Loss of sirt2 further reduces oxygen consumption and ATP levels and further increases mitochondrial protein acetylation in dcerk¹ mutants. (A) Oxygen consumption was measured in freshly isolated mitochondria after addition of ADP (state 3 respiration). It is decreased in both dcerk¹ and dsirt2 mutant mitochondria compared with w¹¹¹⁸. The double mutants show a further decrease in oxygen consumption. (B) ATP level is measured in w¹¹¹⁸, dcerk¹, sirt2, and dcerk¹.dsirt2 fly mitochondria. The amount of ATP is calculated per milligram of mitochondrial protein and normalized to w¹¹¹⁸. The relative level of ATP in individual dcerk¹ and sirt2 is 60%, and the double mutant is 35% of w¹¹¹⁸. (A and B) n = 3; error bars represent SDs. **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001 in Student’s t test. (C) Mitochondrial extracts were prepared from w¹¹¹⁸, dcerk¹, sirt2, and dcerk¹.dsirt2 flies and separated by PAGE followed by Western blotting using an anti–acetyl-Lys antibody. The blot was probed with an antibody to porin as a loading control. dcerk¹.dsirt2 double mutants show a further increase in protein acetylation compared with individual mutants. (D) Wild type and dsirt2 are subjected to starvation and the number of surviving flies is recorded at 6-h intervals. 200 flies divided into 10 groups for each genotype are used in one experiment. The representative graph shows the percentage of survival for each time interval.