Membrane topology of human NPC1L1, a key protein in enterohepatic cholesterol absorption

Jiang Wang, Bei-Bei Chu, Liang Ge, Bo-Liang Li, Yan Yan, and Bao-Liang Song

Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, CAS, Shanghai, Shanghai 200031

Corresponding Author: blsong{at}sibs.ac.cn

The Niemann-Pick C1-like 1 (NPC1L1) is a predicted polytopic membrane protein that is critical for cholesterol absorption. NPC1L1 takes up free cholesterol into cells through vesicular endocytosis. Ezetimibe, a clinically used cholesterol absorption inhibitor, blocks the endocytosis of NPC1L1 thereby inhibiting cholesterol uptake. Human NPC1L1 is a 1332-amino acid protein with a putative sterol-sensing domain (SSD) that shows sequence homology to HMG-CoA reductase (HMGCR), Niemann-Pick C1 (NPC1) and SREBP cleavage-activating protein (SCAP). Here, we use protease protection and immunofluorescence in selectively permeabilized cells to study the topology of NPC1L1. Our data indicate that NPC1L1 contains 13 transmembrane helices. The NH2-terminus of NPC1L1 is in the lumen while the COOH-terminus projects to the cytosol. NPC1L1 contains 7 small cytoplasmic loops, 4 small and 3 large luminal loops, one of which has been reported to bind ezetimibe. Ezetimibe-glucuronide, the major metabolite of ezetimibe in vivo, can block the internalization of NPC1L1 and cholesterol. The membrane topology of NPC1L1 is similar to that of NPC1, and the putative SSD of NPC1L1 is oriented in the same manner as those of HMGCR, SCAP and NPC1. The defined topology of NPC1L1 provides necessary information for further dissecting the functions of different NPC1L1 domains.

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