Sirt4 is a mitochondrial regulator of metabolism and lifespan in Drosophila melanogaster Jason G. Wooda, Bjoern Schwerb,1, Priyan C. Wickremesinghea, Davis A. Hartnetta, Lucas Burhenna, Meyrolin Garciaa, Michael Lia, Eric Verdinb,2, and Stephen L. Helfanda,3 aDepartment of Molecular Biology, Cell Biology, and Biochemistry, Brown University, Providence, RI 02912; and bGladstone Institute of Virology and Immunology, University of California, San Francisco, CA 94158 Edited by Nancy M. Bonini, University of Pennsylvania, Philadelphia, PA, and approved December 29, 2017 (received for review November 28, 2017) Sirtuins are an evolutionarily conserved family of NAD+-dependent response to fasting and starvation as well as longevity. Importantly, deacylases that control metabolism, stress response, genomic sta- we show that transgenic expression of a mitochondrial sirtuin can bility, and longevity. Here, we show the sole mitochondrial sirtuin extend organismal lifespan. in Drosophila melanogaster, Sirt4, regulates energy homeostasis and longevity. Sirt4 knockout flies have a short lifespan, with in- Results creased sensitivity to starvation and decreased fertility and activity. Drosophila Sirt4 Is a Mitochondrial Protein. To study mitochondrial In contrast, flies overexpressing Sirt4 either ubiquitously or specif- sirtuin function in the fly, we examined the sequences of the five ically in the fat body are long-lived. Despite rapid starvation, Sirt4 known Drosophila sirtuin orthologs and found that only one of knockout flies paradoxically maintain elevated levels of energy them, Sirt4, contains a predicted N-terminal mitochondrial tar- reserves, including lipids, glycogen, and trehalose, while fasting, geting sequence (Fig. S1A). Drosophila Sirt4 (hereafter referred suggesting an inability to properly catabolize stored energy. to as dSirt4) is most closely related to the mouse SIRT4 protein Metabolomic analysis indicates several specific pathways are af- (45% identity; Fig. S1B). To test whether dSirt4 is localized to fected in Sirt4 knockout flies, including glycolysis, branched-chain mitochondria in flies, we transfected a dSirt4::Flag construct into amino acid metabolism, and impaired catabolism of fatty acids with S2 cells and performed immunoblot analysis of subcellular fractions. chain length C18 or greater. Together, these phenotypes point to a The dSirt4::Flag signal was strongly enriched in the mitochondrial GENETICS role for Sirt4 in mediating the organismal response to fasting, and fraction relative to other subcellular fractions (Fig. 1A). To further ensuring metabolic homeostasis and longevity. assess the subcellular localization of dSirt4, we expressed either Flag-tagged dSirt4 or GFP-tagged dSirt4 proteins in S2 cells and aging | metabolism | sirtuins | mitochondria | Sirt4 examined subcellular localization via either immunofluorescence (dSirt4::Flag) or direct fluorescence (dSirt4::GFP) analysis (Fig. 1B; + additional images are shown in Fig. S2). Using MitoTracker dye or irtuins are a family of highly conserved NAD -dependent immunostaining of the mitochondrial protein MnSOD, we observed protein deacylases with roles in regulating many cellular S a high degree of overlap of fluorescence signal in the mitochondria processes, including genomic stability, metabolism, and longevity of transfected cells for both dSirt4 constructs, indicating that dSirt4 (1). In mammals, of the seven sirtuin family members, three localizes to mitochondria. (SIRT3, SIRT4, and SIRT5) are localized within mitochondria, where they have wide-ranging and overlapping effects on nu- merous metabolic pathways, including fatty acid metabolism, Significance tricarboxylic acid (TCA) cycle, glycolysis, reactive oxygen species (ROS), oxidative phosphorylation, protein metabolism, and the Sirtuins are a class of proteins known to regulate aspects of urea cycle (reviewed in ref. 2). Although mitochondrial sirtuins genomic stability, metabolism, and lifespan in many organisms. have a range of enzymatic activities and targets, an emerging In this study, we show that the mitochondrial sirtuin Sirt4 plays view suggests they work coordinately to regulate metabolic net- an important role in regulating the organismal response to works in mitochondria in response to changing environmental fasting as well as ensuring normal lifespan in Drosophila. Flies and nutrient conditions. SIRT3 displays robust deacetylase activity, lacking Sirt4 are short-lived, while flies overexpressing Sirt4 are and functions to clear ROS as well as activate fatty acid oxidation long-lived. Flies lacking Sirt4 display a number of metabolic (FAO) in response to fasting (3–7). SIRT5 has desuccinylase, defects, including sensitivity to starvation; decreased