The multidrug resistance half-transporter ABCG2 is purified as a tetramer upon selective extraction from membranes |
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Authors: | Manuela Dezi,Pierre-Fré deric Fribourg,Aurelie Di Cicco,Ophelie Arnaud,Pierre Falson,Daniel Lé vy |
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Affiliation: | a Institut Curie, Centre de Recherche, Paris, F-75231, Franceb CNRS, UMR168, Paris, F-75231, Francec Equipe Labellisée Ligue 2009, Institut de Biologie et Chimie des Protéines, UMR 5086 CNRS-Université Lyon 1 and IFR 128 BioSciences Gerland-Lyon Sud, F-69367 Lyon, Franced Institut Curie, Centre de Recherche, Orsay, F-91405, Francee INSERM, U759, Orsay, F-91405, France |
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Abstract: | ABCG2 is a human membrane ATP-binding cassette half-transporter that hydrolyzes ATP to efflux a large number of chemotherapeutic agents. Several oligomeric states of ABCG2 from homodimers to dodecamers have been reported depending on the overexpression systems and/or the protocols used for purification. Here, we compared the oligomeric state of His6-ABCG2 expressed in Sf9 insect cells and in human Flp-In-293/ABCG2 cells after solubilization in mild detergents. His6-ABCG2 was purified through a new approach involving its specific recognition onto a functionalized lipid layer containing a Ni-NTA lipid. This approach allowed the purification of His-ABCG2 in presence of all solubilized membrane components that might be involved in the stabilisation of native oligomers and without requiring any additional washing or concentration passages. ABCG2 purified onto the NiNTA lipid surfaces were directly analyzed by electron microscopy and by biochemical assays. Altogether, our data are consistent with a tetrameric organization of ABCG2 when expressed in either heterologous Sf9 insect cells or in human homologous cells. |
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Keywords: | ABC, ATP-binding cassette BSA, bovine serum albumin, CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate cmc, critical micelle concentration DDM, dodecyl-β-D-maltoside DOTM, dodecyl-β-D-thiomaltoside DOPC, dioleyl-phospatidyl choline DMPC, dimiristoyl-phosphatidylcholine EM, electron microscopy Fos-choline 16, n-Hexadecylphosphocholine MDR, multidrug resistance NBD, nucleotide-binding domain NEM, N-ethylmaleimide Ni-NTA DOGS, Ni-nitrilotriacetic-1,2-dioleoyl-sn-glycero-3-{[N (5-Amino-1-Carboxypentyl) iminodiacetic Acid]Succinyl TM, transmembrane domain |
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