ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion.
ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [(35)S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in β-lipoprotein secretion by human hepatocytes.
Institute of Biomedicine, Anatomy, PO Box 63, FI-00014 University of Helsinki, Finland.
Eija
E
Nissilä
Yuki
Y
Ohsaki
Marion
M
Weber-Boyvat
Julia
J
Perttilä
Elina
E
Ikonen
Vesa M
VM
Olkkonen
D053299:Apolipoprotein B-100 / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism*; D006056:Golgi Apparatus / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D056945:Hep G2 Cells; D022781:Hepatocytes / Q000378:metabolism / Q000473:pathology; D006801:Humans; D047468:Immunoprecipitation; D018613:Microscopy, Confocal; D008870:Microtubules / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000378:metabolism; D011485:Protein Binding; D034622:RNA Interference; D011987:Receptors, Steroid / Q000235:genetics / Q000378:metabolism*; D000098825:Oxysterol Binding Proteins
D053299:Apolipoprotein B-100; D010716:Phosphatidylinositols; D011987:Receptors, Steroid; D000098825:Oxysterol Binding Proteins; D049452:Green Fluorescent Proteins; D002784:Cholesterol