Abstract
Transport of LDL-derived cholesterol from lysosomes into the cytoplasm requires NPC1 protein; NPC1L1 mediates uptake of dietary cholesterol. We introduced single disulfide bonds into NPC1 and NPC1L1 to explore the importance of inter-domain dynamics in cholesterol transport. Using a sensitive method to monitor lysosomal cholesterol efflux, we found that NPC1's N-terminal domain need not release from the rest of the protein for efficient cholesterol export. Either introducing single disulfide bonds to constrain lumenal/extracellular domains or shortening a cytoplasmic loop abolishes transport activity by both NPC1 and NPC1L1. The widely prescribed cholesterol uptake inhibitor, ezetimibe, blocks NPC1L1; we show that residues that lie at the interface between NPC1L1's three extracellular domains comprise the drug's binding site. These data support a model in which cholesterol passes through the cores of NPC1/NPC1L1 proteins; concerted movement of various domains is needed for transfer and ezetimibe blocks transport by binding to multiple domains simultaneously.
Highlights
NPC1 and NPC1L1 are structurally related, multi-spanning membrane proteins that are important for cholesterol transport in humans
We have shown here that dynamic, inter-domain interactions within NPC1 and NPC1L1 are essential for the ability of these proteins to mediate cholesterol transport out of lysosomes or across the plasma membrane, respectively
Mutants that can lock the MLD to the CTD at the interface of these domains interfere with cholesterol transport; residues that comprise a loop at the cytoplasmic face of the transmembrane domains are needed to provide inter-domain mobility and/or an unconstrained conformation and can be replaced with polyalanine for full function
Summary
NPC1 and NPC1L1 are structurally related, multi-spanning membrane proteins that are important for cholesterol transport in humans. The related NPC1 protein functions in lysosomes to transport LDLderived cholesterol to the cytoplasm (Pfeffer, 2019). Plasma LDL is delivered to the lysosome by endocytosis, and its cholesterol esters are cleaved by acid lipase to release free cholesterol for cellular use (Brown and Goldstein, 1986; Goldstein et al, 1975). NPC2 protein binds this released cholesterol via its iso-octyl group (Xu et al, 2007) and transfers it to the N-terminal domain of NPC1 (Kwon et al, 2009). Mutations in either NPC2 or NPC1 protein can give rise to a severe neurodegenerative disorder called Niemann Pick Type C disease, which leads to massive accumulation of cholesterol in lysosomes of all tissues and premature death (Pentchev et al, 2004)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
