Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation

Abstract

Homologs of the Saccharomyces cerevisiae Sir2 protein, sirtuins, promote longevity in many organisms. Studies of the sirtuin SIRT3 have so far been limited to cell culture systems. Here, we investigate the localization and function of SIRT3 in vivo. We show that endogenous mouse SIRT3 is a soluble mitochondrial protein. To address the function and relevance of SIRT3 in the regulation of energy metabolism, we generated and phenotypically characterized SIRT3 knockout mice. SIRT3-deficient animals exhibit striking mitochondrial protein hyperacetylation, suggesting that SIRT3 is a major mitochondrial deacetylase. In contrast, no mitochondrial hyperacetylation was detectable in mice lacking the two other mitochondrial sirtuins, SIRT4 and SIRT5. Surprisingly, despite this biochemical phenotype, SIRT3-deficient mice are metabolically unremarkable under basal conditions and show normal adaptive thermogenesis, a process previously suggested to involve SIRT3. Overall, our results extend the recent finding of lysine acetylation of mitochondrial proteins and demonstrate that SIRT3 has evolved to control reversible lysine acetylation in this organelle.

FIG. 1.

SIRT3 is a soluble mitochondrial protein highly expressed in tissues rich in mitochondria. (A) Prominent SIRT3 protein expression in mitochondrion-rich tissues. Twenty micrograms of total protein extract from each tissue was analyzed by immunoblotting with antibodies to SIRT3 and the mitochondrial protein MnSOD. SKM, skeletal muscle (quadriceps); WAT, white adipose tissue. (B) Endogenous murine SIRT3 is a mitochondrial protein. Equal amounts of total liver homogenate (total), mitochondria (Mito), light membranes (LM), and cytosol (S-100) were separated by SDS-PAGE and analyzed by immunoblotting using antibodies against SIRT3 and marker proteins of known subcellular distribution. (C) Murine SIRT3 is a soluble mitochondrial protein. Purified mouse liver mitochondria were extracted with sodium carbonate (pH 11.5), and the distribution of SIRT3, the soluble mitochondrial protein GDH, and the integral membrane protein COX-IV was analyzed by immunoblotting. T, total; P, pellet (membranes); S, soluble. (D) SIRT3 is absent from the nucleus in liver. Equal amounts of nuclear and mitochondrial protein extracts were immunoblotted with antibodies against the indicated proteins. RNAP-II, RNA polymerase II; H4, histone H4.

FIG. 2.

Generation of SIRT3-deficient mice. (A) Gene targeting strategy for the SIRT3 locus. Arrows indicate loxP sites, and Neo^r indicates the neomycin resistance cassette used to select targeted ES cell clones. Restriction sites are as shown. WT, wild type. (B) SIRT3 PCR genotyping. KO, knockout; Het, heterozygous. (C) SIRT3 protein is absent from liver and brain mitochondria of knockout mice. Protein extracts were probed with antibodies to SIRT3 and F₁F₀-ATPase subunit α (loading control).

FIG. 3.

SIRT3 is a major mitochondrial protein deacetylase in vivo. (A) Hyperacetylation of mitochondrial proteins in liver of SIRT3-deficient mice. SIRT3-deficient and littermate control mitochondrial extracts from two mice per genotype were fractionated by SDS-PAGE and immunoblotted with polyclonal antibodies to acetyl-lysine (Ac-K), SIRT3, or MnSOD as a loading control. (B) Hyperacetylation of mitochondrial proteins in BAT of SIRT3-deficient mice. Mitochondrial extracts from two SIRT3-deficient mice and two wild-type littermate controls were probed as in panel A except that COX-IV was used as a loading control. (C) Recombinant SIRT3 reverses mitochondrial hyperacetylation associated with SIRT3 deficiency. Mitochondrial extracts were treated with recombinant wild-type (WT) SIRT3 or a SIRT3 catalytic mutant (SIRT3-HY) as shown. NAD⁺, a cofactor required for sirtuin-mediated deacetylation, and nicotinamide (NAM), a sirtuin inhibitor, were added as indicated. Samples were separated by SDS-PAGE and probed with a monoclonal acetyl-lysine antibody. SIRT3 antibodies were used to demonstrate the presence of recombinant SIRT3; mtHsp70 served as a loading control. (D) SIRT3, but not SIRT4 or SIRT5, is responsible for global protein deacetylation in mitochondria. Liver mitochondrial extracts were generated from mice of the indicated genotypes and analyzed as in panel A.

