Soluble NSF attachment protein receptors (SNAREs) in RBL-2H3 mast cells: functional role of syntaxin 4 in exocytosis and identification of a vesicle-associated membrane protein 8-containing secretory compartment

Mast cells upon stimulation through high affinity IgE receptors massively release inflammatory mediators by the fusion of specialized secretory granules (related to lysosomes) with the plasma membrane. Using the RBL-2H3 rat mast cell line, we investigated whether granule secretion involves components of the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) machinery. Several isoforms of each family of SNARE proteins were expressed. Among those, synaptosome-associated protein of 23 kDa (SNAP23) was central in SNARE complex formation. Within the syntaxin family, syntaxin 4 interacted with SNAP23 and all vesicle-associated membrane proteins (VAMPs) examined, except tetanus neurotoxin insensitive VAMP (TI-VAMP). Overexpression of syntaxin 4, but not of syntaxin 2 nor syntaxin 3, caused inhibition of FcepsilonRI-dependent exocytosis. Four VAMP proteins, i.e., VAMP2, cellubrevin, TI-VAMP, and VAMP8, were present on intracellular membrane structures, with VAMP8 residing mainly on mediator-containing secretory granules. We suggest that syntaxin 4, SNAP23, and VAMP8 may be involved in regulation of mast cell exocytosis. Furthermore, these results are the first demonstration that the nonneuronal VAMP8 isoform, originally localized on early endosomes, is present in a regulated secretory compartment.     

α-Synuclein Delays Endoplasmic Reticulum (ER)-to-Golgi Transport in Mammalian Cells by Antagonizing ER/Golgi SNAREs

Toxicity of human α-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson''s disease has not been established. We tested elements of this hypothesis by expressing human α-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant α-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble α-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble α-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that α-synuclein antagonizes SNARE function. Ykt6 reversed α-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified α-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble α-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.     

Analysis of Sec22p in endoplasmic reticulum/Golgi transport reveals cellular redundancy in SNARE protein function

Membrane-bound soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins form heteromeric complexes that are required for intracellular membrane fusion and are proposed to encode compartmental specificity. In yeast, the R-SNARE protein Sec22p acts in transport between the endoplasmic reticulum (ER) and Golgi compartments but is not essential for cell growth. Other SNARE proteins that function in association with Sec22p (i.e., Sed5p, Bos1p, and Bet1p) are essential, leading us to question how transport through the early secretory pathway is sustained in the absence of Sec22p. In wild-type strains, we show that Sec22p is directly required for fusion of ER-derived vesicles with Golgi acceptor membranes. In sec22Delta strains, Ykt6p, a related R-SNARE protein that operates in later stages of the secretory pathway, is up-regulated and functionally substitutes for Sec22p. In vivo combination of the sec22Delta mutation with a conditional ykt6-1 allele results in lethality, consistent with a redundant mechanism. Our data indicate that the requirements for specific SNARE proteins in intracellular membrane fusion are less stringent than appreciated and suggest that combinatorial mechanisms using both upstream-targeting elements and SNARE proteins are required to maintain an essential level of compartmental organization.     

Mapping of R-SNARE function at distinct intracellular GLUT4 trafficking steps in adipocytes

The functional trafficking steps used by soluble NSF attachment protein receptor (SNARE) proteins have been difficult to establish because of substantial overlap in subcellular localization and because in vitro SNARE-dependent binding and fusion reactions can be promiscuous. Therefore, to functionally identify the site of action of the vesicle-associated membrane protein (VAMP) family of R-SNAREs, we have taken advantage of the temporal requirements of adipocyte biosynthetic sorting of a dual-tagged GLUT4 reporter (myc-GLUT4-GFP) coupled with small interfering RNA gene silencing. Using this approach, we confirm the requirement of VAMP2 and VAMP7 for insulin and osmotic shock trafficking from the vesicle storage sites, respectively, and fusion with the plasma membrane. Moreover, we identify a requirement for VAMP4 for the initial biosynthetic entry of GLUT4 from the Golgi apparatus into the insulin-responsive vesicle compartment, VAMP8, for plasma membrane endocytosis and VAMP2 for sorting to the specialized insulin-responsive compartment after plasma membrane endocytosis.     

