Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p
Multiple pools of phosphatidylinositol 4-phosphate detected using the pleckstrin homology domain of Osh2p
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DOI：
10.1074/jbc.m401583200
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发表时间：
2004-10-22
影响因子：
4.8
通讯作者：
Levine, TP
中科院分区：
文献类型：
--
作者：
Roy, A;Levine, TP
磷脂酰肌醇（PtdIns）磷酸（PtdInsP）脂质用作外周膜蛋白募集和激活的细胞内标志。尽管大多数PtdInsPs的分布局限于单个细胞器，但PtdIns（4）P的独特之处在于它存在于几个离散的池中，因此结合PtdIns（4）P的蛋白质必须使用额外的受体才能实现有限的定位。在这里，我们比较了来自Osh 1 p和Osh 2 p的两个高度相关的普列克底物蛋白同源（PH）结构域，Osh 1 p和Osh 2 p是氧固醇结合蛋白（OSBP）的酵母同系物，它们使用PtdIns（4）P靶向膜，并在体外结合PtdIns（4）P和PtdIns（4，5）P（2）。我们表明，高尔基体靶向指定的PH（Osh 1），这是一个额外的网站上的一个面对的结构域之前不知道与受体相互作用。相比之下，PH（Osh 2）没有明显的第二个位点，并且靶向多个PtdInsPs池，每个池依赖于不同的PtdIns 4-激酶。PH（Osh 2）中第二个位点的缺乏使其可以用作4-磷酸化PtdIn分布改变的无偏倚报告基因。例如，在磷酸酶Sac 1 p失活导致PtdIns（4）P过量的细胞中，PH（Osh 2）表明PtdIns（4）P在质膜上积累，而其他高尔基体靶向的PH结构域无法检测到这种变化。
Phosphatidylinositol (PtdIns) phosphate (PtdInsP) lipids are used as intracellular signposts for the recruitment and activation of peripheral membrane proteins. Whereas the distribution of most PtdInsPs is restricted to a single organelle, PtdIns(4)P is unique in that it exists in several discrete pools, and so proteins that bind PtdIns(4) P must use extra receptors to achieve a restricted localization. Here we compare the two highly related pleckstrin homology (PH) domains from Osh1p and Osh2p, yeast homologues of oxysterol-binding protein (OSBP), that target membranes using PtdIns(4) P, and in vitro bind both PtdIns(4) P and PtdIns(4,5) P(2). We show that Golgi targeting is specified by an additional site on PH(Osh1), which lies on a face of the domain not previously known to interact with receptors. In contrast, PH(Osh2) does not have a demonstrable second site, and targets multiple pools of PtdInsPs, each dependent on a different PtdIns 4-kinase. This lack of a second site in PH(Osh2) allows it to be used as an unbiased reporter for altered distribution of 4-phosphorylated PtdIns. For example, in cells with excess PtdIns(4) P caused by inactivation of the phosphatase Sac1p, PH(Osh2) indicates that PtdIns(4) P accumulates on the plasma membrane, whereas other Golgi-targeted PH domains fail to detect this change.