Characterization of a novel phospholipase associated with a coat protein of COPII vesicles

copii包被的囊泡参与蛋白质从内质网（ER）到高尔基体的运输。COPII由三部分组成；一个小的GTP结合蛋白，Sarlp和两个蛋白复合物Sec23p-Sec24p和Sec13p-Sec31p。我们之前已经发现了一个与sec23p相互作用的蛋白p125，它与磷脂酸偏好磷脂酶A_1 （PA-PLA_1）和p125的同源物KIAA0725p具有同源性。PA-PLA_1， p125和KIAA0725p似乎构成了一个新的磷脂酶家族。在本研究中，我们详细表征了p125和KIAA0725P。为了评估KIAA0725P的功能，我们在培养的哺乳动物细胞中过表达KIAA0725P。KIAA0725p的过表达与p125一样，引起高尔基体的分散。与p125不同的是，KIAA0725p的过表达导致系泊蛋白的分散，这些蛋白介导转运囊泡向靶膜的系泊，并引起内质网聚集。采用磷脂脂质体底物测定KIAA0725p的酶活性。我们发现KIAA0725p优先对磷脂酸和Ser-351具有磷脂酶A_1活性，如果需要KIAA0725p的活性。在相同条件下，未检测到p125的活性。这些结果表明KIAA0725p的细胞功能与p125不同，为了确定p125的确切定位，制备了针对p125的单克隆抗体。我们发现p125以及COPII定位于内质网出口位点，其定位通过Sarlp的GTP/GDP循环进行调节。P125结合COPII结合到含有FF基序肽的小珠上，这是已知的从内质网输出的信号。顺式高尔基隔室耗竭。我们的研究结果表明p125通过与COPII的关联参与了早期分泌途径

COPII-coated vesicles are involved in protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. COPII consists of three parts; a small GTP binding protein, Sarlp and the two protein complexes, Sec23p-Sec24p and Sec13p-Sec31p. We have previously identified a Sec23p-interacting protein, p125, which exhibits homology with phosphatidic acid-preferring phospholipase A_1 (PA-PLA_1), and p125's cognate KIAA0725p. PA-PLA_1, p125 and KIAA0725p appear to constitutea novel family of phospholipases. In this study, we charocterized p125 and KIAA0725P in detailTo assess the function of KIAA0725p, we overexpressed it in cultured mammalian cells. Overexpression of KIAA0725p, like that of p125, caused dispersion of the Golgi apparatus. Different from the case of p125, overexpression of KIAA0725p resulted in dispersion of tethering proteins, which mediate the tethering of transport vesicles to the target membranes, and caused aggregation of the ER. The enzyme activity of KIAA0725p was measured using phospholipid liposome substrates. We found that KIAA0725p possesses phospholipase A_1 activity preferentially for phosphatidic acid and Ser-351 if KIAA0725p is required for the activity. Under the same condition, the activity of p125 was not detected. These results suggest that the cellular function of KIAA0725p is different from that of p125To determine the exact localization of p125, monoclonal antibodies against p125 were prepared. We found that p125, as well as the COPII, is localized to ER exit sites and its localization is regulated through the GTP/GDP cycle of Sarlp. P125 in conjunction with the COPII bound to beads bearing a peptide containing a FF motif, which is known to be a signal for export from the ER. Depletion of the cis-Golgi compartment. Our results suggest that p125 is involved in the early secretory pathway through the association with COPII