Lipid raft association of carboxypeptidase E is necessary for its functionas a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting proho rmones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a pr ohormone sorting signal at the trans-Golgi network (TGN) to facilitate targ eting. In this study, we examined the lipid interaction of CPE in bovine pi tuitary secretory granule membranes, which are derived from the TGN. We sho w that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these raft s are enriched in glycosphingolipids and cholesterol. Pulse-chase and subce llular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Choles terol depletion resulted in dissociation of CPE from secretory granule memb ranes and decreased the binding of prohormones to membranes, lit vivo chole sterol depletion using lovastatin resulted in the lack of sorting of CPE an d its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolip id-cholesterol rafts at the TGN, thereby anchoring it in position to bind t o its prohormone cargo.