Figure 7. Acetylation of ATP synthase β at Lys 259 and Lys 480 regulates complex V activity. (A) Nondegradable (non-deg) ATP synthase β (ATP syn β) is resistant to targeted siRNA-mediated degradation. (B) siRNA-resistant versions of ATP synthase β wherein Lys 259 or Lys 480 either individually or together were substituted with Arg or Gln and cotransfected with siRNA to ATP synthase β. Mitochondria were prepared, and complex V activity was measured using an immunocapture assay followed by the amount of ATP synthase β in the same samples. The activity of siRNA-resistant ATP synthase β is taken as 100%. Substitution of either Lys or both with Arg results in increased activity, whereas substitution with Gln results in decreased complex V activity. **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001. (C) An overview of the crystal structure of bovine mitochondrial F1–stator complex is shown on the left in ribbon representation. The F1 domain contains 3α (green), 3β (purple), and a single subunit of γ (pink). The stator complex shows portions of subunit b (teal), oligomycin sensitivity-conferring protein (orange), and F6 (yellowish green). The right shows a closer view of the region around the active site (marked by the black box in the left image). The Lys residues are shown as spheres, and the active site amino acids are shown as stick models. Acetylation of Lys 259 (Lys 206 in the crystal structure) and Lys 480 (430 in the crystal structure) could affect protein conformation near the active site. (D) Endogenous ATP synthase β was immunoprecipitated from human breast cancer cell lines, and its acetylation status was assessed using an acetyl-Lys antibody. ATP synthase β is more acetylated in MDA-MB-231 cells compared with T47D. (E) Complex V activity was measured in mitochondria prepared from human breast cancer cell lines. The activity is significantly less in MDA-MB-231 cells compared with that in T47D cells. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 1.0 × 10−7; T47D–MDA-MB-435: adjusted P = 1.9 × 10−5. (F) Oxygen consumption is less in MDA-MB-231 compared with that in T47D mitochondria. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 2.0 × 10−6; T47D–MDA-MB-435: adjusted P = 1.0 × 10−5. (G) A model depicting Drosophila Sirt2/mammalian SIRT3-mediated deacetylation of ATP synthase β and its impact on complex V activity. Error bars represent SDs. IP, immunoprecipitation; WB, Western blot.
Figure 7.

Acetylation of ATP synthase β at Lys 259 and Lys 480 regulates complex V activity. (A) Nondegradable (non-deg) ATP synthase β (ATP syn β) is resistant to targeted siRNA-mediated degradation. (B) siRNA-resistant versions of ATP synthase β wherein Lys 259 or Lys 480 either individually or together were substituted with Arg or Gln and cotransfected with siRNA to ATP synthase β. Mitochondria were prepared, and complex V activity was measured using an immunocapture assay followed by the amount of ATP synthase β in the same samples. The activity of siRNA-resistant ATP synthase β is taken as 100%. Substitution of either Lys or both with Arg results in increased activity, whereas substitution with Gln results in decreased complex V activity. **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001. (C) An overview of the crystal structure of bovine mitochondrial F1–stator complex is shown on the left in ribbon representation. The F1 domain contains 3α (green), 3β (purple), and a single subunit of γ (pink). The stator complex shows portions of subunit b (teal), oligomycin sensitivity-conferring protein (orange), and F6 (yellowish green). The right shows a closer view of the region around the active site (marked by the black box in the left image). The Lys residues are shown as spheres, and the active site amino acids are shown as stick models. Acetylation of Lys 259 (Lys 206 in the crystal structure) and Lys 480 (430 in the crystal structure) could affect protein conformation near the active site. (D) Endogenous ATP synthase β was immunoprecipitated from human breast cancer cell lines, and its acetylation status was assessed using an acetyl-Lys antibody. ATP synthase β is more acetylated in MDA-MB-231 cells compared with T47D. (E) Complex V activity was measured in mitochondria prepared from human breast cancer cell lines. The activity is significantly less in MDA-MB-231 cells compared with that in T47D cells. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 1.0 × 10−7; T47D–MDA-MB-435: adjusted P = 1.9 × 10−5. (F) Oxygen consumption is less in MDA-MB-231 compared with that in T47D mitochondria. n = 3. Analysis of variance was performed, and Tukey’s honest significance test was applied to determine significance. T47D–MDA-MB-231: adjusted P = 2.0 × 10−6; T47D–MDA-MB-435: adjusted P = 1.0 × 10−5. (G) A model depicting Drosophila Sirt2/mammalian SIRT3-mediated deacetylation of ATP synthase β and its impact on complex V activity. Error bars represent SDs. IP, immunoprecipitation; WB, Western blot.

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