Sterol Carrier Protein-2 Functions in Phosphatidylinositol Transfer and Signaling†

Over 20 years ago, it was reported that liver cytosol contains at least two distinct proteins that transfer phosphatidylinositol in vitro, phosphatidylinositol transfer protein (PITP) and a pH 5.1 supernatant fraction containing sterol carrier protein-2 (SCP-2). In contrast to PITP, there has been minimal progress on the structural and functional significance of SCP-2 in phosphatidylinositol transport. As shown herein, highly purified, recombinant SCP-2 stimulated up to 13-fold the rapid (s) transfer of radiolabeled phosphatidylinositol (PI) from microsomal donor membranes to highly curved acceptor membranes. SCP-2 bound to microsomes in vitro and overexpression of SCP-2 in transfected L-cells resulted in the following: (i) redistribution of phosphatidylinositols from intracellular membranes (mitochondria and microsomes) to the plasma membrane; (ii) enhancement of insulin-mediated inositol-triphosphate production; and (iii) 5.5-fold down regulation of PITP. Like PITP, SCP-2 binds two ligands required for vesicle budding from the Golgi, PI, and fatty acyl CoA. Double immunolabeling confocal microscopy showed SCP-2 significantly colocalized with caveolin-1 in the cytoplasm (punctate) and plasma membrane of SCP-2 overexpressing hepatoma cells (72%), HT-29 cells (58%), and SCP-2 overexpressing L-cells (37%). Taken together, these data show for the first time that SCP-2 plays a hitherto unrecognized role in intracellular phosphatidylinositol transfer, distribution, and signaling. 1 Abbreviations: Fmoc, fluorenylmethoxy-carbonyl; IP3, inositol- triphosphate; L-FABP, liver fatty acid binding protein; LSCM, laser- scanning confocal microscopy; SUV, small unilamellar vesicles; LUV, large unilamellar vesicles; NBD-stearic acid, (7-nitrobenz-2-oxa-1,3- diazol)-octadecanoic acid; PI, phosphatidylinositol; PIP 2, phosphati- dylinositol diphosphate; PITP, phosphatidylinositol transfer protein; POPC, palmitoyloleoyl phosphatidylcholine; SCP-2, sterol carrier protein-2.