FIG. 4.

SIRT3 deacetylates GDH in vivo. (A) Scheme for identifying putative SIRT3 substrates from mouse liver mitochondria. KO, knockout. (B) Representative immunoblot (IB) of protein fractions prepared as illustrated in panel A and probed with monoclonal acetyl-lysine antibody. *, acetyl-lysine antibody-reactive bands analyzed by mass spectrometry. (C and D) GDH is hyperacetylated in SIRT3-deficient mouse liver mitochondria. (C) Acetylated proteins from liver mitochondria of animals with the indicated genotypes were immunoprecipitated (IP) with monoclonal acetyl-lysine antibodies, fractionated by SDS-PAGE, and probed with GDH antibodies. Results shown are representative of five independent experiments. (D) GDH immune complexes from wild-type and SIRT3-deficient liver mitochondria were immunoblotted with monoclonal acetyl-lysine antibodies (Ac-GDH). Probing with GDH antibodies revealed total GDH levels. Results shown are representative of three independent experiments. (E) GDH is a bona fide SIRT3 substrate. Bovine GDH was incubated with recombinant SIRT3, NAD⁺, and nicotinamide (NAM) as indicated. Reactions were stopped by boiling in SDS sample buffer, and levels of total and acetylated GDH were analyzed by immunoblotting. The presence of recombinant SIRT3 was demonstrated by immunoblotting with antibodies to SIRT3.

FIG. 5.

SIRT3-deficient mice are viable and metabolically unremarkable under both fed and fasted conditions. (A) Normal liver histology in wild-type (WT) and SIRT3-deficient (KO) mice subject to an 18-hour fast. (B) SIRT3 deficiency does not affect mitochondrial content. Equal amounts of protein from total liver extracts generated from animals of the indicated genotypes representing two separate litters were probed with antibodies to TFAM to assess mitochondrial content and MnSOD, COX-IV, and cytochrome c as loading controls. (C) Body weight, adiposity, bone mineral density (BMD), and bone mineral content (BMC) are not affected by SIRT3 deficiency. (D) SIRT3-deficient and wild-type littermate control mice display similar measures of energy balance in both the fed and fasted states. Oxygen consumption (), respiratory exchange ratios (/), activity levels, and food intake were measured in metabolic cages. Data represent the means plus SEM for six animals per genotype.

FIG. 6.

SIRT3-deficient mice show normal adaptive thermogenesis. (A) SIRT3-deficient mice show wild-type levels of UCP-1. UCP-1 protein levels were assessed in BAT mitochondrial extracts from wild-type (WT) and SIRT3 knockout (KO) animals from three different litters. Levels of Hsp60 and MnSOD were measured as loading controls. (B) Normal BAT morphology in SIRT3 KO mice. Hematoxylin-eosin staining is shown. Original magnification, ×400. (C) Normal adaptive thermogenesis in SIRT3 KO mice. (Left) SIRT3 KO and WT cohorts are similar in both pre- and post-cold exposure body weights. (Middle) SIRT3 KO and WT mice lose equal percentages of body weight during 6 h of cold exposure (4°C). ns, not significant. (Right) Core body temperatures of 7- to 10-week-old male SIRT3 KO and WT mice during exposure to 4°C for 6 h. Data represent the means ± SEM for three animals per genotype.