complexes, and the role of SM protein is unknown. We investigated syntaxin-SM protein interaction in membrane fusion of Arabidopsis cytokinesis, which involves cytokinesis-specific syntaxin KNOLLE and SM protein KEULE. KEULE interacted with an open conformation of KNOLLE that complemented both knolle and keule mutants. This interaction occurred at the cell division plane and required the KNOLLE linker sequence between helix Hc and SNARE domain. Our results suggest that in cytokinesis, SM protein stabilizes the fusion-competent open form of syntaxin, thereby promoting trans-SNARE complex formation.     

Sly1 protein bound to Golgi syntaxin Sed5p allows assembly and contributes to specificity of SNARE fusion complexes

Fusion of transport vesicles with their target organelles involves specific membrane proteins, SNAREs, which form tight complexes bridging the membranes to be fused. Evidence from yeast and mammals indicates that Sec1 family proteins act as regulators of membrane fusion by binding to the target membrane SNAREs. In experiments with purified proteins, we now made the observation that the ER to Golgi core SNARE fusion complex could be assembled on syntaxin Sed5p tightly bound to the Sec1-related Sly1p. Sly1p also bound to preassembled SNARE complexes in vitro and was found to be part of a vesicular/target membrane SNARE complex immunoprecipitated from yeast cell lysates. This is in marked contrast to the exocytic SNARE assembly in neuronal cells where high affinity binding of N-Sec1/Munc-18 to syntaxin 1A precluded core SNARE fusion complex formation. We also found that the kinetics of SNARE complex formation in vitro with either Sly1p-bound or free Sed5p was not significantly different. Importantly, several presumably nonphysiological SNARE complexes easily generated with Sed5p did not form when the syntaxin was first bound to Sly1p. This indicates for the first time that a Sec1 family member contributes to the specificity of SNARE complex assembly.     

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。     

SNAREpins are functionally resistant to disruption by NSF and alphaSNAP

SNARE (SNAP [soluble NSF {N-ethylmaleimide–sensitive fusion protein} attachment protein] receptor) proteins are required for many fusion processes, and recent studies of isolated SNARE proteins reveal that they are inherently capable of fusing lipid bilayers. Cis-SNARE complexes (formed when vesicle SNAREs [v-SNAREs] and target membrane SNAREs [t-SNAREs] combine in the same membrane) are disrupted by the action of the abundant cytoplasmic ATPase NSF, which is necessary to maintain a supply of uncombined v- and t-SNAREs for fusion in cells. Fusion is mediated by these same SNARE proteins, forming trans-SNARE complexes between membranes. This raises an important question: why doesn''t NSF disrupt these SNARE complexes as well, preventing fusion from occurring at all? Here, we report several lines of evidence that demonstrate that SNAREpins (trans-SNARE complexes) are in fact functionally resistant to NSF, and they become so at the moment they form and commit to fusion. This elegant design allows fusion to proceed locally in the face of an overall environment that massively favors SNARE disruption.     

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.     

YKT6 is a core constituent of membrane fusion machineries at the Arabidopsis trans-Golgi network

SNARE complex formation is essential for membrane fusion in exocytotic and vacuolar trafficking pathways. Vesicle-associated (v-) SNARE associates with a target membrane (t-) SNARE to form a SNARE complex bridging two membranes, which may facilitate membrane fusion. The Arabidopsis genome encodes a large number of predicted SNARE proteins that might function primarily as fusogens for vesicle transport in endomembrane systems. The SNAREs SYP41, SYP61 and VTI12 reside in the trans-Golgi network and have been proposed to function together in vesicle fusion with this organelle. Here, we use a liposome fusion assay to demonstrate that VTI12 and either SYP41 or SYP61, but not both, are required for membrane fusion. This indicates that SYP41 and SYP61 are likely to function in independent vesicle fusion reactions in Arabidopsis. In addition, we have identified two new functionally interchangeable components, YKT61 and YKT62, that show sequence similarity to the multifunctional yeast SNARE YKT6. Both YKT61 and YKT62 interact with SYP41 and are essential for membrane fusion mediated by either SYP41 or SYP61. These results therefore define the core constituents required for membrane fusion at the Arabidopsis trans-Golgi network.     

