SNAREs and the specificity of membrane fusion

A major problem of intracellular membrane traffic concerns the way in which transport vesicles find and fuse with their target organelles. SNARE proteins are involved in fusion, and their mutual recognition could in principle provide the necessary specificity. Alternatively, the preliminary tethering of vesicles, mediated by peripheral membrane proteins, could hold the key. Previous studies of SNARE complex assembly in solution have suggested little specificity, but recent experiments with yeast SNAREs anchored in liposomes show that their interactions can be highly selective. It is likely that both tethering and SNARE engagement contribute to the accuracy of membrane transport.     

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.     

Effects of synaptotagmin 2 on membrane fusion between liposomes that contain SNAREs involved in exocytosis in mast cells

Mast cells play a pivotal role in allergic responses. Antigen stimulation causes elevation of the intracellular Ca(2+) concentration, which triggers the exocytotic release of inflammatory mediators such as histamine. Recent research, including our own, has revealed that SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins such as syntaxin-3, -4, SNAP-23, and VAMP-8 are involved in exocytosis. Although exocytosis in mast cells is Ca(2+) dependent, the target molecule that interacts with Ca(2+) is not clear. Synaptotagmin is a Ca(2+) sensor and regulates exocytosis in neuronal cells. However, the role of synaptotagmin 2, a member of the synaptotagmin family, in exocytosis in mast cells remains controversial. In this study, we investigated the role of synaptotagmin 2 by a liposome-based fusion assay. SNARE proteins (SNAP-23, syntaxin-3, VAMP-8) and synaptotagmin 2 were expressed in Escherichia coli and purified as GST-tagged or His-tagged fusion proteins. These SNARE proteins were incorporated by a detergent dialysis method. Membrane fusion between liposomes was monitored by fluorescence resonance energy transfer between fluorescent-labeled phospholipids. In the presence of Ca(2+), low synaptotagmin 2 concentration inhibited membrane fusion between SNARE-containing liposomes, while high synaptotagmin 2 concentration enhanced membrane fusion. This enhancement required phosphatidylserine as a membrane component. These results suggest that synaptotagmin 2 regulates membrane fusion of SNARE-containing liposomes involved in exocytosis in mast cells, and that this regulation is dependent on synaptotagmin 2 concentration, Ca(2+), and phosphatidylserine.     

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.     

SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis

During exocytosis, SNARE proteins of secretory vesicles interact with the corresponding SNARE proteins in the plasmalemma to initiate the fusion reaction. However, it is unknown whether SNAREs are uniformly distributed in the membrane or whether specialized fusion sites exist. Here we report that in the plasmalemma, syntaxins are concentrated in 200 nm large, cholesterol-dependent clusters at which secretory vesicles preferentially dock and fuse. The syntaxin clusters are distinct from cholesterol-dependent membrane rafts since they are Triton X-100-soluble and do not co-patch with raft markers. Synaptosomal-associated protein (SNAP)-25 is also clustered in spots, which partially overlap with syntaxin. Cholesterol depletion causes dispersion of these clusters, which is associated with a strong reduction in the rate of secretion, whereas the characteristics of individual exocytic events are unchanged. This suggests that high local concentrations of SNAREs are required for efficient fusion.     

i-SNAREs: inhibitory SNAREs that fine-tune the specificity of membrane fusion

A new functional class of SNAREs, designated inhibitory SNAREs (i-SNAREs), is described here. An i-SNARE inhibits fusion by substituting for or binding to a subunit of a fusogenic SNAREpin to form a nonfusogenic complex. Golgi-localized SNAREs were tested for i-SNARE activity by adding them as a fifth SNARE together with four other SNAREs that mediate Golgi fusion reactions. A striking pattern emerges in which certain subunits of the cis-Golgi SNAREpin function as i-SNAREs that inhibit fusion mediated by the trans-Golgi SNAREpin, and vice versa. Although the opposing distributions of the cis- and trans-Golgi SNAREs themselves could provide for a countercurrent fusion pattern in the Golgi stack, the gradients involved would be strongly sharpened by the complementary countercurrent distributions of the i-SNAREs.     

