Extended Data Figure 1: Functional studies of liposome-reconstituted ABCG2.

a, Transport curves showing E₁S uptake by ABCG2 in the presence and absence of ATP. b, The rate of E₁S transport was measured at different E₁S concentrations and the Michaelis constant was determined. c, ATP-dependent transport curves showing E₁S uptake in the presence and absence of Ko143 or 5D3-Fab. d, The 30 s to 2 min linear portion of the curves from c used to determine the initial rates of E₁S transport. e, Representative SDS–PAGE to determine the orientation of liposome-reconstituted YFP–3C–ABCG2 using in-gel fluorescence, comparing the intensity of free YFP after the addition of 3C protease in disrupted and non-disrupted samples; 54 ± 2% of ABCG2 was oriented with the NBDs on the outside of the proteoliposomes and all assays were subsequently corrected for this value; error, s.d. (n = 3). Non-disrupted supernatant (+3C) (lane 1); disrupted supernatant (+3C) (lane 2); non-disrupted supernatant (−3C) (lane 3); disrupted supernatant (−3C) (lane 4). f, The basal ATPase activity of nanodisc-reconstituted ABCG2 is equivalent to the E₁S-stimulated activity of liposome-reconstituted ABCG2 and cannot be stimulated further. ABCG2 is not responsive to cholate in the presence or absence of E₁S. The curves have been normalized to the basal ATPase activity of liposome-reconstituted ABCG2. In a–d and f, two separate experiments were performed in triplicate (error bars, s.d.).