Dhanvantari S, Loh Y P
Section on Cellular Neurobiology, Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 2000 Sep 22;275(38):29887-93. doi: 10.1074/jbc.M005364200.
Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.
膜羧肽酶E（CPE）是一种分选受体，可将促激素（如阿黑皮素原）靶向内分泌细胞中的调节性分泌途径。其膜结合对于它在反式高尔基体网络（TGN）结合促激素分选信号以促进靶向是必要的。在本研究中，我们检测了CPE在源自TGN的牛垂体分泌颗粒膜中的脂质相互作用。我们发现CPE与分泌颗粒膜中的抗去污剂脂质结构域（即脂筏）相关联。脂质分析表明，这些脂筏富含糖鞘脂和胆固醇。AtT-20细胞中的脉冲追踪和亚细胞分级实验表明，CPE与膜脂筏的关联仅在其到达高尔基体后才发生。胆固醇耗竭导致CPE从分泌颗粒膜上解离，并减少了促激素与膜的结合。使用洛伐他汀进行体内胆固醇耗竭导致CPE及其货物缺乏向调节性分泌途径的分选。我们提出，CPE的分选受体功能需要其在TGN与糖鞘脂 - 胆固醇脂筏相互作用，从而将其锚定在能够结合其促激素货物的位置。