Towards reconstitution of membrane fusion mediated by SNAREs and other synaptic proteins

Abstract

Proteoliposomes have been widely used for in vitro studies of membrane fusion mediated by synaptic proteins. Initially, such studies were made with large unsynchronized ensembles of vesicles. Such ensemble assays limited the insights into the SNARE-mediated fusion mechanism that could be obtained from them. Single particle microscopy experiments can alleviate many of these limitations but they pose significant technical challenges. Here we summarize various approaches that have enabled studies of fusion mediated by SNAREs and other synaptic proteins at a single-particle level. Currently available methods are described and their advantages and limitations are discussed.     

Identification of SNAREs involved in synaptotagmin VII-regulated lysosomal exocytosis

Ca2+-regulated exocytosis of lysosomes has been recognized recently as a ubiquitous process, important for the repair of plasma membrane wounds. Lysosomal exocytosis is regulated by synaptotagmin VII, a member of the synaptotagmin family of Ca2+-binding proteins localized on lysosomes. Here we show that Ca2+-dependent interaction of the synaptotagmin VII C(2)A domain with SNAP-23 is facilitated by syntaxin 4. Specific interactions also occurred in cell lysates between the plasma membrane t-SNAREs SNAP-23 and syntaxin 4 and the lysosomal v-SNARE TI-VAMP/VAMP7. Following cytosolic Ca2+ elevation, SDS-resistant complexes containing SNAP-23, syntaxin 4, and TI-VAMP/VAMP7 were detected on membrane fractions. Lysosomal exocytosis was inhibited by the SNARE domains of syntaxin 4 and TI-VAMP/VAMP7 and by cleavage of SNAP-23 with botulinum neurotoxin E, thereby functionally implicating these SNAREs in Ca2+-regulated exocytosis of conventional lysosomes.     

Reconstituted membrane fusion requires regulatory lipids, SNAREs and synergistic SNARE chaperones

The homotypic fusion of yeast vacuoles, each with 3Q- and 1R-SNARE, requires SNARE chaperones (Sec17p/Sec18p and HOPS) and regulatory lipids (sterol, diacylglycerol and phosphoinositides). Pairs of liposomes of phosphatidylcholine/phosphatidylserine, bearing three vacuolar Q-SNAREs on one and the R-SNARE on the other, undergo slow lipid mixing, but this is unaffected by HOPS and inhibited by Sec17p/Sec18p. To study these essential fusion components, we reconstituted proteoliposomes of a more physiological composition, bearing vacuolar lipids and all four vacuolar SNAREs. Their fusion requires Sec17p/Sec18p and HOPS, and each regulatory lipid is important for rapid fusion. Although SNAREs can cause both fusion and lysis, fusion of these proteoliposomes with Sec17p/Sec18p and HOPS is not accompanied by lysis. Sec17p/Sec18p, which disassemble SNARE complexes, and HOPS, which promotes and proofreads SNARE assembly, act synergistically to form fusion-competent SNARE complexes, and this synergy requires phosphoinositides. This is the first chemically defined model of the physiological interactions of these conserved fusion catalysts.     

SNAREs in neurons--beyond synaptic vesicle exocytosis (Review)

The paradigm for soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) function in mammalian cells has been built on advancements in our understanding of structural and biochemical aspects of synaptic vesicle exocytosis, involving specifically synaptobrevin, syntaxin 1 and SNAP25. Interestingly, a good number of SNAREs which are not directly involved in neurotransmitter exocytosis, are either brain-enriched or have distinct neuron-specific functions. Syntaxins 12/13 regulates glutamate receptor recycling via its interaction with neuron-enriched endosomal protein of 21 kDa (NEEP21). TI-VAMP/VAMP7 is essential for neuronal morphogenesis and mediates the vesicular transport processes underlying neurite outgrowth. Ykt6 is highly enriched in the cerebral cortex and hippocampus and is targeted to a novel compartment in neurons. Syntaxin 16 has a moderate expression level in many tissues, but is rather enriched in the brain. Here, we review and discuss the neuron-specific physiology and possible pathology of these and other (such as SNAP-29 and Vti1a-beta) members of the SNARE family.     

