FIGURE 3.

Purified ABCB6 was efficiently reconstituted into liposomes. Efficiency of ABCB6 reconstitution in liposomes was analyzed by (A) Western blot analysis of a flotation assay of ABCB6-FLAG proteoliposomes (2 μg of each fraction) in a Nycodenz gradient (B), Coomassie Blue staining of equal amounts of proteoliposomes (25 μg of protein) obtained from the 5% fraction of the gradient, and (C) immunoblotting of equal amounts of proteoliposomes obtained from the 5% fraction with ABCB6-specific antibody. Flotation assay shows co-migration of majority of ABCB6 proteoliposomes into the 5% fraction of the gradient, whereas unincorporated protein is found at the bottom of the gradient (40%). Coomassie staining and immunoblotting show comparable efficiency of reconstitution of both ABCB6-wildtype and ABCB6-mutant protein. (D) Membrane orientation of reconstituted ABCB6-FLAG (4 μg protein) was determined by protease protection assay utilizing the FLAG-tag at the C terminus and orientation of ABCB6 was followed by a FLAG-antibody. SDS was used to permeabilize the proteoliposomes (almost 100% cleavage). Data in all panels are derived from three independent measurements. a.u., arbitrary units; WT, ABCB6 wild type (functional) protein; MT, ABCB6 mutant (non-functional) protein; Con-Lip, control liposomes; WT-Lip, ABCB6 wild type liposomes; MT-Lip, ABCB6 mutant liposomes.