Reconstitution of ORP-mediated lipid exchange process coupled to PI(4)P metabolism.
Reconstitution of ORP-mediated lipid exchange process coupled to PI(4)P metabolism.
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ORP 介导的脂质交换过程与 PI(4)P 代谢耦合的重建。
DOI：
10.1101/2023.08.04.551917
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发表时间：
2023
期刊：
影响因子：
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通讯作者：
Drin,Guillaume
中科院分区：
文献类型：
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作者：
Fuggetta,Nicolas;Rigolli,Nicola;Magdeleine,Maud;Seminara,Agnese;Drin,Guillaume
脂质在真核细胞中的分布依赖于脂质转移和脂质代谢之间的紧密耦合。然而，这些耦合仍然没有得到很好的描述。值得注意的是，目前尚不清楚与PI（4）P代谢偶联的OSBP相关蛋白（ORP）家族的脂质交换剂在多大程度上有助于内质网（ER）和其他细胞区域之间的固醇和磷脂酰丝氨酸梯度的形成。为了解决这个问题，我们已经在体外检测了Osh 4p（一种代表性的ORP）在高尔基体模拟膜（其中PI（4）P由PI 4-激酶产生）和ER模拟膜（其中PI（4）P由磷酸酶Sac 1 p水解）之间的活性。使用定量，实时测定，我们证明，Osh 4p创建一个甾醇/PI（4）P交换之间的两个膜的甾醇梯度，一旦在这个界面上产生的PI（4）P梯度后，ATP添加，并定义多少PI（4）P必须合成这个过程。然后，使用由我们的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，我们表明，Sec 14 p，通过转移膜之间的磷脂酰肌醇，可以支持PI（4）P的合成和创造的甾醇梯度的Osh 4p。这些结果表明，在PI（4）P代谢的控制下，ORP在细胞内分配脂质的程度。
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.