Reconstitution of ORP-mediated lipid exchange process coupled to PI(4)P metabolism

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.