Figure 1: Transport activity and thermostability of wild-type APC1 in the presence or absence of calcium.

(a) Uptake of radiolabelled ATP (red) into proteoliposomes measured with different unlabelled internal and external substrates and effectors (black). Proteoliposomes were made with 2.5 mM ATP and 1 mM calcium chloride (circles), 2.5 mM ATP and 1 mM EGTA (triangles) or no ATP and 1 mM EGTA (squares). Uptake was initiated by addition of 1.5 μM [¹⁴C]-ATP to the outside of liposomes. (b) The initial uptake rates in the conditions indicated in the table were calculated from the fit of the curves in panel A. (c) Thermostability assays with CPM. Purified carrier protein was diluted to 50 μg mL⁻¹ in assay buffer, and pre-equilibrated CPM was added to 1 μg mL⁻¹. Either 2.5 mM calcium chloride (blue curve) or 2.5 mM EGTA (black curve) was added prior to temperature ramping. A sample without protein was included for baseline reference (grey). (d) The apparent melting temperature can be derived from the inflection point of the unfolding curve from the first derivative. (e) APC1 proteoliposomes loaded with 2.5 mM ATP and uptake of 1.5 μM [¹⁴C]-ATP stimulated by the addition of 0, 0.05, 0.1, 0.5, 1, 2, 3 or 4 mM calcium chloride on the outside. Free calcium concentration was determined against a standard curve in the presence of 1 μM of fluo-5N (Supplementary Fig. 3). The relationship between initial ATP uptake rate and the calcium concentration was plotted using specific initial rates (Supplementary Fig. 3) and fitted with a curve describing hyperbolic saturation kinetics. (f) Uptake from APC1 proteoliposomes loaded with 2.5 mM ATP, with increasing concentrations of ATP (1.5, 6, 12, 20, 38, 60, 80 and 100 μM) added to the outside and in the presence of either 0.25 mM or 4 mM calcium chloride. The Michaelis-Menten relationship between initial ATP uptake rate (Supplementary Fig. 4) and the ATP concentration was plotted for 0.25 mM (filled squares) and 4 mM (open squares) concentrations of external calcium using the specific initial rates. Error bars represent standard deviation for measurements taken from three independent liposome preparations.