ZW10, a dynamitin-interacting protein associated with kinetochores, is known to participate directly in turning off of the spindle checkpoint. In the present study, we show that ZW10 is located in the endoplasmic reticulum as well as in the cytosol during interphase, and forms a subcomplex with RINT-1 (Rad50-interacting protein) and p31 in a large complex comprising syntaxin 18, an endoplasmic reticulum-localized t-SNARE implicated in membrane trafficking. Like conventional syntaxin-binding proteins, ZW10, RINT-1 and p31 dissociated from syntaxin 18 upon Mg 2 þ -ATP treatment in the presence of NSF and a-SNAP, whereas the subcomplex was not disassembled. Overexpression, microinjection and knockdown experiments revealed that ZW10 is involved in membrane trafficking between the endoplasmic reticulum and Golgi. The present results disclose an unexpected role for a spindle checkpoint protein, ZW10, during interphase.

Synaptic exocytosis requires the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins syntaxin 1, SNAP-25, and synaptobrevin (VAMP). Assembly of the SNAREs into a stable core complex is supposed to catalyze membrane fusion, and proteoliposomes reconstituted with synaptic SNARE proteins spontaneously fuse with each other. We now show that liposome fusion mediated by synaptic SNAREs is inhibited by botulinum neurotoxin E (BoNT͞E) but can be rescued by supplementing the C-terminal portion of SNAP-25. Furthermore, fusion is prevented by a SNAP-25-specific antibody known to block exocytosis in chromaffin cells, and it is competed for by soluble fragments of the R-SNAREs synaptobrevin 2, endobrevin͞VAMP-8, and tomosyn. No accumulation of clustered vesicles is observed during the reaction. Rapid artificial clustering of SNARE-containing proteoliposomes enhances the fusion rate at low but not at saturating liposome concentrations. We conclude that the rate of liposome fusion is dominated by the intrinsic properties of the SNAREs rather than by the preceding docking step. E xocytosis of synaptic vesicles requires the N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) proteins syntaxin 1 and SNAP-25 on the synaptic plasma membrane, and synaptobrevin (also referred to as VAMP) on the vesicle membrane. Syntaxin 1 and synaptobrevin possess a single transmembrane domain at the C-terminal end, whereas SNAP-25 is membrane-anchored by palmitoyl side chains attached in the middle of the molecule. Although the essential function of the SNAREs for neurotransmitter release is well established, it is still debated whether these proteins operate as fusion catalysts or whether they act upstream of the actual fusion reaction (for review, see refs. 1-3).Each SNARE-protein contains one (syntaxin, synaptobrevin) or two (SNAP-25) characteristic stretches of 60-70 aa arranged in heptad repeats, referred to as SNARE motifs (4). Although isolated SNARE motifs are unstructured, they spontaneously assemble into stable core complexes consisting of four helix bundles. Each helix is contributed by a different SNARE motif (5, 6) representing a separate subfamily, referred to as Qa-, Qb-, Qc-, and R-SNARE motif (4, 7). Disassembly requires ATP and the action of the AAA-ATPase the N-ethylmaleimide-sensitive factor in conjunction with cofactors (8). Because membrane fusion requires that SNAREs are initially present on both membranes, assembly of the core complex would pull the membranes closely together, resulting in fusion, with the energy being provided by the assembly reaction (9, 10).Although studies on soluble recombinant SNAREs have been instrumental in developing our current thinking about how SNAREs fuse membranes, it still needs to be clarified how the speed and efficiency of biological fusion reactions is brought about at the molecular level. As a step toward this goal, it is necessary to reconstitute exocytotic membrane fusion by using purified proteins and artificial membranes. Recently, R...

Tomosyn is a 130-kDa syntaxin-binding protein that contains a large N-terminal domain with WD40 repeats and a C-terminal domain homologous to R-SNAREs. Here we show that tomosyn forms genuine SNARE core complexes with the SNAREs syntaxin 1 and SNAP-25. In vitro studies with recombinant proteins revealed that complex formation proceeds from unstructured monomers to a stable four-helical bundle. The assembled complex displayed features typical for SNARE core complexes, including a profound hysteresis upon unfoldingrefolding transitions. No stable complexes were formed between the SNARE motif of tomosyn and either syntaxin or SNAP-25 alone. Furthermore, both native tomosyn and its isolated C-terminal domain competed with synaptobrevin for binding to endogenous syntaxin and SNAP-25 on inside-out sheets of plasma membranes. Tomosyn-SNARE complexes were effectively disassembled by the ATPase N-ethylmaleimide-sensitive factor together with its cofactor ␣-SNAP. Moreover, the C-terminal domain of tomosyn was as effective as the cytoplasmic portion of synaptobrevin in inhibiting evoked exocytosis in a cell-free preparation derived from PC12 cells. Similarly, overexpression of tomosyn in PC12 cells resulted in a massive reduction of exocytosis, but the release parameters of individual exocytotic events remained unchanged. We conclude that tomosyn is a soluble SNARE that directly competes with synaptobrevin in the formation of SNARE complexes and thus may function in down-regulating exocytosis.

