Transport vesicles coated with the COPII complex, which is assembled from Sar1p, Sec23p-Sec24p, and Sec13p-Sec31p, are involved in protein export from the endoplasmic reticulum (ER). We previously identified and characterized a novel Sec23p-interacting protein, p125, that is only expressed in mammals and exhibits sequence homology with phosphatidic acid-preferring phospholipase A 1 (PA-PLA 1 ). In this study, we examined the localization and function of p125 in detail. By using immunofluorescence and electron microscopy, we found that p125 is principally localized in ER exit sites where COPII-coated vesicles are produced. Analyses of chimeric proteins comprising p125 and two other members of the mammalian PA-PLA 1 family (PA-PLA 1 and KIAA0725p) showed that, for localization to ER exit sites, the p125-specific N-terminal region is critical, and the putative lipase domain is interchangeable with KIAA0725p but not with PA-PLA 1 . RNA interferencemediated depletion of p125 affected the organization of ER exit sites. The structure of the cis-Golgi compartment was also substantially disturbed, whereas the medial-Golgi was not. Protein export from the ER occurred without a significant delay in p125-depleted cells. Our study suggests that p125 is a mammalian-specific component of ER exit sites and participates in the organization of this compartment.

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.