Coat protein complex II (COPII)-coated vesicles/carriers, which mediate export of proteins from the endoplasmic reticulum (ER), are formed at special ER subdomains in mammals, termed ER exit sites or transitional ER. The COPII coat consists of a small GTPase, Sar1, and two protein complexes, Sec23-Sec24 and Sec13-Sec31. Sec23-Sec24 and Sec13-Sec31 appear to constitute the inner and the outermost layers of the COPII coat, respectively. We previously isolated two mammalian proteins (p125 and p250) that bind to Sec23. p125 was found to be a mammalian-specific, phospholipase A 1 -like protein that participates in the organization of ER exit sites. Here we show that p250 is encoded by the KIAA0310 clone and has sequence similarity to yeast Sec16 protein. Although KIAA0310p was found to be localized at ER exit sites, subcellular fractionation revealed its predominant presence in the cytosol. Cytosolic KIAA0310p was recruited to ER membranes in a manner dependent on Sar1. Depletion of KIAA0310p mildly caused disorganization of ER exit sites and delayed protein transport from the ER, suggesting its implication in membrane traffic out of the ER. Overexpression of KIAA0310p affected ER exit sites in a manner different from that of p125. Binding experiments suggested that KIAA0310p interacts with both the inner and the outermost layer coat complexes, whereas p125 binds principally to the inner layer complex. Our results suggest that KIAA0310p, a mammalian homologue of yeast Sec16, builds up ER exit sites in cooperation with p125 and plays a role in membrane traffic from the ER.

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...