Novel members of the human oxysterol-binding protein family bind phospholipids and regulate vesicle transport

Oxysterol-binding proteins (OSBPs) are a family of eukaryotic intracellular lipid receptors. Mammalian OSBP1 binds oxygenated derivatives of cholester ol and mediates sterol and phospholipid synthesis through as yet poorly und efined mechanisms. The precise cellular roles for the remaining members of the oxysterol-binding protein family remain to be elucidated. In yeast, a f amily of OSBPs has been identified based on primary sequence similarity to the ligand binding domain of mammalian OSBP1, Yeast Kes1p, an oxysterol-bin ding protein family member that consists of only the ligand binding domain, has been demonstrated to regulate the Sec14p pathway for Golgi-derived ves icle transport. Specifically, inactivation of the KES1 gene resulted in the ability of yeast to survive in the absence of Sec14p, a phosphatidylinosit ol/phosphatidylcholine transfer protein that is normally required for cell viability due to its essential requirement in transporting vesicles from th e Golgi, We cloned the two human members of the OSBP family, ORP1 and ORP2, with the highest degree of similarity to yeast Kes1p, We expressed ORP1 an d ORP2 in yeast lacking Sec14p and Kes1p function and found that ORP1 compl emented Kes1p function with respect to cell growth and Golgi vesicle transp ort, whereas ORP2 was unable to do so. Phenotypes associated with overexpre ssion of ORP2 in yeast were a dramatic decrease in cell growth and a block in Golgi-derived vesicle transport distinct from that of ORP1. Purification of ORP1 and ORP2 for ligand binding studies demonstrated ORP1 and ORP2 did not bind 25-hydroxycholesterol but instead bound phospholipids with both p roteins exhibiting strong binding to phosphatidic acid and weak binding to phosphatidylinositol 3-phosphate. In Chinese hamster ovary cells, ORP1 loca lized to a cytosolic location, whereas ORP2 was associated with the Golgi a pparatus, consistent with our vesicle transport studies that indicated ORP1 and ORP2 function at different steps in the regulation of vesicle transpor t.