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）到高尔基体的转运。COPII由三个部分组成：一个小GTP结合蛋白Sarlp和两个蛋白质复合物Sec 23 p-Sec 24 p和Sec 13 p-Sec 31 p。我们以前已经鉴定了一个与Sec 23 p相互作用的蛋白质p125，它与磷脂酸偏好型磷脂酶A_1（PA-PLA_1）和p125的同源物KIAA 0725 p具有同源性。PA-PLA_1、p125和KIAA 0725 p可能构成一个新的磷脂酶家族。本研究对p125和KIAA 0725 p进行了详细的研究。KIAA 0725 p的过表达，如p125，造成分散的高尔基体。与p125的情况不同，KIAA 0725 p的过表达导致了介导转运囊泡与靶膜的束缚的束缚蛋白的分散，并引起了ER的聚集。使用磷脂脂质体底物测量KIAA 0725 p的酶活性。我们发现KIAA 0725 p具有磷脂酶A_1的活性，如果该活性需要KIAA 0725 p的话，那么KIAA 0725 p优先对磷脂酸和Ser-351具有活性。在相同条件下，未检测到p125活性。这些结果表明KIAA 0725 p的细胞功能与p125不同。我们发现，p125，以及COPII，是本地化的ER出口网站，其本地化是通过GTP/GDP循环的Sarlp调节。P125与COPII结合到带有含有FF基序的肽的珠子上，已知FF基序是从ER输出的信号。顺式高尔基体区室的耗竭。我们的研究结果表明，p125通过与COPII的关联参与早期分泌途径

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