Members of the syntaxin family are target-soluble Nethylmaleimide-sensitive factor-attachment protein receptors involved in vesicle docking and/or fusion within the exocytic and endocytotic pathways. By using the yeast two-hybrid system, we have identified a novel member of the syntaxin family, syntaxin 18, that binds to ␣-soluble N-ethylmaleimide-sensitive factor-attachment protein. Subcellular fractionation and immunocytochemical analysis revealed that syntaxin 18 is principally located in the endoplasmic reticulum. We examined the effect of overexpression of FLAG-tagged syntaxin 18 and a mutant lacking the N-terminal 81 amino acid residues on protein transport and organelles in the early secretory pathway. Both expressed proteins localized to the endoplasmic reticulum, and the expressed FLAG-syntaxin 18 caused remarkable aggregation of endoplasmic reticulum membranes. Although expression of the FLAG-syntaxin 18 lacking the N-terminal region produced less effect on the morphology of the endoplasmic reticulum, dispersion of the endoplasmic reticulum-Golgi intermediate compartment and cisGolgi was elicited. Moreover, overexpression of the FLAG-syntaxin 18 mutant inhibited protein export from the endoplasmic reticulum. These results taken together suggest that syntaxin 18 functions in transport between the endoplasmic reticulum and Golgi.

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COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum to the Golgi apparatus. COPII consists of three parts: Sar1p and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p. Using a glutathione S-transferase fusion protein with mouse Sec23p, we identified a novel mammalian Sec23p-interacting protein, p125, which is clearly distinct from Sec24p. The N-terminal region of p125 is rich in proline residues, and the central and C-terminal regions exhibit significant homology to phospholipid-modifying proteins, especially phosphatidic acid preferring-phospholipase A 1 . We transiently expressed p125 and mouse Sec23p in mammalian cells and examined their interaction. The results showed that the N-terminal region of p125 is important for the interaction with Sec23p. We confirmed the interaction between the two proteins by a yeast two-hybrid assay. Overexpression of p125, like that of mammalian Sec23p, caused disorganization of the endoplasmic reticulum-Golgi intermediate compartment and Golgi apparatus, suggesting its role in the early secretory pathway.

The endoplasmic reticulum (ER) is thought to play an important structural and functional role in phagocytosis. According to this model, direct membrane fusion between the ER and the plasma or phagosomal membrane must precede further invagination, but the exact mechanisms remain elusive. Here, we investigated whether various ER-localized SNARE proteins are involved in this fusion process. When phagosomes were isolated from murine J774 macrophages, we found that ER-localized SNARE proteins (syntaxin 18, D12, and Sec22b) were significantly enriched in the phagosomes. Fluorescence and immuno-EM analyses confirmed the localization of syntaxin 18 in the phagosomal membranes of J774 cells stably expressing this protein tagged to a GFP variant. To examine whether these SNARE proteins are required for phagocytosis, we generated 293T cells stably expressing the Fc␥ receptor, in which phagocytosis occurs in an IgGmediated manner. Expression in these cells of dominant-negative mutants of syntaxin 18 or D12 lacking the transmembrane domain, but not a Sec22b mutant, impaired phagocytosis. Syntaxin 18 small interfering RNA (siRNA) selectively decreased the efficiency of phagocytosis, and the rate of phagocytosis was markedly enhanced by stable overexpression of syntaxin 18 in J774 cells. Therefore, we conclude that syntaxin 18 is involved in ER-mediated phagocytosis, presumably by regulating the specific and direct fusion of the ER and plasma or phagosomal membranes. INTRODUCTIONPhagocytosis is the coordinated process by which large foreign particles are internalized into a newly formed organelle, the phagosome. In professional phagocytes of the immune system, including macrophages, neutrophils, and dendritic cells, the internalization is triggered by activation of various types of cell surface receptors in the course of innate and adaptive immune responses. For example, in the case of immunoglobulin (Ig)-mediated phagocytosis, Fc receptors (Fc␥Rs) cluster where they contact an Ig-opsonized solid surface; this induces actin polymerization, resulting in the formation of pseudopods that engulf the particle . Subsequently, the phagosomes mature by fusing with other organelles of the endocytic pathway, leading to the formation of phagolysosomes (Downey et al., 1999;Vieira et al., 2002).In macrophages, every membrane fusion event during phagocytosis is thought to be mediated by soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (Coppolino et al., 2001). It is currently believed that the assembly of SNARE proteins leads to a tight connection between the vesicle and the target membrane, which initiates the opening of the fusion pore . The SNARE complex forms an extended parallel four-helix bundle in order to fuse the two membranes Antonin et al., 2002). Three helices are extended from one membrane by proteins of the syntaxin and SNAP-25 families (Q-SNAREs) that contain a conserved glutamine residue at a central position called the "0" layer, and the remaining helix, extended from the opposite membr...

Using macrophages overexpressing or reducing SNAP-23, this study shows that SNAP-23 is implicated in phagosome formation and maturation, presumably by mediating SNARE-based membrane traffic. Indeed, a conformational change in SNAP-23 structure based on FRET signal is observed on the phagosome membrane of cells overexpressing the lysosomal SNARE VAMP7.