Syntaxin 1A is a nervous system-specific protein thought to function during the late steps of the regulated secretory pathway by mediating the docking of secretory vesicles with the plasma membrane. The effects of transiently overexpressing syntaxin 1A were examined on protein secretion in constitutively secreting cell lines that do not normally express the protein. Syntaxin 1A slows the constitutive release of two marker proteins, human growth hormone (hGH) and vesicular stomatitis virus glycoprotein from COS-1 cells, increasing the intracellular half-life of human growth hormone from 90 min to 18 h. These secretory proteins are concentrated in the periphery of the cell. The effect is specific for the full-length neuronal protein. Neither a syntaxin 1A variant that lacks a membrane attachment domain nor syntaxin 2 causes the cells to retain human growth hormone. The effect of syntaxin 1A is partially reversed by incubating the cells with botulinum type C1 neurotoxin, which specifically cleaves syntaxin 1A. Release of human growth hormone from syntaxin 1A-expressing cells is maintained during a blockade of protein synthesis, suggesting that the hormone is being released from a pool of stored vesicles that accumulate before the addition of cycloheximide. The existence of a post-Golgi storage compartment in syntaxin 1A-expressing cells was confirmed using brefeldin A to collapse the Golgi stacks. Brefeldin A rapidly blocks growth hormone release in control cultures while having no effect on release in cells expressing syntaxin 1A. Reducing the temperature to 19 degrees C, which inhibits transport from the trans-Golgi network, also inhibits hGH secretion from cells without syntaxin 1A but has little effect on hGH secretion from cells with syntaxin 1A. The present experiments indicate that syntaxin 1A enables the storage of vesicles which would otherwise be immediately released (Bittner, 1996).

Cortical vesicles (CV) possess components critical to the mechanism of exocytosis. The homotypic fusion of CV centrifuged or settled into contact has a sigmoidal Ca2+ activity curve comparable to exocytosis (CV-PM fusion). Sr2+ and Ba2+ also trigger CV-CV fusion, and agents affecting different steps of exocytotic fusion block Ca2+, Sr2+, and Ba2+-triggered CV-CV fusion. The maximal number of active fusion complexes per vesicle, Max, was quantified by NEM inhibition of fusion, showing that CV-CV fusion satisfies many criteria of a mathematical analysis developed for exocytosis. Both Max and the Ca2+ sensitivity of fusion complex activation are comparable to that determined for CV-PM fusion. Using Ca2+-induced SNARE complex disruption, the relationship between membrane fusion (CV-CV and CV-PM) and the SNARE complex has been analyzed. Fusion and complex disruption have different sensitivities to Ca2+, Sr2+, and Ba2+: the complex remains Ca2+- sensitive on fusion-incompetent CV, and disruption does not correlate with the quantified activation of fusion complexes. Under conditions that disrupt the SNARE complex, CV on the PM remain docked and fusion competent, and isolated CV still dock and fuse, but with a markedly reduced Ca2+ sensitivity. Thus, in this system, neither the formation, presence, nor disruption of the SNARE complex is essential to the Ca2+-triggered fusion of exocytotic membranes. Therefore the SNARE complex alone cannot be the universal minimal fusion machine for intracellular fusion. It is suggest that this complex modulates the Ca2+ sensitivity of fusion (Coorssen, 1998).

Syntaxins are cytoplasmically oriented integral membrane soluble NEM-sensitive factor receptors (SNAREs; soluble NEM-sensitive factor attachment protein receptors) thought to serve as targets for the assembly of protein complexes important in regulating membrane fusion. The SNARE hypothesis predicts that the fidelity of vesicle traffic is controlled in part by the correct recognition of vesicle SNAREs with their cognate target SNARE partner. In the exocrine acinar cells of the pancreas, multiple syntaxin isoforms are expressed and that they appear to reside in distinct membrane compartments. Syntaxin 2 is restricted to the apical plasma membrane whereas syntaxin 4 is found most abundantly on the basolateral membranes. Surprisingly, syntaxin 3 was found to be localized to a vesicular compartment, the zymogen granule membrane. These proteins are capable of specific interaction with vesicle SNARE proteins. Their nonoverlapping locations support the general principle of the SNARE hypothesis and provide new insights into the mechanisms of polarized secretion in epithelial cells (Gaisano, 1996).

