PDF(Pubmed)
Abstract：
Oxysterol-binding protein-related proteins (ORPs) play key roles in the distribution of lipids in eukaryotic cells by exchanging sterol or phosphatidylserine for PI4P between the endoplasmic reticulum (ER) and other cell regions. However, it is unclear how their exchange capacity is coupled to PI4P metabolism. To address this question quantitatively, we analyze the activity of a representative ORP, Osh4p, in an ER/Golgi interface reconstituted with ER- and Golgi-mimetic membranes functionalized with PI4P phosphatase Sac1p and phosphatidylinositol (PI) 4-kinase, respectively. Using real-time assays, we demonstrate that upon adenosine triphosphate (ATP) addition, Osh4p creates a sterol gradient between these membranes, relying on the spatially distant synthesis and hydrolysis of PI4P, and quantify how much PI4P is needed for this process. Then, we develop a quantitatively accurate kinetic model, validated by our data, and extrapolate this to estimate to what extent PI4P metabolism can drive ORP-mediated sterol transfer in cells. Finally, we show that Sec14p can support PI4P metabolism and Osh4p activity by transferring PI between membranes. This study establishes that PI4P synthesis drives ORP-mediated lipid exchange and that ATP energy is needed to generate intermembrane lipid gradients. Furthermore, it defines to what extent ORPs can distribute lipids in the cell and reassesses the role of PI-transfer proteins in PI4P metabolism.
摘要：
氧化固醇结合蛋白相关蛋白（ORP）通过在内质网（ER）和其他细胞区域之间将甾醇或磷脂酰丝氨酸交换为PI4P，在真核细胞中的脂质分布中起关键作用。然而,目前尚不清楚它们的交换能力如何与PI4P代谢相关.为了定量地解决这个问题，我们分析代表性ORP的活性，Osh4p,在用PI4P磷酸酶Sac1p和磷脂酰肌醇（PI）4-激酶功能化的ER和高尔基体模拟膜重建的ER/高尔基体界面中，分别。使用实时检测，我们证明，在三磷酸腺苷（ATP）添加后，Osh4p在这些膜之间产生甾醇梯度，依靠PI4P的空间距离的合成和水解，并量化此过程需要多少PI4P。然后，我们开发了一个定量精确的动力学模型，通过我们的数据验证，并对此进行推断以估计PI4P代谢在多大程度上可以驱动ORP介导的固醇在细胞中的转移。最后,我们表明Sec14p可以通过在膜之间转移PI来支持PI4P代谢和Osh4p活性。这项研究确定PI4P合成驱动ORP介导的脂质交换，并且需要ATP能量来产生膜间脂质梯度。此外，它定义了ORP可以在细胞中分布脂质的程度，并重新评估了PI转移蛋白在PI4P代谢中的作用。
