Sterol liganding of OSBP-related proteins (ORPs) regulates the subcellular distribution of ORP–VAPA complexes and their impacts on organelle structure

Abstract

Oxysterol-binding protein (OSBP) and its homologues (ORPs) are lipid-binding/transfer proteins with affinity for oxysterols, cholesterol and glycerophospholipids. In addition to a ligand-binding domain, a majority of the ORPs carry a pleckstrin homology domain that targets organelle membranes via phosphoinositides, and a motif targeting the endoplasmic reticulum (ER) via VAMP-associated proteins (VAPs).We employed here Bimolecular Fluorescence Complementation (BiFC) to systematically assess the effects of sterol manipulation of HuH7 cells on complexes of established sterol-binding ORPs with their ER receptor, VAMP-associated protein A (VAPA).Depletion of cellular cholesterol with lipoprotein-deficient medium and Mevastatin caused concentration of OSBP–VAPA complexes and Golgi complex markers at a juxtanuclear position, an effect reversed by low-density lipoprotein treatment. A similar redistribution of OSBP–VAPA but not of sterol-binding deficient mutant OSBP(ΔELSK)–VAPA, occurred upon treatment with the high-affinity ligand, 25-hydroxycholesterol (25OHC), which reduced total and free cholesterol. ORP2–VAPA complexes, which localize in untreated cells at blob-like ER structures with associated lipid droplets, were redistributed upon treatment with the ORP2 ligand 22(R)OHC to a diffuse cytoplasmic/ER pattern and the plasma membrane. Analogously, distribution of ORP4L–VAPA complexes between the plasma membrane and vimentin intermediate filament associated compartments was modified by statin or 25OHC treatment. The treatments resulted in loss of vimentin co-localization, and sterol-binding deficient ORP4L(ΔELSR)–VAPA localized predominantly to the plasma membrane.In conclusion, treatment with statin or oxysterol ligands modify the subcellular targeting of ORP–VAPA complexes, consistent with the notion that this machinery controls lipid homeostasis and signaling at organelle interfaces.