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.Conserved from bacteria to humans, the sirtuin family of NAD ϩ -dependent protein deacetylase/mono-ADP-ribosyltransferase enzymes controls a variety of cellular processes such as aging, metabolism, and gene silencing (18,24). It has been proposed that sirtuins mediate the longevity-promoting effects of calorie restriction (CR) in yeast, worms, flies, and mice (4,17,22,24). Seven mammalian sirtuins (SIRT1 to -7) are known (11,12,18,24). At least three sirtuins (SIRT3, SIRT4, and SIRT5) localize to mitochondria, suggesting the existence of sirtuin substrates in that organelle (19,26,28,(31)(32)(33). Several lines of evidence link SIRT3 to metabolism: SIRT3 is down-regulated in muscle from diabetic animals (37) and upregulated in white and brown adipose tissue in response to CR (33). Overexpression of SIRT3 in cells affects expression of genes involved in mitochondrial function (33). SIRT3 regulates the acetylation level and activity of acetyl-coenzyme A synthetase 2, a protein that may play a role in energy production in mammals under starvation conditions (20,31). SIRT4 is an ADP-ribosyltransferase that has been implicated in regulating amino acid-stimulated insulin secretion in mice via modification of glutamate dehydrogenase (GDH) (19). No functions have been reported for SIRT5.

Mitochondria are tailored to meet the metabolic and signaling needs of each cell. To explore its molecular composition, we performed a proteomic survey of mitochondria from mouse brain, heart, kidney, and liver and combined the results with existing gene annotations to produce a list of 591 mitochondrial proteins, including 163 proteins not previously associated with this organelle. The protein expression data were largely concordant with large-scale surveys of RNA abundance and both measures indicate tissue-specific differences in organelle composition. RNA expression profiles across tissues revealed networks of mitochondrial genes that share functional and regulatory mechanisms. We also determined a larger "neighborhood" of genes whose expression is closely correlated to the mitochondrial genes. The combined analysis identifies specific genes of biological interest, such as candidates for mtDNA repair enzymes, offers new insights into the biogenesis and ancestry of mammalian mitochondria, and provides a framework for understanding the organelle's contribution to human disease.

We report that human acetyl-CoA synthetase 2 (AceCS2) is a mitochondrial matrix protein. AceCS2 is reversibly acetylated at Lys-642 in the active site of the enzyme. The mitochondrial sirtuin SIRT3 interacts with AceCS2 and deacetylates Lys-642 both in vitro and in vivo. Deacetylation of AceCS2 by SIRT3 activates the acetylCoA synthetase activity of AceCS2. This report identifies the first acetylated substrate protein of SIRT3. Our findings show that a mammalian sirtuin directly controls the activity of a metabolic enzyme by means of reversible lysine acetylation. Because the activity of a bacterial ortholog of AceCS2, called ACS, is controlled via deacetylation by a bacterial sirtuin protein, our observation highlights the conservation of a metabolic regulatory pathway from bacteria to humans.eversible lysine acetylation is a highly regulated posttranslational protein modification, which is controlled by protein deacetylases and acetyltransferases (1, 2). Although the importance of reversible lysine acetylation of nuclear nonhistone and histone proteins is well established, the role of protein modification by reversible lysine acetylation in mitochondria is unknown.The nicotinamide (NAM) adenine dinucleotide (NAD ϩ )-dependent deacetylase silent information regulator 2 (sir2) is an important mediator of longevity in response to caloric restriction signals in Saccharomyces cerevisiae, Caenorhabditis elegans, and Drosophila melanogaster (3-9). Seven mammalian Sir2 homologs (SIRT1-7) are known (10-13). The recent discovery that SIRT3, SIRT4, and SIRT5 are found in mitochondria (14-17) suggests the existence of mitochondrial sirtuin substrate proteins. Results and DiscussionAs a strategy to identify lysine-acetylated mitochondrial proteins that could be targeted by SIRT3, -4, or -5, we searched for human proteins with sequence similarity to the region surrounding the acetylated lysine residue found in acetyl-CoA synthetase (ACS) from Salmonella enterica. We chose the acetyl-lysine-containing region of S. enterica ACS, because it is a known substrate for the sirtuin CobB (18,19). In a second step, human proteins showing high similarity were analyzed for their mitochondrial localization probability by using MITOPROT II and PREDOTAR Ver.

Identifying the genes responsible for human diseases requires combining information about gene position with clues about biological function. The recent availability of whole-genome data sets of RNA and protein expression provides powerful new sources of functional insight. Here we illustrate how such data sets can expedite disease-gene discovery, by using them to identify the gene causing Leigh syndrome, French-Canadian type (LSFC, Online Mendelian Inheritance in Man no. 220111), a human cytochrome c oxidase deficiency that maps to chromosome 2p16-21. Using four public RNA expression data sets, we assigned to all human genes a ''score'' reflecting their similarity in RNA-expression profiles to known mitochondrial genes. Using a large survey of organellar proteomics, we similarly classified human genes according to the likelihood of their protein product being associated with the mitochondrion. By intersecting this information with the relevant genomic region, we identified a single clear candidate gene, LRPPRC. Resequencing identified two mutations on two independent haplotypes, providing definitive genetic proof that LRPPRC indeed causes LSFC. LRPPRC encodes an mRNA-binding protein likely involved with mtDNA transcript processing, suggesting an additional mechanism of mitochondrial pathophysiology. Similar strategies to integrate diverse genomic information can be applied likewise to other disease pathways and will become increasingly powerful with the growing wealth of diverse, functional genomics data.