Characterization of a novel phospholipase A_1 family

由Sar1p、Sec23p-Sec24p和sec3p - sec31p组装而成的COPII复合物包裹的转运囊泡参与了内质网（ER）的蛋白质输出。我们之前发现了一种新的sec23p相互作用蛋白p125，该蛋白仅在哺乳动物中表达，并与磷脂酸偏好磷脂酶A_1 （PA-PLA_1）序列同源。PA-PLA_1、p125和KIAA0725p似乎构成了细胞内磷脂酶A_1家族。在这项研究中，我们分析了PA-PLA_1和由p125和其他两个成员组成的嵌合蛋白的定位。p125的功能也已经使用RNA干扰方法进行了详细的研究。我们之前的研究表明，p125主要定位于产生copii包被囊泡的内质网出口位点，而KIAA0725p定位于高尔基体。免疫荧光显微镜分析PA-PLA_1的定位。结果表明，PA-PLA_1完全定位于细胞质中，与p125或KIAA0725p不同。由p125和KIAA0725p的n端区域组成的嵌合蛋白定位在内质网出口位点，而由p125和PA-PLA_1的n端区域组成的嵌合蛋白则定位在细胞质中，这表明对于内质网出口位点的定位，p125特异性的n端区域至关重要，并且假定的脂肪酶结构域与KIAA0725p可互换，而与PA-PLA_1不可互换。为了深入了解p125的功能，p125 mRNA被RNAi靶向降解。在p125缺失的细胞中，内质网出口位点的组织受到影响。顺-高尔基室的结构也受到了很大的干扰，而中-高尔基室则没有。在p125缺失的细胞中，VSV-G蛋白从内质网输出无明显延迟。这些结果表明p125是哺乳动物内质网出口位点的特异性成分，并参与了该区室的组织。

Transport vesicles coated with the COPII complex, which is assembled from Sar1p, Sec23p-Sec24p, and Secl3p-Sec31p, are involved in protein export from the endoplasmic reticulum(ER). We previously identified a novel Sec23p-interacting protein, p125, which is only expressed in mammals and exhibits sequence homology with phosphatidic acid-preferring phospholipase A_1 (PA-PLA_1). PA-PLA_1,p125, and KIAA0725p appear to constitute an intracellular phospholipase A_1 family. In this study, we have analyzed the localization of PA-PLA_1 and chimeric proteins comprising p125 and two other members. The function of p125 has also been examined in detail using an RNA interference approach.Our previous study showed that p125 is principally localized in ER exit sites where COPII-coated vesicles are produced, and that KIAA0725p is localized in the Golgi. Localization of PA-PLA_1 was analyzed by immunofluorescence microscopy. The results showed that PA-PLA_1 is exclusively localized in the cytosol, differing from that of p125 or KIAA0725p. A chimeric protein composed of the N-terminal region of p125 and KIAA0725p was localized in ER exit sites, but one composed of the N-terminal region of p125 and PA-PLA_1 was in cytosol, indicating that for localization to ER exit sites, the p125-specific N-terminal region is critical, and that the putative lipase domain is interchangeable with KIAA0725p but not with PA-PLA_1. To gain an insight into the function of p125, p125 mRNA was targeted for degradation by RNAi. In p125-depleted cells, the organization of ER exit sites was affected. The structure of the cis-Golgi compartment was also substantially disturbed, whereas the medial-Golgi was not. VSV-G Protein export from the ER occurred without a significant delay in p125-depleted cells. These results suggest that p125 is a mammalian-specific component of ER exit sites and participates in the organization of this compartment.