The proposed cis-Golgi vesicle receptor syntaxin 5 is found in a complex with Golgi-associated SNARE of 28 kDa (GOS-28), rbet1, rsly1, and two novel proteins characterized in this study: rat sec22b and membrin, both cytoplasmically oriented integral membrane proteins. The complex appears to recapitulate vesicle docking interactions of proteins originating from distinct compartments, since syntaxin 5, rbet1, and GOS-28 localize to Golgi membranes, whereas mouse sec22b and membrin accumulate in the endoplasmic reticulum. Protein interactions in the complex are dramatically rearranged by N-ethylmaleimide-sensitive factor. The complex consists of two or more subcomplexes with some members (rat sec22b and syntaxin 5) in common and others (rbet1 and GOS-28) mutually exclusively associated. It is proposed that these protein interactions determine vesicle docking/fusion fidelity between the endoplasmic reticulum and Golgi (Hay, 1997).

A major physiological role of insulin is the regulation of glucose uptake into skeletal and cardiac muscle and adipose tissue, mediated by an insulin-stimulated translocation of GLUT4 glucose transporters from an intracellular vesicular pool to the plasma membrane. This process is similar to the regulated docking and fusion of vesicles in neuroendocrine cells, a process that involves SNARE-complex proteins. Several SNARE proteins are found in adipocytes: vesicle-associated membrane protein (VAMP-2), its related homolog cellubrevin, and syntaxin-4. Treatment of permeabilized 3T3-L1 adipocytes with botulinum neurotoxin D, which selectively cleaves VAMP-2 and cellubrevin, inhibits the ability of insulin to stimulate translocation of GLUT4 vesicles to the plasma membrane. Treatment of the permeabilized adipocytes with glutathione S-transferase fusion proteins encoding soluble forms of VAMP-2 or syntaxin-4 also effectively block insulin-regulated GLUT4 translocation. These results provide evidence of a functional role for SNARE-complex proteins in insulin-stimulated glucose uptake and suggest that adipocytes utilize a mechanism of regulating vesicle docking and fusion analogous to that found in neuroendocrine tissues (Cheatham, 1996).

Reconstitution of synaptic vesicle formation in vitro has revealed a pathway of synaptic vesicle biogenesis from endosomes that requires the heterotetrameric adaptor complex AP3. Adaptor protein 3 has been implicated in Golgi-to-vacuole traffic. Because synaptic vesicles have a distinct protein composition, the AP3 complex should selectively recognize some or all of the synaptic vesicle proteins. One element of this recognition process is the v-SNARE known as VAMP-2. Tetanus toxin, which cleaves VAMP-2, inhibits the formation of synaptic vesicles and their coating with AP3 in vitro. Mutant tetanus toxin and botulinum toxins, which cleave t-SNAREs, do not inhibit synaptic vesicle production. AP3-containing complexes isolated from coated vesicles could be immunoprecipitated by a VAMP-2 antibody. These data imply that AP3 recognizes a component of the fusion machinery, which may prevent the production of inert synaptic vesicles (Salem, 1998).

How is the Golgi apparatus reconstructed after mitosis? Multiple components are involved, but a common one appears to be Syntaxin 5, a t- or target-SNARE that forms a docking (and fusion) component for vesicular targeting. Two other components are involved: NSF and p97 are both ATPases that constitute one component; SNAPs that interact with the SNAREs (SNAP receptors) constitute another. NSF and p97 appear to act in two different pathways that have different consequences for the Golgi apparatus. Each golgi cisterna has two domains: an inner core containing resident Golgi enzymes, and a peripheral rim that is involved in the budding and fusion of transport vesicles. It is thought that fusion events at the rim are controlled by NSF and those at the core by p97. NSF and SNAPs are thought to break up a SNARE pair, but it is unclear at what step this occurs. In the original model, NSF/SNAPs bind to the SNAREs that have docked the vesicle to the target membrane; the break-up of the SNARE pair is somehow coupled to membrane fusion. More recently it has been suggested that NSF acts before docking and fusion to break up the SNARE pair formed during the previous fusion event and prime it ready for the next. Several studies point in this direction, the most recent being work on yeast vacuole fusion. The yeast homologs of NSF and -SNAP are needed to prime the vacuoles but not for their subsequent fusion. A cell-free system that mimics the reassembly of Golgi stacks at the end of mitosis requires the two ATPases, NSF and p97, to rebuild Golgi cisternae. Morphological studies now show that alpha-SNAP can inhibit the p97 pathway, whereas p47, a component of the p97 pathway, can inhibit the NSF pathway. Anti-syntaxin 5 antibodies and a soluble, recombinant syntaxin 5 inhibit both pathways, suggesting that this t-SNARE is a common component. Biochemical studies confirm this, showing that p47 binds directly to syntaxin 5 and competes for binding with alpha-SNAP. p47 also mediates the binding of p97 to syntaxin 5 and so plays an analogous role to alpha-SNAP, which mediates the binding of NSF (Rabouille, 1998).

