Figure 5. dSdhaf3 mutants are sensitive to oxidative stress and display reduced levels of SdhB, reduced SDH activity, and motility defects.

Five-day old w¹¹¹⁸ control (solid line) and dSdhaf3 mutant (dotted line) males were transferred to vials with (A) 5% ethanol, 1% agar in PBS, (B) 30 mM paraquat in semi-defined medium, or (C) 100% O₂ with standard medium, and living animals were scored daily. Homozygous SdhB¹²⁰⁸¹ mutants (dashed line) were included in the hyperoxia experiment. Each graph was compiled from 3-5 experiments, using a total of 15-21 vials with 20 animals per vial. Error bars represent ±SEM. dSdhaf3 mutants are significantly more sensitive than controls under each condition, p<0.001. (D) GC/MS was used to compare the relative levels of small metabolites in wild-type controls (grey boxes) and dSdhaf3 mutants (white boxes). N=12 samples from two independent experiments with 20 flies/sample (5-day old). ***p<0.001. (E) Proteins were extracted from mitochondria isolated from w¹¹¹⁸ controls, dSdhaf3 mutants, or UAS-dSdhaf3/+ transformants, and analyzed by immunoblotting to detect SdhA, SdhB, and ATPα, (subunit of complex V). (F) A continuous colorimetric assay was used to measure SDH enzyme activity in extracts of purified mitochondria from w¹¹¹⁸ controls, dSdhaf3 mutants, and UASdSdhaf3/+ transformants. ***p<0.001. (G) Proteins from purified mitochondria were extracted from w¹¹¹⁸ controls and dSdhaf3 mutants, fractionated by non-denaturing PAGE, and analyzed for SDH and Complex IV activity. (H) Control w¹¹¹⁸ flies and dSdhaf3 mutants were tested for motility in three independent experiments using a total of 18 vials with 20 adults/vial at 1, 2, 3, or 4-weeks of age. Climbing ability is reported as the number of flies that climbed above a line drawn 4 cm above the bottom of the vial five seconds after being tapped to the bottom. *p<0.05, ***p<0.001