In Vitro Strategy to Measure Sterol/Phosphatidylinositol-4-Phosphate Exchange Between Membranes.

Abstract

Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for phosphatidylinositol-4-phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificial membranes using Förster resonance energy transfer (FRET)-based assays and a standard spectrofluorometer. Such an approach can serve to better characterize the activity of known sterol/PI4P exchangers, but also toreveal whether ill-defined ORP/Osh proteins or LTPs belonging to other families have such an exchange activity. Besides, this protocol is amenable to test whether molecules can act as Orphilins, which have been found to inhibit the sterol/PI4P exchange activity of certain ORPs. Last, our strategy to measure in real-time PI4P transport using a known lipid-binding domain can serve as a basis for the design of novel in vitro protocols aiming to detect other lipid species.