fertility demalonylase, and deglutarylase activities and up-regulates en- and activity; and an inability to utilize energy stores, particu- zymes in the urea cycle during fasting (2, 8). The targets and larly long-chain fatty acids, suggesting Sirt4 is important for enzymatic activity of SIRT4 remain enigmatic relative to the rest maintaining metabolic homeostasis. Our results suggest that of the sirtuins. Reported enzymatic activities for SIRT4 include boosting mitochondrial sirtuin activity may be an important both ADP ribosylation (9, 10) as well as a number of deacylase avenue for treating age-related metabolic decline and pre- activities, including removal of acetyl (11), lipoyl (12), glutaryl, serving healthy lifespan. methylglutaryl, and hydroxymethylglutaryl (13) adducts from ly- Author contributions: J.G.W., B.S., E.V., and S.L.H. designed research; J.G.W., B.S., P.C.W., sine residues. Likewise, metabolic targets of SIRT4 activity are D.A.H., L.B., M.G., and M.L. performed research; J.G.W. and B.S. analyzed data; and wide-ranging, including reported roles in insulin signaling, lipid J.G.W., B.S., and S.L.H. wrote the paper. metabolism, TCA cycle, pyruvate metabolism, and amino acid The authors declare no conflict of interest. – oxidation (9 13). The Drosophila melanogaster genome contains This article is a PNAS Direct Submission. five sirtuins named Sirt1, Sirt2, Sirt4, Sirt6, and Sirt7 after their Published under the PNAS license. closest mammalian orthologs. Of these five sirtuins, only one, Sirt4, 1Present addresses: Department of Neurological Surgery and Eli and Edythe Broad Center contains a predicted mitochondrial targeting sequence, suggesting of Regeneration Medicine and Stem Cell Research, University of California, San Francisco, that Sirt4 may act as a more general mitochondrial sirtuin in this CA 94158. organism and perform functions distributed across other mito- 2Present address: Buck Institute for Research on Aging, Novato, CA 94945. chondrial sirtuins in mammals. Here, we report a genetic charac- 3To whom correspondence should be addressed. Email: [email protected]. terization of the Drosophila sirtuin Sirt4, and show it localizes to This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. the mitochondria and plays a role in regulating the metabolic 1073/pnas.1720673115/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1720673115 PNAS Latest Articles | 1of6 Downloaded by guest on September 26, 2021 AB dSirt4 Mediates Response to Fasting and Starvation. In mammalian cells, mitochondrial sirtuins have been implicated in regulation of metabolic homeostasis via several different mitochondrial pathways (6–14, 16). To assess the importance of dSirt4 in maintaining normal metabolism during fasting and starvation, we performed a starvation sensitivity assay in dSirt4 knockout or dSirt4-overexpressing flies. We found that flies lacking dSirt4 were sensitive to starvation, and died much sooner than the genetically matched wild-type cohort (Fig. 3A and Fig. S5A). Conversely, we found that flies overexpressing dSirt4 were re- sistant to starvation, and able to survive longer than genetically Fig. 1. dSirt4 is localized to mitochondria. (A) Subcellular fractionation of matched uninduced controls in the absence of food (Fig. 3B and dSirt4::Flag cells shows mitochondrial localization. Homogenates from S2 cells expressing dSirt4::Flag were fractionated into mitochondria-enriched Fig. S5B). To complement the whole-organism starvation assays, heavy membrane (HM), light membrane (LM), and cytosolic (Cyt) fractions, we next examined whether dSirt4 expression was induced under and analyzed by Western blot. The mitochondrial protein MnSOD and cy- fasting or starvation conditions in wild-type flies. In mammals, tosolic proteins tubulin and Hsp90α are shown as fractionation controls. expression of both SIRT1 and SIRT3 is induced upon fasting (B) Immunofluorescence of dSirt4 constructs confirms mitochondrial localization. (18). Similarly, we observed a transcriptional up-regulation of dSirt4::Flag (Top) or dSirt4::GFP (Bottom) colocalizes with MitoTracker dye (Top) dSirt4 upon overnight fasting in the fat body of wild-type flies, μ or MnSOD (Bottom) in mitochondria of S2 cells. (Scale bar, 10 m.) indicating activation of dSirt4 under these conditions (Fig. 3C). Together, these results suggest that dSirt4 is responsive to nu- Modulating dSirt4 Levels Influences Organismal Lifespan. To facili- tritional inputs, and that functional dSirt4 in the fly is important tate physiological studies and examine the function of dSirt4 in a for mediating metabolic changes coincident with a fasting or whole-organism context, we used an available dSirt4 knockout starved state in the animal. line, and additionally