Syntaxin 18, a soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor (SNARE) protein implicated in endoplasmic reticulum (ER) membrane fusion, forms a complex with other SNAREs (BNIP1, p31, and Sec22b) and several peripheral membrane components (Sly1, ZW10, and RINT-1). In the present study, we showed that a peripheral membrane protein encoded by the neuroblastoma-amplified gene (NAG) is a subunit of the syntaxin 18 complex. NAG encodes a protein of 2371 amino acids, which exhibits weak similarity to yeast Dsl3p/Sec39p, an 82-kDa component of the complex containing the yeast syntaxin 18 orthologue Ufe1p. Under conditions favoring SNARE complex disassembly, NAG was released from syntaxin 18 but remained in a p31-ZW10-RINT-1 subcomplex. Binding studies showed that the extreme N-terminal region of p31 is responsible for the interaction with NAG and that the N- and the C-terminal regions of NAG interact with p31 and ZW10-RINT-1, respectively. Knockdown of NAG resulted in a reduction in the expression of p31, confirming their intimate relationship. NAG depletion did not substantially affect Golgi morphology and protein export from the ER, but it caused redistribution of Golgi recycling proteins accompanied by a defect in protein glycosylation. These results together suggest that NAG links between p31 and ZW10-RINT-1 and is involved in Golgi-to-ER transport.

In Syn18(390)-transfected cells, we frequently (40-50% of cells at 72 hours after transfection) observed large patches positive for an ER membrane protein, Bap31 (Annaert et al., 1997) (Fig. 1B, middle row, left). Albeit much less frequently, similar patches were observed in cells transfected with the less efficient siRNA Syn18(770) (bottom row, left), suggesting that the redistribution of Bap31 is a consequence of syntaxin 18 depletion, and not a consequence of off target effect of Syn18(390). The different frequencies of the Bap31-positive patches are probably the result of the different knockdown efficiency of the two siRNAs. Fig. 1B also shows that silencing of syntaxin 18 causes a substantial dispersion of the Golgi complex marked by a cis-Golgi marker, p115 (Waters et al., 1992), without affecting microtubules. Other Golgi proteins, such as GM130, mannosidase II (Man II), β-COP and the KDEL receptor (KDEL-R), were also dispersed (supplementary material Fig. S1). The time course of morphological changes of the ER and Golgi structures concomitant with syntaxin 18 depletion is shown in supplementary material Fig. S2.To investigate in detail the morphology of the ER and the Golgi complex in syntaxin-18-depleted cells, we performed electron microscopy. In Syn18(390)-transfected cells, vesiculated membrane structures, instead of the Golgi stacks, were observed at the perinuclear region ( Fig. 2B,C; supplementary material Fig. S3). Furthermore, there were well-defined membrane aggregates consisting of a convoluted network of branching tubules, as well as dilated ER structures, in Syn18(390)-transfected cells ( Fig. 2B-D; supplementary material Fig. S3). Similar results were obtained with Syn18(770)-transfected cells, although ER aggregates were observed only in some cells (data not shown). Quantitative analysis showed that the area and length of the ER normalized to the cytoplasmic area of Syn18(390)-transfected cells are higher than those of mock-transfected cells (Tables 1 and 2), suggesting a proliferation of the ER membrane concomitant with syntaxin 18 depletion.Immunoelectron microscopy confirmed Golgi disassembly and the formation of ER membrane aggregates in syntaxin-18-depleted cells. In Syn18(390)-transfected cells, a cis-Golgi marker, p115, and . At 72 hours after transfection, the cells were stained with an antibody against syntaxin 18 (right) or solubilized in phosphate-buffered saline with 0.5% SDS. The lysates (10 μg each) were separated by SDS-PAGE and analyzed by immunoblotting with the indicated antibodies (left). (B) HeLa cells were treated as described in A and stained for Bap31, p115 or α-tubulin. The distributions of the proteins investigated were indistinguishable between mock-transfected cells and lamin A/C siRNA-treated cells (data not shown). Scale bars: 10 μm. The boxed area in B is shown enlarged in C. G, Golgi complex; M, mitochondria; ER, endoplasmic reticulum; N, nucleus. Arrows, arrowheads and asterisks indicate vesiculated membrane structures, ER patches and dilated ER, respectiv...