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。     

The polybasic juxtamembrane region of Sso1p is required for SNARE function in vivo

Exocytosis in Saccharomyces cerevisiae requires the specific interaction between the plasma membrane t-SNARE complex (Sso1/2p;Sec9p)and a vesicular v-SNARE (Snc1/2p). While SNARE proteins drive membrane fusion, many aspects of SNARE assembly and regulation are ill defined. Plasma membrane syntaxin homologs (including Sso1p) contain a highly charged juxtamembrane region between the transmembrane helix and the "SNARE domain" or core complex domain. We examined this region in vitro and in vivo by targeted sequence modification, including insertions and replacements. These modified Sso1 proteins were expressed as the sole copy of Sso in S. cerevisiae and examined for viability. We found that mutant Sso1 proteins with insertions or duplications show limited function, whereas replacement of as few as three amino acids preceding the transmembrane domain resulted in a nonfunctional SNARE in vivo. Viability is also maintained when two proline residues are inserted in the juxtamembrane of Sso1p, suggesting that helical continuity between the transmembrane domain and the core coiled-coil domain is not absolutely required. Analysis of these mutations in vitro utilizing a reconstituted fusion assay illustrates that the mutant Sso1 proteins are only moderately impaired in fusion. These results suggest that the sequence of the juxtamembrane region of Sso1p is vital for function in vivo, independent of the ability of these proteins to direct membrane fusion.     

神经递质释放过程中的可溶性NSF附着蛋白受体(SNARE)和相关蛋白

近年来,对突触小泡释放神经递质分子机制的研究迅速发展,发现了大量位于神经末梢的蛋白质.它们之间的相互作用与突触小泡释放神经递质相关,特别是位于突触小泡膜上的突触小泡蛋白/突触小泡相关膜蛋白(synaptobrevin/VAMP),位于突触前膜上的syntaxin和突触小体相关蛋白(synaptosome-associated protein of 25 ku),三者聚合形成的可溶性NSF附着蛋白受体(SNARE)核心复合体在突触小泡的胞裂外排、释放递质过程中有重要作用.而一些已知及未知的与SNARE蛋白有相互作用的蛋白质,可通过调节SNARE核心复合体的形成与解离来影响突触小泡的胞裂外排,从而可以调节突触信号传递的效率及强度,在突触可塑性的形成中起重要作用.     

Negative Regulation of Syntaxin4/SNAP-23/VAMP2-Mediated Membrane Fusion by Munc18c In Vitro

Background

Translocation of the facilitative glucose transporter GLUT4 from an intracellular store to the plasma membrane is responsible for the increased rate of glucose transport into fat and muscle cells in response to insulin. This represents a specialised form of regulated membrane trafficking. Intracellular membrane traffic is subject to multiple levels of regulation by conserved families of proteins in all eukaryotic cells. Notably, all intracellular fusion events require SNARE proteins and Sec1p/Munc18 family members. Fusion of GLUT4-containing vesicles with the plasma membrane of insulin-sensitive cells involves the SM protein Munc18c, and is regulated by the formation of syntaxin 4/SNAP23/VAMP2 SNARE complexes.

Methodology/Principal Findings

Here we have used biochemical approaches to characterise the interaction(s) of Munc18c with its cognate SNARE proteins and to examine the role of Munc18c in regulating liposome fusion catalysed by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. We demonstrate that Munc18c makes contacts with both t- and v-SNARE proteins of this complex, and directly inhibits bilayer fusion mediated by the syntaxin 4/SNAP23/VAMP2 SNARE complex.

Conclusion/Significance

Our reductionist approach has enabled us to ascertain a direct inhibitory role for Munc18c in regulating membrane fusion mediated by syntaxin 4/SNAP23/VAMP2 SNARE complex formation. It is important to note that two different SM proteins have recently been shown to stimulate liposome fusion mediated by their cognate SNARE complexes. Given the structural similarities between SM proteins, it seems unlikely that different members of this family perform opposing regulatory functions. Hence, our findings indicate that Munc18c requires a further level of regulation in order to stimulate SNARE-mediated membrane fusion.     