The competition between the NSF and p97 pathways may have important implications for the biogenesis and functioning of the Golgi apparatus. The fusion events at the rim are likely controlled by NSF and those in the core by p97. Therefore, during interphase, NSF would control the flux of cargo through the Golgi apparatus whereas p97 would control its biogenesis. The ratio of these two activities would then depend on the cell type. At one extreme would be the differentiated secretory cell with a high flux of cargo but only sufficient biogenesis to compensate for Golgi turnover. At the other extreme would be the nonsecreting but rapidly dividing tumour cell with much less cargo but a high rate of organelle biogenesis. By utilizing a common component of the fusion machinery (syntaxin 5), the cell would be able to integrate the flux of cargo through the Golgi apparatus with its biogenesis. Calculation shows that there are roughly similar amounts of alpha-SNAP and p47 in cytosol, suggesting that competition in vivo is a real possibility. Efforts are now focused on trying to adjust the levels of these two regulatory components so as to determine the consequences for the Golgi apparatus (Rabouille, 1998).

The homotypic fusion of endoplasmic reticulum (ER) membranes is crucial for normal cell division and maintenance of an intact organelle. The fusion of ER membranes in yeast does not require Sec18p/NSF and Sec17p, two proteins needed for docking of vesicles with their target membrane (heterotypic fusion). Instead, ER membranes require an NSF-related ATPase: Cdc48p. Since both vesicular and organelle fusion events use related ATPases, an investigation was carried out to determine whether both fusion events are also SNARE mediated. Evidence is presented that the fusion of ER membranes requires Ufe1p, a t-SNARE that localizes to the ER, but that no known v-SNARE is involved. It is proposed that the Ufe1 protein acts in the dual capacity of an organelle membrane fusion-associated SNARE by undergoing direct t-t-SNARE and Cdc48p interactions during organelle membrane fusion, as well as a t-SNARE for vesicular traffic (Patel, 1998).

Several lines of evidence suggest that Ufe1p does not function in ER membrane fusion by pairing to a known v-SNARE but instead most likely binds to Ufe1p on the opposite membrane. No known v-SNARE that localizes in part to the ER affects ER fusion when conditionally defective. However, the same mutations exhibit conditional defects in other membrane fusion events that are not related to ER membrane fusion, such as in anterograde and retrograde traffic. The fact that Ufe1p is able to form higher multimers is a function that is consistent with its proposed role in homotypic SNARE pairing. Together with data that show that Ufe1p functions in heterotypic membrane docking/fusion of retrograde carriers with the v-SNARE Sec22p and the Sec20p/Tip20p complex, these findings suggest that the same t-SNARE can function in heterotypic vesicle-mediated fusion events as well as in homotypic organelle membrane fusion events. In addition, reciprocal genetic interactions are found between Ufe1p and Cdc48p, an ATPase that functions in the Sec18p-independent ER membrane fusion, as well as a direct physical association between Cdc48p and Ufe1p. It is concluded that the Ufe1 protein can act in the capacity of a heterotypic as well as homotypic membrane fusion SNARE molecule dependent on whether it associates with a v-SNARE or another t-SNARE (Patel, 1998).

