Figure 3.

Reduced cytochrome bc₁ complex enzymatic activity and increased proguanil sensitivity in PfmRPL13 knockdown parasites. A, a schematic presentation of the Q cycle in the bc₁ complex, modified from Ref. 33. In the assay, the rate of reduction of oxidized cyt c by reduced Qd catalyzed by the bc₁ complex was measured in mitochondria isolated from P. falciparum cultures with a light-scatter rejecting UV/VIS spectrometer (OLIS, Bogart, GA). B, the cyt c reduction activity of the bc₁ complex is dramatically reduced in the PfmRPL13 knockdown parasites. In each measurement, activity of the bc₁ complex was determined by recording the absorbance change at 550 nm per microgram of protein in the mix. The bc₁ enzymatic activity of the knockdown cultures was normalized to that of the culture maintained constantly under aTc. Statistical analysis is done by a Student's t test in n = 3 experiments. **, p < 0.001; *, p < 0.05. C, hypersensitivity to proguanil in the PfmRPL13 knockdown parasites. The knockdown parasites were grown for three cycles in the absence of aTc before exposure to proguanil. EC₅₀ values of proguanil ([μm]) are D10 aTc ON (10.7), D10 aTc OFF (10.6), yDHODH line Atv OFF (6.99), yDHODH line Atv ON (0.11), PfmRPL13 aTc ON (9.56), PfmRPL13 aTc OFF (0.17). Atv, atovaquone. Data shown are the mean ± S.D. of three replicates and are representative of n = 5 independent experiments.