A search for synthetic peptides that inhibit soluble N-ethylmaleimide sensitive-factor attachment receptor-mediated membrane fusion

Soluble N-ethylmaleimide sensitive-factor attachment receptor (SNARE) proteins have crucial roles in driving exocytic membrane fusion. Molecular recognition between vesicle-associated (v)-SNARE and target membrane (t)-SNARE leads to the formation of a four-helix bundle, which facilitates the merging of two apposing membranes. Synthetic peptides patterned after the SNARE motifs are predicted to block SNARE complex formation by competing with the parental SNAREs, inhibiting neuronal exocytosis. As an initial attempt to identify the peptide sequences that block SNARE assembly and membrane fusion, we created thirteen 17-residue synthetic peptides derived from the SNARE motifs of v- and t-SNAREs. The effects of these peptides on SNARE-mediated membrane fusion were investigated using an in vitro lipid-mixing assay, in vivo neurotransmitter release and SNARE complex formation assays in PC12 cells. Peptides derived from the N-terminal region of SNARE motifs had significant inhibitory effects on neuroexocytosis, whereas middle- and C-terminal-mimicking peptides did not exhibit much inhibitory function. N-terminal mimicking peptides blocked N-terminal zippering of SNAREs, a rate-limiting step in SNARE-driven membrane fusion. Therefore, the results suggest that the N-terminal regions of SNARE motifs are excellent targets for the development of drugs to block SNARE-mediated membrane fusion and neurotransmitter release.     

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.     

Membrane fusion by VAMP3 and plasma membrane t-SNAREs

Pairing of SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins on vesicles (v-SNAREs) and SNARE proteins on target membranes (t-SNAREs) mediates intracellular membrane fusion. VAMP3/cellubrevin is a v-SNARE that resides in recycling endosomes and endosome-derived transport vesicles. VAMP3 has been implicated in recycling of transferrin receptors, secretion of alpha-granules in platelets, and membrane trafficking during cell migration. Using a cell fusion assay, we examined membrane fusion capacity of the ternary complexes formed by VAMP3 and plasma membrane t-SNAREs syntaxin1, syntaxin4, SNAP-23 and SNAP-25. VAMP3 forms fusogenic pairing with t-SNARE complexes syntaxin1/SNAP-25, syntaxin1/SNAP-23 and syntaxin4/SNAP-25, but not with syntaxin4/SNAP-23. Deletion of the N-terminal domain of syntaxin4 enhanced membrane fusion more than two fold, indicating that the N-terminal domain negatively regulates membrane fusion. Differential membrane fusion capacities of the ternary v-/t-SNARE complexes suggest that transport vesicles containing VAMP3 have distinct membrane fusion kinetics with domains of the plasma membrane that present different t-SNARE proteins.     

The multi-functional SNARE protein Ykt6 in autophagosomal fusion processes

Autophagy is a degradative pathway in which cytosolic material is enwrapped within double membrane vesicles, so-called autophagosomes, and delivered to lytic organelles. SNARE (Soluble N-ethylmaleimide sensitive factor attachment protein receptor) proteins are key to drive membrane fusion of the autophagosome and the lytic organelles, called lysosomes in higher eukaryotes or vacuoles in plants and yeast. Therefore, the identification of functional SNARE complexes is central for understanding fusion processes and their regulation. The SNARE proteins Syntaxin 17, SNAP29 and Vamp7/VAMP8 are responsible for the fusion of autophagosomes with lysosomes in higher eukaryotes. Recent studies reported that the R-SNARE Ykt6 is an additional SNARE protein involved in autophagosome-lytic organelle fusion in yeast, Drosophila, and mammals. These current findings point to an evolutionarily conserved role of Ykt6 in autophagosome-related fusion events. Here, we briefly summarize the principal mechanisms of autophagosome-lytic organelle fusion, with a special focus on Ykt6 to highlight some intrinsic features of this unusual SNARE protein.