Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.
Phospholipase D stimulates release of nascent secretory vesicles from the trans-Golgi network.
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DOI：
10.1083/jcb.138.3.495
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发表时间：
1997-08-11
期刊：
影响因子：
--
通讯作者：
Shields D
中科院分区：
文献类型：
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作者：
Chen YG;Siddhanta A;Austin CD;Hammond SM;Sung TC;Frohman MA;Morris AJ;Shields D
磷脂酶D（PLD）是一种磷脂水解酶，其活化与哺乳动物细胞中的信号转导途径、细胞生长和膜运输有关。一些实验室已经证明，包括ADP-核糖基化因子（ARF）在内的小GTP结合蛋白可以在体外刺激PLD活性，并且已经在高尔基体膜中发现了ARF激活的PLD活性。由于ARF-1也被证明可以增强内分泌细胞TGN中新生分泌囊泡的释放，因此我们假设该反应是通过PLD激活而发生的。使用来自生长激素和催乳素分泌垂体GH 3细胞的透化细胞系统，我们证明了免疫亲和纯化的人PLD 1刺激新生的分泌囊泡从TGN出芽约两倍。相比之下，类似的纯化，但酶失活的突变形式的PLD 1，指定的Lys 898 Arg，没有影响囊泡出芽时，加入到透化的细胞。从TGN的新生分泌囊泡的释放是敏感的1%的1-丁醇，浓度抑制PLD催化的磷脂酸的形成。此外，ARF-1刺激内源性PLD活性在高尔基体膜约三倍，这种激活与其增强囊泡出芽。我们的研究结果表明，ARF的PLD活性的调节起着重要的作用，在新生的分泌囊泡从TGN的释放。
Phospholipase D (PLD) is a phospholipid hydrolyzing enzyme whose activation has been implicated in mediating signal transduction pathways, cell growth, and membrane trafficking in mammalian cells. Several laboratories have demonstrated that small GTP-binding proteins including ADP-ribosylation factor (ARF) can stimulate PLD activity in vitro and an ARF-activated PLD activity has been found in Golgi membranes. Since ARF-1 has also been shown to enhance release of nascent secretory vesicles from the TGN of endocrine cells, we hypothesized that this reaction occurred via PLD activation. Using a permeabilized cell system derived from growth hormone and prolactin-secreting pituitary GH3 cells, we demonstrate that immunoaffinity-purified human PLD1 stimulated nascent secretory vesicle budding from the TGN approximately twofold. In contrast, a similarly purified but enzymatically inactive mutant form of PLD1, designated Lys898Arg, had no effect on vesicle budding when added to the permeabilized cells. The release of nascent secretory vesicles from the TGN was sensitive to 1% 1-butanol, a concentration that inhibited PLD-catalyzed formation of phosphatidic acid. Furthermore, ARF-1 stimulated endogenous PLD activity in Golgi membranes approximately threefold and this activation correlated with its enhancement of vesicle budding. Our results suggest that ARF regulation of PLD activity plays an important role in the release of nascent secretory vesicles from the TGN.