Neuronal SNAREs do not trigger fusion between synthetic membranes but do promote PEG-mediated membrane fusion

At low surface concentrations that permit formation of impermeable membranes, neuronal soluble N-ethyl maleimide sensitive factor attachment protein receptor (SNARE) proteins form a stable, parallel, trans complex when vesicles are brought into contact by a low concentration of poly(ethylene glycol) (PEG). Surprisingly, formation of a stable SNARE complex does not trigger fusion under these conditions. However, neuronal SNAREs do promote fusion at low protein/lipid ratios when triggered by higher concentrations of PEG. Promotion of PEG-triggered fusion required phosphatidylserine and depended only on the surface concentration of SNAREs and not on the formation of a trans SNARE complex. These results were obtained at protein surface concentrations reported for synaptobrevin in synaptic vesicles and with an optimally fusogenic lipid composition. At a much higher protein/lipid ratio, vesicles joined by SNARE complex slowly mixed lipids at 37 degrees C in the absence of PEG, in agreement with earlier reports. However, vesicles containing syntaxin at a high protein/lipid ratio (>or=1:250) lost membrane integrity. We conclude that the neuronal SNARE complex promotes fusion by joining membranes and that the individual proteins syntaxin and synaptobrevin disrupt membranes so as to favor formation of a stalk complex and to promote conversion of the stalk to a fusion pore. These effects are similar to the effects of viral fusion peptides and transmembrane domains, but they are not sufficient by themselves to produce fusion in our in vitro system at surface concentrations documented to occur in synaptic vesicles. Thus, it is likely that proteins or factors other than the SNARE complex must trigger fusion in vivo.     

Evaluation of the annexins as potential mediators of membrane fusion in exocytosis

Membrane fusion is a central event in the process of exocytosis. It occurs between secretory vesicle membranes and the plasma membrane and also among secretory vesicle membranes themselves during compound exocytosis. In many cells the fusion event is regulated by calcium. Since the relevant membranes do not undergo fusion in vitro when highly purified, much attention has been paid to possible protein mediators of these calcium-dependent fusion events. The annexins comprise a group of calcium-dependent membrane-aggregating proteins, of which synexin is the prototype, which can initiate contacts between secretory vesicle membranes which will then fuse if the membranes are further perturbed by the addition of exogenous free fatty acids. This review discusses the secretory pathway and the evidence obtained fromin vitro studies that suggests the annexins may be mediators or regulators of membrane fusion in exocytosis.     

Cytotoxic T lymphocyte exocytosis: bring on the SNAREs!

Despite our general understanding of membrane traffic, the molecular machinery at the immunological synapse (IS) that regulates exocytosis of lytic granules from cytotoxic T lymphocytes (CTLs) remains elusive. The identification of disease-causing mutations in the small GTPase Rab27a, priming factor Munc13-4 and fusion protein syntaxin11 has defined an important role for these proteins in CTL exocytosis. In addition, the demonstration of a direct interaction in vitro between Rab27a and Munc13-4 suggests the possibility that the Rab27a-Munc13-4 cascade might regulate CTL exocytosis by engaging SNAREs such as syntaxin11. We propose that these SNAREs are likely to mediate the fusion of lytic granules with the plasma membrane of the IS.     

Identification of SNAREs involved in regulated exocytosis in the pancreatic acinar cell.

The molecular basis of exocytotic membrane fusion in the pancreatic acinar cell was investigated using an in vitro assay that measures both zymogen granule-plasma membrane fusion and granule-granule fusion. These two fusion events were differentially sensitive to Ca(2+), suggesting that they are controlled by different Ca(2+)-sensing mechanisms. Botulinum neurotoxin C (BoNT/C) treatment of the plasma membranes caused cleavage of syntaxin 2, the apical isoform of this Q-SNARE, but did not affect syntaxin 4, the basolateral isoform. BoNT/C also cleaved syntaxin 3, the zymogen granule isoform. BoNT/C treatment of plasma membranes abolished granule-plasma membrane fusion, whereas toxin treatment of the granules reduced granule-plasma membrane fusion and abolished granule-granule fusion. Tetanus toxin cleaved granule-associated synaptobrevin 2 but caused only a small reduction in both granule-plasma membrane fusion and granule-granule fusion. Our results indicate that syntaxin 2 is the isoform that mediates fusion between zymogen granules and the apical plasma membrane of the acinar cell. Syntaxin 3 mediates granule-granule fusion, which might be involved in compound exocytosis. In contrast, the major R-SNARE on the zymogen granule remains to be identified.     

