title: Characterization of the sterol and phosphatidylinositol 4-phosphate binding properties of Golgi-associated OSBP-related protein 9 (ORP9).
abstract: Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [32P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.
authors.affiliation: Departments of Pediatrics, Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Xinwei
authors.initials: X
authors.lastname: Liu
authors.affiliation: Departments of Pediatrics, Biochemistry and Molecular Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
authors.forename: Neale D
authors.initials: ND
authors.lastname: Ridgway
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D016466:CHO Cells; D002460:Cell Line; D002782:Cholestenes / Q000378:metabolism; D003412:Cricetulus; D015870:Gene Expression; D006056:Golgi Apparatus / Q000378:metabolism*; D006801:Humans; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D011485:Protein Binding; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D013261:Sterols / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
chemical_list: D002782:Cholestenes; D018129:Phosphatidylinositol Phosphates; D011987:Receptors, Steroid; D013261:Sterols; D000098825:Oxysterol Binding Proteins; C037178:phosphatidylinositol 4-phosphate; C025498:cholesta-5,7,9-trien-3 beta-ol