Abstract
Niemann–Pick C1 (NPC1) is a membrane protein required for the transport of low-density lipoprotein (LDL)-derived cholesterol from endosomes and lysosomes to the other organelles. Here, we describe the recombinant protein expression, purification, and characterization of the human NPC1. The protein is transiently expressed in human embryonic kidney (HEK) cells. Our purification protocol describes the steps to obtain a pure and homogeneous NPC1 protein. Niemann–Pick C2 (NPC2) is a small soluble protein, which mediates cholesterol transport in tandem with NPC1. Finally, we also describe two biochemical approaches to characterize NPC1 function in vitro—a cholesterol transfer assay from purified NPC2 to NPC1 and a binding assay between NPC1 and NPC2.
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References
Vanier MT (2015) Complex lipid trafficking in Niemann-Pick disease type C. J Inherit Metab Dis 38:187–199
Carstea ED, Morris JA, Coleman KG et al (1997) Niemann-Pick C1 disease gene: homology to mediators of cholesterol homeostasis. Science 277:228–231
Davies JP, Chen FW, Ioannou YA (2000) Transmembrane molecular pump activity of Niemann-Pick C1 protein. Science 290:2295–2298
Sleat DE, Wiseman JA, El-Banna M et al (2004) Genetic evidence for nonredundant functional cooperativity between NPC1 and NPC2 in lipid transport. Proc Natl Acad Sci U S A 101:5886–5891
Infante RE, Wang ML, Radhakrishnan A et al (2008) NPC2 facilitates bidirectional transfer of cholesterol between NPC1 and lipid bilayers, a step in cholesterol egress from lysosomes. Proc Natl Acad Sci U S A 105:15287–15292
Davies JP, Ioannou YA (2000) Topological analysis of Niemann-Pick C1 protein reveals that the membrane orientation of the putative sterol-sensing domain is identical to those of 3-hydroxy-3-methylglutaryl-CoA reductase and sterol regulatory element binding protein cleavage-activating protein. J Biol Chem 275:24367–24374
Kwon HJ, Abi-Mosleh L, Wang ML et al (2009) Structure of N-terminal domain of NPC1 reveals distinct subdomains for binding and transfer of cholesterol. Cell 137:1213–1224
Wang ML, Motamed M, Infante RE et al (2010) Identification of surface residues on Niemann-Pick C2 essential for hydrophobic handoff of cholesterol to NPC1 in lysosomes. Cell Metab 12:166–173
Deffieu MS, Pfeffer SR (2011) Niemann-Pick type C 1 function requires lumenal domain residues that mediate cholesterol-dependent NPC2 binding. Proc Natl Acad Sci U S A 108:18932–18936
Gong X, Qian HW, Zhou XH et al (2016) Structural insights into the Niemann-Pick C1 (NPC1)-mediated cholesterol transfer and ebola infection. Cell 165:1467–1478
Li XC, Wang JW, Coutavas E et al (2016) Structure of human Niemann-Pick C1 protein. Proc Natl Acad Sci U S A 113:8212–8217
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Gong, X., Qian, H. (2019). Purification and Characterization of Human Niemann–Pick C1 Protein. In: Drin, G. (eds) Intracellular Lipid Transport. Methods in Molecular Biology, vol 1949. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9136-5_18
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DOI: https://doi.org/10.1007/978-1-4939-9136-5_18
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Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-9135-8
Online ISBN: 978-1-4939-9136-5
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