The basic reaction mechanisms for membrane fusion in the trafficking of intracellular membranes and in exocytosis are probably identical. But in contrast to regulated exocytosis, intracellular fusion reactions are referred to as 'constitutive', since no final Ca2+-dependent triggering step has been observed. Although transport from the endoplasmic reticulum to the Golgi apparatus in the cell depends on Ca2+, as does endosome fusion and assembly of the nuclear envelope, it is unclear whether Ca2+ triggers these events. Membrane fusion involves several subreactions: priming, tethering and docking. Proteins that are needed for fusion include p115, SNAPs, NSF, SNAREs and small GTPases, all of which operate in these early reactions, but the machinery that catalyses the final mixing of biological membranes is still unknown. Ca2+ is released from the vacuolar lumen following completion of the docking step. Calmodulin is identified as the putative Ca2+ sensor and as the first component required in the post-docking phase of vacuole fusion. Calmodulin binds tightly to vacuoles upon Ca2+ release. Unlike synaptotagmin or syncollin in exocytosis, calmodulin does not act as a fusion clamp but actively promotes bilayer mixing. Hence, activation of SNAREs is not sufficient to drive bilayer mixing between physiological membranes. It is proposed that Ca2+ control of the latest phase of membrane fusion may be a conserved feature, relevant not only for exocytosis, but also for intracellular, 'constitutive' fusion reactions. However, the origin of the Ca2+ signal, its receptor and its mode of processing differ (Peters, 1998).

Endocytosis-mediated recycling of plasma membrane is a critical vesicle trafficking step important in diverse biological processes. The membrane trafficking decisions and sorting events take place in a series of heterogeneous and highly dynamic organelles, the endosomes. Syntaxin 13, a recently discovered member of the syntaxin family, has been suggested to play a role in mediating endosomal trafficking. To better understand the function of syntaxin 13 its intracellular distribution in nonpolarized cells was examined. By confocal immunofluorescence and electron microscopy, syntaxin 13 is primarily found in tubular early and recycling endosomes, where it colocalizes with transferrin receptor. Additional labeling is also present in endosomal vacuoles, where it is often found in clathrin-coated membrane areas. Furthermore, anti-syntaxin 13 antibody inhibits transferrin receptor recycling in permeabilized PC12 cells. Immunoprecipitation of syntaxin 13 reveals that, in Triton X-100 extracts, syntaxin 13 is present in a complex(es) comprised of betaSNAP, VAMP 2/3, and SNAP-25. This complex(es) binds exogenously added alphaSNAP and NSF and dissociates in the presence of ATP, but not ATPgammaS. These results support a role for syntaxin 13 in membrane fusion events during the recycling of plasma membrane proteins (Prekeris, 1998).

Intracellular membrane fusion is mediated by the formation of a four-helix bundle comprised of SNARE proteins. Every cell expresses large numbers 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, it is shown 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 (Scales, 2000).

SNARE function in the cell is much more specific than is suggested by the promiscuous interactions observed in vitro. In all cases, the known cognate SNAREs for the fusion event (syntaxin 1a, VAMP2, and SNAP-25) are the most effective at inhibiting or rescuing fusion, but in a few cases, similarly localized SNAREs are also able to function, albeit at lower efficiencies (Scales, 2000).

While it is not possible to test the activity of these SNARE proteins in other transport steps in cracked PC12 cells, they do function in other assays. For example, the soluble forms of the endosomal syntaxin 7 and VAMP8, which have no effect in the assay used here, do indeed inhibit the endosomal transport steps in which they are involved, demonstrating the specificity of these SNAREs. In MDCK cells, overexpression of syntaxin 3, but not syntaxins 2 or 4, inhibits transport to the apical plasma membrane from the TGN and endosomes, demonstrating a functional difference between these syntaxins that is also reflected in the assay used here, where syntaxin 3 is significantly less efficient at inhibiting dense core vesicle-plasma membrane fusion. Conversely, overexpression of plasma membrane syntaxin 1a has no effect on ER-to-Golgi transport, whereas soluble syntaxin 5 does inhibit this fusion reaction, as expected from its function. Similarly, homotypic mitotic Golgi fusion and homotypic ER fusion events, mediated by syntaxin 5 and Ufe1p, respectively, are inhibited by their soluble counterparts. The ER-Golgi R-SNARE Sec22b does not inhibit dense core vesicle-plasma membrane fusion, but its overexpression disrupts the cis-Golgi localization of syntaxin 5 (Scales, 2000).