Dedicated SNAREs and specialized TRIM cargo receptors mediate secretory autophagy

Autophagy is a process delivering cytoplasmic components to lysosomes for degradation. Autophagy may, however, play a role in unconventional secretion of leaderless cytosolic proteins. How secretory autophagy diverges from degradative autophagy remains unclear. Here we show that in response to lysosomal damage, the prototypical cytosolic secretory autophagy cargo IL‐1β is recognized by specialized secretory autophagy cargo receptor TRIM16 and that this receptor interacts with the R‐SNARE Sec22b to recruit cargo to the LC3‐II⁺ sequestration membranes. Cargo secretion is unaffected by downregulation of syntaxin 17, a SNARE promoting autophagosome–lysosome fusion and cargo degradation. Instead, Sec22b in combination with plasma membrane syntaxin 3 and syntaxin 4 as well as SNAP‐23 and SNAP‐29 completes cargo secretion. Thus, secretory autophagy utilizes a specialized cytosolic cargo receptor and a dedicated SNARE system. Other unconventionally secreted cargo, such as ferritin, is secreted via the same pathway.     

Genetic interactions with the yeast Q-SNARE VTI1 reveal novel functions for the R-SNARE YKT6.

SNARE proteins are required for fusion of transport vesicles with target membranes. Previously, we found that the yeast Q-SNARE Vti1p is involved in transport to the cis-Golgi, to the prevacuole/late endosome, and to the vacuole. Here we identified a previously uncharacterized gene, VTS1, and the R-SNARE YKT6 both as multicopy and as low copy suppressors of the growth and vacuolar transport defect in vti1-2 cells. Ykt6p was known to function in retrograde traffic to the cis-Golgi and homotypic vacuolar fusion. We found that VTI1 and YKT6 also interacted in traffic to the prevacuole and vacuole, indicating that these SNARE complexes contain Ykt6p, Vti1p, plus Pep12p and Ykt6p, Vti1p, Vam3p, plus Vam7p, respectively. As Ykt6p was required for several transport steps, R-SNAREs cannot be the sole determinants of specificity. To study the role of the 0 layer in the SNARE motif, we introduced the mutations vti1-Q158R and ykt6-R165Q. SNARE complexes to which Ykt6p contributed a fourth glutamine residue in the 0 layer were nonfunctional, suggesting an essential function for arginine in the 0 layer of these complexes. vti1-Q158R cells had severe defects in several transport steps, indicating that the second arginine in the 0 layer interfered with function.     

Systematic analysis of SNARE molecules in Arabidopsis: dissection of the post-Golgi network in plant cells

In all eucaryotic cells, specific vesicle fusion during vesicular transport is mediated by membrane-associated proteins called SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors). Sequence analysis identified a total of 54 SNARE genes (18 Qa-SNAREs/Syntaxins, 11 Qb-SNAREs, 8 Qc-SNAREs, 14 R-SNAREs/VAMPs and 3 SNAP-25) in the Arabidopsis genome. Almost all of them were ubiquitously expressed through out all tissues examined. A series of transient expression assays using green fluorescent protein (GFP) fused proteins revealed that most of the SNARE proteins were located on specific intracellular compartments: 6 in the endoplasmic reticulum, 9 in the Golgi apparatus, 4 in the trans-Golgi network (TGN), 2 in endosomes, 17 on the plasma membrane, 7 in both the prevacuolar compartment (PVC) and vacuoles, 2 in TGN/PVC/vacuoles, and 1 in TGN/PVC/plasma membrane. Some SNARE proteins showed multiple localization patterns in two or more different organelles, suggesting that these SNAREs shuttle between the organelles. Furthermore, the SYP41/SYP61-residing compartment, which was defined as the TGN, was not always located along with the Golgi apparatus, suggesting that this compartment is an independent organelle distinct from the Golgi apparatus. We propose possible combinations of SNARE proteins on all subcellular compartments, and suggest the complexity of the post-Golgi membrane traffic in higher plant cells.     

Stability profile of the neuronal SNARE complex reflects its potency to drive fast membrane fusion

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) form the SNARE complex to mediate most fusion events of the secretory pathway. The neuronal SNARE complex is featured by its high stability and half-zippered conformation required for driving robust and fast synaptic exocytosis. However, these two features seem to be thermodynamically mutually exclusive. In this study, we have employed temperature-dependent disassociation assays and single-molecule Förster resonance energy transfer (FRET) experiments to analyze the stability and conformation of the neuronal SNARE complex. We reclassified the amino acids of the SNARE motif into four sub-groups (core, core-side I and II, and non-contact). Our data showed that the core residues predominantly contribute to the complex stability to meet a basal requirement for SNARE-mediated membrane fusion, while the core-side residues exert an unbalanced effect on the N- and C-half bundle stability that determines the half-zippered conformation of the neuronal SNARE complex, which would accommodate essential regulations by complexins and synaptotagmins for fast Ca²⁺-triggered membrane fusion. Furthermore, our data confirmed a strong coupling of folding energy between the N- and C-half assembly of the neuronal SNARE complex, which rationalizes the strong potency of the half-zippered conformation to conduct robust and fast fusion. Overall, these results uncovered that the stability profile of the neuronal SNARE complex reflects its potency to drive fast and robust membrane fusion. Based on these results, we also developed a new parameter, the stability factor (F_s), to characterize the overall stability of the neuronal SNARE complex and resolved a linear correlation between the stability and inter-residue coulombic interactions of the neuronal SNARE complex, which would help rationally design artificial SNARE complexes and remold functional SNARE complexes with desirable stability.     

