ATPase activity of C-terminal mutants in the presence of varying amounts of MQ (A), QN (B), and CQ (C). Activity for 3D7, Dd2 and 7G8 is also reproduced in ref [10]. The ATPase activity of purified PM fractions was measured using the colorimetric determination of orthophosphate released from ATP as described in detail previously [8]. Briefly, plates were set up on ice: assay buffer pH 7.5 (180 mM NH₄Cl/100 mM Mes-Tris/10 mM MgCl₂/.01% NaN₃) was added to each well followed by relevant drug solutions, and finally membrane samples, to a total volume of 100 μL. The plate was shaken, warmed to 37°C, and ATP was then added. After shaking at 37°C for an additional 15 minutes, the reaction was stopped, stabilized 10 minutes later, and absorbance at 720 nm read after 30 minutes. Results shown are the average (+/− s.d.) for at least 2 independent membrane preparations, each done at least in triplicate, and values are normalized to PfMDR1 content via densitometry as described in detail elsewhere [8]. Single mutants, solid lines, closed symbols: S1034C, closed squares; N1042D, closed triangles; D1246Y, closed circles. Strain isoforms, dashed lines, open symbols: 3D7, open squares; 7G8, open circles. Double mutant data are omitted for clarity but are available from the authors upon request: 1034/1042 ATPase activity is very similar to that of D1246Y when plotted vs. these concentrations of MQ, QN or CQ, whereas 1034/1246 and 1042/1246 show behavior that lies between that of D1246Y vs. 7G8 and S1034C. Note behavior for D1246Y is reminiscent of that seen for isoform Dd2 [8] indicating that single site mutations at two widely separate positions (1246 and 86 respectively) are capable of approximately doubling V_max.

Representative polyHis blot showing approximately equal inducible expression of new PfMDR1 mutants in the constructed yeast strains. Expression levels are similar to those of 3D7, Dd2 and 7G8 isoforms reported previously [8]. “ZC” indicates negative control carrying empty zeocin selectable vector. Yeast were grown under standard conditions, induced with methanol for ~20 hours and plasma membranes purified by acid precipitation as described [8]. Protein was quantified by amido black and an equivalent amount of membrane protein was loaded into wells of a 7.5% polyacrylamide gel, which was run at 110V for 100 min and subsequently transferred to a PVDF membrane overnight at 40 mA. The blot was developed using the PentaHis detection kit from Qiagen according to manufacturer’s instructions. After averaging densitometry from two such plots, we calculate that for each mutant, PfMDR1 constitutes 3.63, 2.45, 2.71, 1.94, 2.85, 2.05, and 2.46 % of total membrane protein, respectively.