SNARE (soluble N-ethylmaleimide-sensitive fusion protein-attachment protein receptor) proteins involved in membrane fusion usually contain a conserved alpha-helix (SNARE motif) that is flanked by a C-terminal transmembrane domain. They can be classified into Q-SNARE and R-SNARE based on the structural property of their motifs. Assembly of four SNARE motifs (Qa, b, c and R) is supposed to trigger membrane fusion. We have previously shown that ER (endoplasmic reticulum)-localized syntaxin 18 (Qa) forms a complex with BNIP1 (Qb), p31/Use1 (Qc), Sec22b (R) and several peripheral membrane proteins. In the present study, we examined the interaction of syntaxin 18 with other SNAREs using pulldown assays and CD spectroscopy. We found that the association of syntaxin 18 with Sec22b induces an increase in alpha-helicity of their SNARE motifs, which results in the formation of high-affinity binding sites for BNIP1 and p31. This R-SNARE-dependent Q-SNARE assembly is quite different from the assembly mechanisms of SNAREs localized in organelles other than the ER. The implication of the mechanism of ER SNARE assembly is discussed in the context of the physiological roles of the syntaxin 18 complex.

p31, the mammalian orthologue of yeast Use1p, is an endoplasmic reticulum (ER)-localized soluble Nethylmaleimide-sensitive factor attachment protein (SNAP) receptor (SNARE) that forms a complex with other SNAREs, particularly syntaxin 18. However, the role of p31 in ER function remains unknown. To determine the role of p31 in vivo, we generated p31 conditional knockout mice. We found that homozygous deletion of the p31 gene led to early embryonic lethality before embryonic day 8.5. Conditional knockout of p31 in brains and mouse embryonic fibroblasts (MEFs) caused massive apoptosis accompanied by upregulation of ER stressassociated genes. Microscopic analysis showed vesiculation and subsequent enlargement of the ER membrane in p31-deficient cells. This type of drastic disorganization in the ER tubules has not been demonstrated to date. This marked change in ER structure preceded nuclear translocation of the ER stress-related transcription factor C/EBP homologous protein (CHOP), suggesting that ER stress-induced apoptosis resulted from disruption of the ER membrane structure. Taken together, these results suggest that p31 is an essential molecule involved in the maintenance of ER morphology and that its deficiency leads to ER stress-induced apoptosis.The endoplasmic reticulum (ER) consists of a network of tubules and sheets that extends from the cell center to the periphery in eukaryotic cells. It synthesizes secretory and membrane proteins as well as lipids. In addition, the ER has many diverse functions, including folding, posttranslational modification, export of secretory and membrane proteins, and calcium storage. Various intracellular and extracellular stimuli, including reduction of disulfide bonds, calcium depletion from the ER lumen, inhibition of glycosylation, and impairment of protein transport from the ER to the Golgi complex, affect functions of the ER, and disturbance in ER functions causes ER stress. In case of prolonged ER stress, cellular signaling leading to cell death are activated. ER stress has been suggested to be involved in various disorders (12,19,36).The ER maintains several functionally and morphologically distinct subdomains, such as rough and smooth membranes and ER exit sites. Despite the structural complexity, the ER is a dynamic organelle, and ER tubules dynamically detach and fuse with each other to form three-way junctions in a microtubule (MT)-dependent fashion (1, 17, 18, 34).Several proteins have been implicated in the regulation of ER structure. The loss of function of molecules including BNIP1, p97, and p37 involved in ER morphology leads to the loss of three-way junctions; however, the tubular structure of the ER is relatively unaffected (20,28,29). Vedrenne and Hauri proposed the mechanisms underlying ER network formation as follows (31): ER membranes are pulled along MTs by MT plus end-directed kinesin-type motor kinesin-1 (8), and the resulting membrane extensions are stabilized by the cytoskeleton-linking ER membrane protein of 63 kDa (13,30). If ER membranes get close...

ZW10 interacts with dynamitin, a subunit of the dynein accessory complex dynactin, and functions in termination of the spindle checkpoint during mitosis and in membrane transport between the endoplasmic reticulum (ER) and Golgi apparatus during interphase. Its associations with kinetochores and ER membranes are mediated by Zwint‐1 and RINT‐1, respectively. A previous yeast two‐hybrid study showed that the C‐terminal region of ZW10 interacts with dynamitin, and part of this region has been used as an inhibitor of ZW10 function. In the present study, we reinvestigated the interaction between ZW10 and dynamitin, and showed that the N‐terminal region of ZW10 is the major binding site for dynamitin and, like full‐length ZW10, could potentially move along microtubules to the centrosomal area in a dynein‐dynactin‐dependent manner. Competitive binding experiments demonstrated that dynamitin and RINT‐1 occupy the same N‐terminal region of ZW10 in a mutually exclusive fashion. Consistent with this, over‐expression of RINT‐1 interfered with the dynein‐dynactin‐mediated movement of ZW10 to the centrosomal area. Given that the N‐terminal region of ZW10 also interacts with Zwint‐1, this region may be important for switching partners; one partner is a determinant for localization (kinetochore and ER) and the other links ZW10 to dynein function.