title: Efficient purification and reconstitution of ATP binding cassette transporter B6 (ABCB6) for functional and structural studies.
abstract: The mitochondrial ATP binding cassette transporter ABCB6 has been associated with a broad range of physiological functions, including growth and development, therapy-related drug resistance, and the new blood group system Langereis. ABCB6 has been proposed to regulate heme synthesis by shuttling coproporphyrinogen III from the cytoplasm into the mitochondria. However, direct functional information of the transport complex is not known. To understand the role of ABCB6 in mitochondrial transport, we developed an in vitro system with pure and active protein. ABCB6 overexpressed in HEK293 cells was solubilized from mitochondrial membranes and purified to homogeneity. Purified ABCB6 showed a high binding affinity for MgATP (Kd = 0.18 μM) and an ATPase activity with a Km of 0.99 mM. Reconstitution of ABCB6 into liposomes allowed biochemical characterization of the ATPase including (i) substrate-stimulated ATPase activity, (ii) transport kinetics of its proposed endogenous substrate coproporphyrinogen III, and (iii) transport kinetics of substrates identified using a high throughput screening assay. Mutagenesis of the conserved lysine to alanine (K629A) in the Walker A motif abolished ATP hydrolysis and substrate transport. These results suggest a direct interaction between mitochondrial ABCB6 and its transport substrates that is critical for the activity of the transporter. Furthermore, the simple immunoaffinity purification of ABCB6 to near homogeneity and efficient reconstitution of ABCB6 into liposomes might provide the basis for future studies on the structure/function of ABCB6.
authors.affiliation: Department of Pharmacology, Toxicology and Therapeutics, University of Kansas Medical Center, Kansas City, Kansas 66160, USA.
authors.forename: Hemantkumar
authors.initials: H
authors.lastname: Chavan
authors.forename: Mohiuddin Md Taimur
authors.initials: MM
authors.lastname: Khan
authors.forename: George
authors.initials: G
authors.lastname: Tegos
authors.forename: Partha
authors.initials: P
authors.lastname: Krishnamurthy
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D003306:Coproporphyrins / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008563:Membrane Lipids / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: C107991:ABCB6 protein, human; D018528:ATP-Binding Cassette Transporters; D003306:Coproporphyrins; D008563:Membrane Lipids; D020033:Protein Isoforms; C026971:coproporphyrin III; D000255:Adenosine Triphosphate
keywords: ABC Transporter; Liposomes; Membrane Transport; Protein Purification; Reconstitution of Membrane Transporters