Figure 6. Human ATP synthase β is an acetylated protein, and its deacetylation is regulated by SIRT3. (A) pCMV vector or ATP synthase β (DDK tagged) was transfected in HEK293T cells, immunoprecipitated using an antibody to DDK tag, and probed with an antibody to acetyl-Lys (Ac-Lys). (B) HEK293T cells were cotransfected with ATP synthase β (ATP syn β) and either SIRT3 siRNA or scrambled siRNA. ATP synthase β was immunoprecipitated, and its acetylation status was assessed. The bottom blot shows reduction of SIRT3 protein upon siRNA treatment. Knockdown of SIRT3 increases acetylation of ATP synthase β. (C) Expression vector for wild-type SIRT3 was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. Overexpression of SIRT3 decreases acetylation of ATP synthase β. (D) HEK293T cells were cotransfected with ATP synthase β and either SIRT4 siRNA or scrambled siRNA. SIRT4 knockdown does not affect acetylation of ATP synthase β. (E) Wild-type SIRT4 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT4 overexpression does not affect acetylation of ATP synthase β. (F) HEK293T cells were cotransfected with ATP synthase β and either SIRT5 siRNA or scrambled siRNA. SIRT5 knockdown does not affect acetylation of ATP synthase β. (G) Wild-type SIRT5 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT5 overexpression does not affect acetylation of ATP synthase β. (H) HEK293T cells were cotransfected with ATP synthase β and either SIRT1 siRNA or scrambled siRNA. SIRT1 knockdown does not affect acetylation of ATP synthase β. (I) Wild-type SIRT1 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT1 overexpression does not affect acetylation of ATP synthase β. (J) Mitochondria were prepared from SIRT3 siRNA–treated or scrambled siRNA–treated cells, and complex V activity was measured. The activity of mitochondria from scrambled siRNA treatment was taken as 100%. SIRT3 knockdown results in an ∼40% decrease in complex V activity. n = 3; error bars represent SDs. **, P ≤ 0.01–0.001 in Student’s t test. (K) Endogenous ATP synthase β was immunoprecipitated from HEK293T cells overexpressing SIRT3, and the immunoprecipitate was probed with antibodies to ATP synthase β and SIRT3. SIRT3 can coimmunoprecipitate with ATP synthase β. IP, immunoprecipitation; WB, Western blot.
Figure 6.

Human ATP synthase β is an acetylated protein, and its deacetylation is regulated by SIRT3. (A) pCMV vector or ATP synthase β (DDK tagged) was transfected in HEK293T cells, immunoprecipitated using an antibody to DDK tag, and probed with an antibody to acetyl-Lys (Ac-Lys). (B) HEK293T cells were cotransfected with ATP synthase β (ATP syn β) and either SIRT3 siRNA or scrambled siRNA. ATP synthase β was immunoprecipitated, and its acetylation status was assessed. The bottom blot shows reduction of SIRT3 protein upon siRNA treatment. Knockdown of SIRT3 increases acetylation of ATP synthase β. (C) Expression vector for wild-type SIRT3 was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. Overexpression of SIRT3 decreases acetylation of ATP synthase β. (D) HEK293T cells were cotransfected with ATP synthase β and either SIRT4 siRNA or scrambled siRNA. SIRT4 knockdown does not affect acetylation of ATP synthase β. (E) Wild-type SIRT4 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT4 overexpression does not affect acetylation of ATP synthase β. (F) HEK293T cells were cotransfected with ATP synthase β and either SIRT5 siRNA or scrambled siRNA. SIRT5 knockdown does not affect acetylation of ATP synthase β. (G) Wild-type SIRT5 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT5 overexpression does not affect acetylation of ATP synthase β. (H) HEK293T cells were cotransfected with ATP synthase β and either SIRT1 siRNA or scrambled siRNA. SIRT1 knockdown does not affect acetylation of ATP synthase β. (I) Wild-type SIRT1 expression vector was cotransfected in HEK293T cells with ATP synthase β, and its acetylation status was assessed after immunoprecipitation. SIRT1 overexpression does not affect acetylation of ATP synthase β. (J) Mitochondria were prepared from SIRT3 siRNA–treated or scrambled siRNA–treated cells, and complex V activity was measured. The activity of mitochondria from scrambled siRNA treatment was taken as 100%. SIRT3 knockdown results in an ∼40% decrease in complex V activity. n = 3; error bars represent SDs. **, P ≤ 0.01–0.001 in Student’s t test. (K) Endogenous ATP synthase β was immunoprecipitated from HEK293T cells overexpressing SIRT3, and the immunoprecipitate was probed with antibodies to ATP synthase β and SIRT3. SIRT3 can coimmunoprecipitate with ATP synthase β. IP, immunoprecipitation; WB, Western blot.

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