Focal exocytosis by eosinophils--compound exocytosis and cumulative fusion.

We have investigated the granule fusion events during exocytosis in horse eosinophils by time-resolved patch-clamp capacitance measurements. Stimulation with intracellular GTP gamma S leads to a stepwise capacitance increase by 4.0 +/- 0.9 pF. At GTP gamma S concentrations < 20 microM the step size distribution is in agreement with the granule size distribution in resting cells. Above 80 microM the number of steps is reduced and very large steps occur. The total capacitance increase, however, is unaffected. These results show that at high GTP gamma S concentrations granule--granule fusion occurs inside the cell forming large compound granules, which then fuse with the plasma membrane (compound exocytosis). The electrical equivalent circuit of the cell during degranulation indicates the formation of a degranulation sac by cumulative fusion events. Fusion of the first granule with the plasma membrane induces fusion of further granules with this granule directing the release of all the granular material to the first fusion pore. The physiological function of eosinophils is the killing of parasites. Compound exocytosis and cumulative fusion enable the cells to focus the release of cytotoxic proteins to well defined target regions and prevent uncontrolled diffusion of this material, which would damage intact host cells.     

Screening of enzymatic mechanisms possible involved in membrane fusion during exocytosis in Paramecium cells

Microinjected alkaline phosphatase triggered exocytosis of trichocysts. Among a variety of inhibitors and stimulators interfering with fusogenic mechanism discussed in the literature, only phosphatase inhibitors inhibited exocytosis. Various other enzymes were also tested with a new in vitro system (Vilmart-Seuwen et al., 1986), but results were mostly negative. (The possible involvement of proteases remains questionable.) Positive results obtained with micro-injected alkaline phosphatase are in line with our previous results: (a) The occurrence of a cytochemical reaction for phosphatase activity precisely at fusion sites (Plattner et al., 1980); (b) the occurrence of protein dephosphorylation during exocytosis (Gilligan and Satir, 1982; Zieseniss and Plattner, 1985) and (c) the negative modulatory effect of ATP on exocytotic membrane fusion (Vilmart-Seuwen et al., 1986).     

Structure of membrane tethers and their role in fusion

Vesicular transport between different membrane compartments is a key process in cell biology required for the exchange of material and information. The complex machinery that executes the formation and delivery of transport vesicles has been intensively studied and yielded a comprehensive view of the molecular principles that underlie the budding and fusion process. Tethering also represents an essential step in each trafficking pathway. It is mediated by Rab GTPases in concert with so‐called tethering factors, which constitute a structurally diverse family of proteins that share a similar role in promoting vesicular transport. By simultaneously binding to proteins and/or lipids on incoming vesicles and the target compartment, tethers are thought to bridge donor and acceptor membrane. They thus provide specificity while also promoting fusion. However, how tethering works at a mechanistic level is still elusive. We here discuss the recent advances in the structural and biochemical characterization of tethering complexes that provide novel insight on how these factors might contribute the efficiency of fusion.     

SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.     

A role of complexin-lipid interactions in membrane fusion

Complexins (Cpxs) and synaptotagmins regulate calcium-dependent exocytosis. A central helix in Cpx confers specific binding to the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor (SNARE) fusion machinery. An accessory helix in the amino-terminal region inhibits membrane fusion by blocking SNAREpin zippering. We now show that an amphipathic helix in the carboxy-terminal region of CpxI binds lipid bilayers and affects SNARE-mediated lipid mixing in a liposome fusion assay. The substitution of a hydrophobic amino acid within the helix by a charged residue abolishes the lipid interaction and the stimulatory effect of CpxI in liposome fusion. In contrast, the introduction of the bulky hydrophobic amino acid tryptophan stimulates lipid binding and liposome fusion. This data shows that local Cpx-lipid interactions can play a role in membrane fusion.