Endobrevin/VAMP8 mediates exocytotic release of hexosaminidase from rat basophilic leukaemia cells

Mast cells are important players in innate immunity and mediate allergic responses. Upon stimulation, they release biologically active mediators including histamine, cytokines and lysosomal hydrolases. We used permeabilized rat basophilic leukaemia cells as model to identify R-SNAREs (soluble NSF (N-ethylmaleimide-sensitive fusion protein)) mediating exocytosis of hexosaminidase from mast cells. Of a complete set of recombinant mammalian R-SNAREs, only vesicle associated membrane protein (VAMP8)/endobrevin consistently blocked hexosaminidase release, which was also insensitive to treatment with clostridial neurotoxins. Thus, VAMP8, which also mediates fusion of late endosomes and lysosomes, plays a major role in hexosaminidase release, strengthening the view that mast cell granules share properties of both secretory granules and lysosomes.     

Ordering the final events in yeast exocytosis

In yeast, assembly of exocytic soluble N-ethylmaleimide-sensitive fusion protein (NSF) attachment protein receptor (SNARE) complexes between the secretory vesicle SNARE Sncp and the plasma membrane SNAREs Ssop and Sec9p occurs at a late stage of the exocytic reaction. Mutations that block either secretory vesicle delivery or tethering prevent SNARE complex assembly and the localization of Sec1p, a SNARE complex binding protein, to sites of secretion. By contrast, wild-type levels of SNARE complexes persist in the sec1-1 mutant after a secretory block is imposed, suggesting a role for Sec1p after SNARE complex assembly. In the sec18-1 mutant, cis-SNARE complexes containing surface-accessible Sncp accumulate in the plasma membrane. Thus, one function of Sec18p is to disassemble SNARE complexes on the postfusion membrane.     

Cellular and molecular mechanism for secretory autophagy

Macroautophagy/autophagy plays a role in unconventional secretion of leaderless cytosolic proteins. Whether and how secretory autophagy diverges from conventional degradative autophagy is unclear. We have shown that the prototypical secretory autophagy cargo IL1B/IL-1β (interleukin 1 β) is recognized by TRIM16, and that this first to be identified secretory autophagy receptor interacts with the R-SNARE SEC22B to jointly deliver cargo to the MAP1LC3B-II-positive sequestration membranes. Cargo secretion is unaffected by knockdowns of STX17, a SNARE catalyzing autophagosome-lysosome fusion as a prelude to cargo degradation. Instead, SEC22B in combination with plasma membrane syntaxins completes cargo secretion. Thus, secretory autophagy diverges from degradative autophagy by using specialized receptors and a dedicated SNARE machinery to bypass fusion with lysosomes.     

Subunit structure of a mammalian ER/Golgi SNARE complex

SNAP receptor (SNARE) complexes bridge opposing membranes to promote membrane fusion within the secretory and endosomal pathways. Because only the exocytic SNARE complexes have been characterized in detail, the structural features shared by SNARE complexes from different fusion steps are not known. We now describe the subunit structure, assembly, and regulation of a quaternary SNARE complex, which appears to mediate an early step in endoplasmic reticulum (ER) to Golgi transport. Purified recombinant syntaxin 5, membrin, and rbet1, three Q-SNAREs, assemble cooperatively to create a high affinity binding site for sec22b, an R-SNARE. The syntaxin 5 amino-terminal domain potently inhibits SNARE complex assembly. The ER/Golgi quaternary complex is remarkably similar to the synaptic complex, suggesting that a common pattern is followed at all transport steps, where three Q-helices assemble to form a high affinity binding site for a fourth R-helix on an opposing membrane. Interestingly, although sec22b binds to the combination of syntaxin 5, membrin, and rbet1, it can only bind if it is present while the others assemble; sec22b cannot bind to a pre-assembled ternary complex of syntaxin 5, membrin, and rbet1. Finally, we demonstrate that the quaternary complex containing sec22b is not an in vitro entity only, but is a bona fide species in living cells.     

