PDF(Pubmed)
Abstract：
Lipid distribution in the eukaryotic cells depends on tight couplings between lipid transfer and lipid metabolism. Yet these couplings remain poorly described. Notably, it is unclear to what extent lipid exchangers of the OSBP-related proteins (ORPs) family, coupled to PI(4)P metabolism, contribute to the formation of sterol and phosphatidylserine gradient between the endoplasmic reticulum (ER) and other cell regions. To address this question, we have examined in vitro the activity of Osh4p, a representative ORP, between Golgi mimetic membranes in which PI(4)P is produced by a PI 4-kinase and ER mimetic membranes in which PI(4)P is hydrolyzed by the phosphatase Sac1p. Using quantitative, real-time assays, we demonstrate that Osh4p creates a sterol gradient between the two membranes by sterol/PI(4)P exchange as soon as a PI(4)P gradient is generated at this interface following ATP addition, and define how much PI(4)P must be synthesized for this process. Then, using a kinetic model supported by our in vitro data, we estimate to what extent PI(4)P metabolism can drive lipid transfer in cells. Finally, we show that Sec14p, by transferring phosphatidylinositol between membranes, can support the synthesis of PI(4)P and the creation of a sterol gradient by Osh4p. These results indicate to what extent ORPs, under the control of PI(4)P metabolism, can distribute lipids in the cell.
摘要：
真核细胞中的脂质分布取决于脂质转移和脂质代谢之间的紧密耦合。然而，这些联轴器的描述仍然很差。值得注意的是,目前尚不清楚OSBP相关蛋白（ORP）家族的脂质交换剂在多大程度上，与PI(4)P代谢相结合，有助于在内质网（ER）和其他细胞区域之间形成甾醇和磷脂酰丝氨酸梯度。为了解决这个问题，我们在体外检测了Osh4p的活性，代表ORP，在由PI4激酶产生PI（4）P的高尔基体模拟膜和由磷酸酶Sac1p水解PI（4）P的ER模拟膜之间。使用定量，实时分析，我们证明了Osh4p通过甾醇/PI（4）P交换在两个膜之间产生甾醇梯度，一旦PI（4）P梯度在ATP添加后在该界面处产生，并定义该过程必须合成多少PI(4)P。然后，使用我们体外数据支持的动力学模型，我们估计PI（4）P代谢在多大程度上可以驱动细胞中的脂质转移。最后,我们展示了Sec14p，通过在膜之间转移磷脂酰肌醇，可以支持PI（4）P的合成和Osh4p生成甾醇梯度。这些结果表明ORP在多大程度上，在PI（4）P代谢的控制下，可以在细胞中分布脂质。
