Drosophila Sirt2/mammalian SIRT3 deacetylates ATP synthase β and regulates complex V activity

Abstract

Adenosine triphosphate (ATP) synthase β, the catalytic subunit of mitochondrial complex V, synthesizes ATP. We show that ATP synthase β is deacetylated by a human nicotinamide adenine dinucleotide (NAD(+))-dependent protein deacetylase, sirtuin 3, and its Drosophila melanogaster homologue, dSirt2. dsirt2 mutant flies displayed increased acetylation of specific Lys residues in ATP synthase β and decreased complex V activity. Overexpression of dSirt2 increased complex V activity. Substitution of Lys 259 and Lys 480 with Arg in human ATP synthase β, mimicking deacetylation, increased complex V activity, whereas substitution with Gln, mimicking acetylation, decreased activity. Mass spectrometry and proteomic experiments from wild-type and dsirt2 mitochondria identified the Drosophila mitochondrial acetylome and revealed dSirt2 as an important regulator of mitochondrial energy metabolism. Additionally, we unravel a ceramide-NAD(+)-sirtuin axis wherein increased ceramide, a sphingolipid known to induce stress responses, resulted in depletion of NAD(+) and consequent decrease in sirtuin activity. These results provide insight into sirtuin-mediated regulation of complex V and reveal a novel link between ceramide and Drosophila acetylome.

Figure 1.

Increase in ceramide levels results in depletion of NAD⁺ and decrease in sirtuin activity leading to hyperacetylation of proteins in different cellular compartments. (A) dcerk¹ fly extracts show 65% reduction in NAD⁺ level compared with w¹¹¹⁸ control. n = 3. (B) NAD synthesis and salvage pathways. TDO, tryptophan-2,3-dioxygenase; KMO, kynurenine 3-monooxygenase; QPRTase, quinolinate phosphoribosyltransferase; NaMNAT, nicotinic acid mononucleotide adenyltransferase; NADS, NAD synthetase; NMNAT, nicotinamide mononucleotide adenyltransferase; NAmPRTase, nicotinamide phosphoribosyl transferase; NDase, nicotinamidase; NaPRTase, nicotinic acid phosphoribosyltransferase. (C) Mass spectrometric measurements of metabolites in the salvage and the de novo pathways for synthesis of NAD⁺. n = 3. (D) Soluble, mitochondrial, and nuclear extracts were prepared from w¹¹¹⁸ and dcerk¹ mutant flies and separated by PAGE. Protein acetylation was monitored by Western blotting using an anti–acetyl-Lys antibody. The individual blots were probed with antibodies to actin, porin, and H2A as loading controls. dcerk¹ mutants show protein hyperacetylation in the different cellular compartments. Arrows indicate proteins that are hyperacetylated in dcerk¹ compared with w¹¹¹⁸. MM, molecular mass. (E) Mitochondrial NAD⁺ levels are decreased in dcerk¹ compared with control. (F) d14 long chain base ceramides with different fatty acids were estimated by MS in sphingolipid-enriched fractions prepared from w¹¹¹⁸ and dcerk¹ mitochondria. C denotes the carbon chain length of fatty acids in the different ceramides. The amount of ceramide is normalized to total carbon content, and the level in w¹¹¹⁸ is taken as 100%. Many ceramides show significant increase in the mutant mitochondria compared with w¹¹¹⁸. n = 3. Error bars represent SDs. *, P ≤ 0.05–0.01; **, P ≤ 0.01–0.001; ***, P ≤ 0.001–0.0001 in Student’s t test.

Figure 2.

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.

Figure 3.

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.

Figure 4.

Analyses of the Drosophila mitochondrial acetylome and dSirt2 acetylome reveal extensive acetylation of proteins engaged in OXPHOS and metabolic pathways involved in energy production. (A) GO analysis (cellular component) of the acetylome shows significant enrichment of mitochondria-related terms. (B) Distribution of acetyl-Lys sites identified per protein in the mitochondrial acetylome. (C) Pathway analysis of the mitochondrial acetylome with the number of proteins identified per pathway indicated. (D) Consensus sequence logo plot for acetylation sites, ±6 amino acids from all acetyl-Lys identified in the mitochondrial acetylome. (E) GO analysis (cellular component) of the acetylated proteins that increase in the dsirt2 mutant. (F) Pathway analysis of the acetylated proteins that increase in dsirt2 with the number of proteins identified per pathway indicated. (G) Consensus sequence logo plot for acetylation sites, ±6 amino acids from all acetyl-Lys identified in proteins that increase in dsirt2.

Figure 5.

Identification of complex V subunits with the Lys residues that are acetylated in dcerk¹ and dsirt2 mutants. (A) GO analysis (biological process component) of the Drosophila mitochondrial acetylome shows significant enrichment of OXPHOS complexes, particularly, complex I and complex V. The numbers indicate the number of acetylated subunits out of the total number of OXPHOS subunits in each complex. (B) Distribution of acetyl-Lys sites identified in each acetylated protein of the OXPHOS complexes shows 70% of the proteins have more than one site of acetylation. (C) GO analysis (biological process component) of the acetylated proteins that increase in dsirt2 features OXPHOS complex I and complex V prominently. The numbers indicate the number of acetylated subunits out of the total number of OXPHOS subunits in each complex in the dsirt2 mutant. (D) Mass spectrometric identification of the Lys residues that are acetylated in dcerk¹ and dsirt2 (1.5-fold or more) in different subunits of complex V. For Lys residues that are conserved, the corresponding human Lys is shown. Asterisks denote Lys residues that have been identified as acetylated in other proteomic surveys. The blue numbers indicate modified Lys residues identified both in dcerk¹ and dsirt2 mutants.

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 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.