The longin domain regulates subcellular targeting of VAMP7 in Arabidopsis thaliana

SNAREs (soluble N-ethyl-maleimide sensitive factor attachment protein receptors) which locate on the specific organelle membrane assure the correct vesicular transport by mediating specific membrane fusions. SNAREs are referred to as R- or Q-SNAREs on the basis of the amino acid sequence similarities and specific conserved residues. All of the Arabidopsis R-SNAREs have a N-terminal domain, called the longin domain (LD). In this study, we investigated the vacuolar targeting mechanism of Arabidopsis R-SNAREs. The vacuolar localized AtVAMP711 was used as the mother protein of GFP-tagged chimeric proteins joined to several domains such as the LD, the SNARE motif (SNM) and the transmembrane domain (TMD) of other organelle-localized R-SNAREs. The results showed that, whereas the TMD is not relevant for the vacuolar targeting, a complete LD is essential for the vacuolar and subcellular targeting.     

The molecular basis for the endocytosis of small R-SNAREs by the clathrin adaptor CALM

SNAREs provide a large part of the specificity and energy needed for membrane fusion and, to do so, must be localized to their correct membranes. Here, we show that the R-SNAREs VAMP8, VAMP3, and VAMP2, which cycle between the plasma membrane and endosomes, bind directly to the ubiquitously expressed, PtdIns4,5P(2)-binding, endocytic clathrin adaptor CALM/PICALM. X-ray crystallography shows that the N-terminal halves of their SNARE motifs bind the CALM(ANTH) domain as helices in a manner that mimics SNARE complex formation. Mutation of residues in the CALM:SNARE interface inhibits binding in vitro and prevents R-SNARE endocytosis in vivo. Thus, CALM:R-SNARE interactions ensure that R-SNAREs, required for the fusion of endocytic clathrin-coated vesicles with endosomes and also for subsequent postendosomal trafficking, are sorted into endocytic vesicles. CALM's role in directing the endocytosis of small R-SNAREs may provide insight into the association of CALM/PICALM mutations with growth retardation, cognitive defects, and Alzheimer's disease.     

A multigene family encoding R-SNAREs in the ciliate Paramecium tetraurelia

SNARE proteins (soluble N-ethylmaleimide-sensitive factor attachment protein receptors) mediate membrane interactions and are conventionally divided into Q-SNAREs and R-SNAREs according to the possession of a glutamine or arginine residue at the core of their SNARE domain. Here, we describe a set of R-SNAREs from the ciliate Paramecium tetraurelia consisting of seven families encoded by 12 genes that are expressed simultaneously. The complexity of the endomembrane system in Paramecium can explain this high number of genes. All P. tetraurelia synaptobrevins (PtSybs) possess a SNARE domain and show homology to the Longin family of R-SNAREs such as Ykt6, Sec22 and tetanus toxin-insensitive VAMP (TI-VAMP). We localized four exemplary PtSyb subfamilies with GFP constructs and antibodies on the light and electron microscopic level. PtSyb1-1, PtSyb1-2 and PtSyb3-1 were found in the endoplasmic reticulum, whereas PtSyb2 is localized exclusively in the contractile vacuole complex. PtSyb6 was found cytosolic but also resides in regularly arranged structures at the cell cortex (parasomal sacs), the cytoproct and oral apparatus, probably representing endocytotic compartments. With gene silencing, we showed that the R-SNARE of the contractile vacuole complex, PtSyb2, functions to maintain structural integrity as well as functionality of the osmoregulatory system but also affects cell division.     

Interactions within the yeast t-SNARE Sso1p that control SNARE complex assembly

In the eukaryotic secretory and endocytic pathways, transport vesicles shuttle cargo among intracellular organelles and to and from the plasma membrane. Cargo delivery entails fusion of the transport vesicle with its target, a process thought to be mediated by membrane bridging SNARE protein complexes. Temporal and spatial control of intracellular trafficking depends in part on regulating the assembly of these complexes. In vitro, SNARE assembly is inhibited by the closed conformation adopted by the syntaxin family of SNAREs. To visualize this closed conformation directly, the X-ray crystal structure of a yeast syntaxin, Sso1p, has been determined and refined to 2.1 A resolution. Mutants designed to destabilize the closed conformation exhibit accelerated rates of SNARE assembly. Our results provide insight into the mechanism of SNARE assembly and its intramolecular and intermolecular regulation.     

