dcerk¹ mutants show acetylation of many OXPHOS subunits and decrease in complex V activity, which is rescued by supplementing NAD⁺ and inhibited by nicotinamide. dSirt2 regulates complex V activity in dcerk¹ mutants. (A) BN-PAGE–separated bands from dcerk¹ were digested with trypsin and subjected to LC-MS/MS to identify the different subunits of the complexes and the subunits that are acetylated. (B) dcerk¹ mitochondria show a 40% reduction in complex V activity. Supplementing with NAD⁺ restores complex V activity in dcerk¹. Complex V activity was normalized to the activity of citrate synthase, a mitochondrial marker. The ATPase activity of untreated w¹¹¹⁸ was taken as 100%. (C) Nicotinamide treatment further inhibits complex V activity in dcerk¹. The ATPase activity of untreated w¹¹¹⁸ was taken as 100%. n = 3. (D) Mitochondria were isolated from different sirtuin-null mutants, and complex V activity was measured. Complex V activity was normalized to the activity of citrate synthase. The ATPase activity of w¹¹¹⁸ was taken as 100%. dsirt2 mutants show 30% reduction in activity. n = 3. (E) Mitochondria were isolated from w¹¹¹⁸, dcerk¹, dsirt2, dcerk¹.dsirt2, and dcerk¹.dsirt2 raised on food supplemented with NAD⁺, and complex V activity was measured. The ATPase activity of w¹¹¹⁸ was taken as 100%. dcerk¹.dsirt2 mutants show a further reduction in complex V activity compared with the single mutants. Supplementing with NAD⁺ does not alter this activity. n = 3. (F) The wild-type Sirt2 transgene was ubiquitously overexpressed using the actin-GAL4 driver in dsirt2 and dcerk¹ mutants. The UAS-Sirt2 transgenic and GAL4 driver in each genetic background were additional controls. Mitochondria were prepared, and complex V activity was measured. The activity of w¹¹¹⁸ was taken as 100%. Overexpression of the Sirt2 transgene significantly rescues complex V activity in the dsirt2 mutant and partially in the dcerk¹ mutant. Error bars represent SDs. *, P ≤ 0.05–0.01; ** P ≤ 0.01–0.001; *** P ≤ 0.001–0.0001 in Student’s t test.