The results presented here demonstrate that the specificity of core complex function in driving membrane fusion is not simply reflected in the ability of the complexes to form in vitro. In fact, no activity could be measured for most VAMPs and syntaxins in inhibiting dense core vesicle-plasma membrane fusion, although all these SNAREs form highly stable complexes in vitro. Complex thermostability appears only to correlate with membrane fusion for the SNAP-25 C- and N-terminal hydrophobic layer mutants, with lower fusion achieved by less stable mutants. These data demonstrate that fusion complex surface residues are a determinant of the specificity of fusion that may not greatly affect the thermostability of the complexes. It is concluded that SNARE proteins play an important role in the fidelity of membrane trafficking. The specific localization of SNARE proteins throughout the cell is therefore likely to be critical in the organization of the membrane compartments of cells. The information for SNARE pairing specificity is not, however, completely determined by the ability to form stable complexes, but is probably determined through interactions with other proteins, perhaps chaperones for formation of the core fusion complex. The nature of these other specificity determinants is not yet clear, but rabs, rab effector proteins, and sec1 family members are potential candidates (Scales, 2000).

Septins are GTPases required for the completion of cytokinesis in diverse organisms, yet their roles in cytokinesis or other cellular processes remain unknown. A newly identified septin, CDCrel-1, is predominantly expressed in the nervous system. This protein is associated with membrane fractions, and a significant fraction of the protein copurifies and coprecipitates with synaptic vesicles. In detergent extracts, CDCrel-1 and another septin, Nedd5, immunoprecipitates with the SNARE protein syntaxin by directly binding to syntaxin via the SNARE interaction domain. Transfection of HIT-T15 cells with wild-type CDCrel-1 inhibits secretion, whereas GTPase dominant-negative mutants enhance secretion. These data suggest that septins may regulate vesicle dynamics through interactions with syntaxin (Beites, 1999).

A single family member homolog of syntaxin has been identified in the sea urchin. Syntaxin is present throughout development, and in rapidly dividing cleavage stage embryos it is present on numerous vesicles at the cell cortex. It is hypothesized that syntaxin mediates essential membrane fusion events during early embryogenesis, reasoning that the vesicles and/or their contents are important for development. Functional inactivation of syntaxin with either Botulinum neurotoxin C1 (BoNT-C1), which specifically proteolyzes syntaxin, or antibodies against syntaxin results in an inhibition of cell division. These observations suggest that syntaxin is essential for membrane fusion events critical for cell division (Conner, 1999).

BoNT-C1 cleaves syntaxin family members at an amino acid sequence specific site near the transmembrane domain at the C terminus. Because the sea urchin syntaxin contains the conserved BoNT-C1 cleavage site, and BoNT-C1 cleaves sea urchin syntaxin in vitro, it is hypothesized that antibodies to the N-terminal region of syntaxin should no longer localize to vesicles in toxin-injected cells in vivo, because BoNT-C1 cleavage would release the syntaxin N-terminal region from the vesicle membrane. To test this hypothesis, a single blastomere of a two-cell embryo was injected with BoNT-C1 and allowed to develop until phenotypic difference in cell division was observed. It was asked whether syntaxin localized to vesicles at the cell cortex by injecting fluorochrome-labeled antibodies against syntaxin. In toxin-free cells, syntaxin localizes to vesicles enriched at the cell cortex, whereas in toxin-treated cells, vesicle-associated syntaxin signals are dramatically decreased. To quantify the effects of BoNT-C1 on syntaxin vesicle immunolocalization at the cortex, fluorescence measurements of at least 15 confocal sections were taken for each embryo examined. As the concentration of BoNT-C1 is increases, syntaxin immunolocalization decreases compared with toxin-free blastomeres of the same embryo. Injection of 3.9 and 5.3 nM BoNT-C1 results in an ~30% and 70% decrease in syntaxin immunolocalization, respectively, suggesting that proper cell division requires at least ~70% intact syntaxin. It is hypothesized that treatment with syntaxin antibodies or BoNT-C1 could halt cell progression through the cell cycle at a checkpoint that monitors membrane status within the cell (Conner, 1999).