SNARE assembly and membrane fusion, a kinetic analysis

SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) assembly may promote intracellular membrane fusion, an essential process for vesicular transport in cells. Core complex formation between vesicle-associated SNARE and target membrane SNARE perhaps drives the merging of two membranes into a single bilayer. Using spin-labeling EPR, trans-SNARE complex formation was monitored "locally" at four different core locations of recombinant yeast SNAREs, which are individually reconstituted into phospholipid vesicles. The results indicate that the time scales of core formation are virtually the same at all four locations throughout the core region, indicating the possibility of a single step core assembly, which appears to be somewhat different from what has been postulated by the "zipper" model. The EPR data were then compared with the kinetics of the lipid mixing measured with the fluorescence assay. The analysis suggests that SNARE core assembly occurs on a much faster time scale than the lipid mixing, providing a new insight into the timing of individual events in SNARE-induced membrane fusion.     

Regulation of exocytosis by the exocyst subunit Sec6 and the SM protein Sec1

Trafficking of protein and lipid cargo through the secretory pathway in eukaryotic cells is mediated by membrane-bound vesicles. Secretory vesicle targeting and fusion require a conserved multisubunit protein complex termed the exocyst, which has been implicated in specific tethering of vesicles to sites of polarized exocytosis. The exocyst is directly involved in regulating soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) complexes and membrane fusion through interactions between the Sec6 subunit and the plasma membrane SNARE protein Sec9. Here we show another facet of Sec6 function-it directly binds Sec1, another SNARE regulator, but of the Sec1/Munc18 family. The Sec6-Sec1 interaction is exclusive of Sec6-Sec9 but compatible with Sec6-exocyst assembly. In contrast, the Sec6-exocyst interaction is incompatible with Sec6-Sec9. Therefore, upon vesicle arrival, Sec6 is proposed to release Sec9 in favor of Sec6-exocyst assembly and to simultaneously recruit Sec1 to sites of secretion for coordinated SNARE complex formation and membrane fusion.     

Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties.

Assembly of soluble N-ethylmaleimide-sensitive fusion attachment protein receptor (SNARE) proteins between two opposing membranes is thought to be the key event that initiates membrane fusion. Many new SNARE proteins have recently been localized to distinct intracellular compartments, supporting the view that sets of specific SNAREs are specialized for distinct trafficking steps. We have now investigated whether other SNAREs can form complexes with components of the synaptic SNARE complex including synaptobrevin/VAMP 2, SNAP-25, and syntaxin 1. When the Q-SNAREs syntaxin 2, 3, and 4, and the R-SNARE endobrevin/VAMP 8 were used in various combinations, heat-resistant complexes were formed. Limited proteolysis revealed that these complexes contained a protease-resistant core similar to that of the synaptic complex. All complexes were disassembled by the ATPase N-ethylmaleimide-sensitive fusion protein and its cofactor alpha-SNAP. Circular dichroism spectroscopy showed that major conformational changes occur during assembly, which are associated with induction of structure from unstructured monomers. Furthermore, no preference for synaptobrevin was observed during the assembly of the synaptic complex when endobrevin/VAMP 8 was present in equal concentrations. We conclude that cognate and non-cognate SNARE complexes are very similar with respect to biophysical properties, assembly, and disassembly, suggesting that specificity of membrane fusion in intracellular membrane traffic is not due to intrinsic specificity of SNARE pairing.     

Membrane‐directed molecular assembly of the neuronal SNARE complex

Since the discovery and implication of N‐ethylmaleimide‐sensitive factor (NSF)‐attachment protein receptor (SNARE) proteins in membrane fusion almost two decades ago, there have been significant efforts to understand their involvement at the molecular level. In the current study, we report for the first time the molecular interaction between full‐length recombinant t‐SNAREs and v‐SNARE present in opposing liposomes, leading to the assembly of a t‐/v‐SNARE ring complex. Using high‐resolution electron microscopy, the electron density maps and 3D topography of the membrane‐directed SNARE ring complex was determined at nanometre resolution. Similar to the t‐/v‐SNARE ring complex formed when 50 nm v‐SNARE liposomes meet a t‐SNARE‐reconstituted planer membrane, SNARE rings are also formed when 50 nm diameter isolated synaptic vesicles (SVs) meet a t‐SNARE‐reconstituted planer lipid membrane. Furthermore, the mathematical prediction of the SNARE ring complex size with reasonable accuracy, and the possible mechanism of membrane‐directed t‐/v‐SNARE ring complex assembly, was determined from the study. Therefore in the present study, using both lipososome‐reconstituted recombinant t‐/v‐SNARE proteins, and native v‐SNARE present in isolated SV membrane, the membrane‐directed molecular assembly of the neuronal SNARE complex was determined for the first time and its size mathematically predicted. These results provide a new molecular understanding of the universal machinery and mechanism of membrane fusion in cells, having fundamental implications in human health and disease.