These readouts activate or inhibit cytosolic signalling pathways that make sure the appropriate changes in nuclear gene expression occur to maintain mitochondrial homeostasis2-6

These readouts activate or inhibit cytosolic signalling pathways that make sure the appropriate changes in nuclear gene expression occur to maintain mitochondrial homeostasis2-6. Mitochondrial dysfunction is usually a hallmark of ageing and alterations in mitochondrial activity affect the lifespan of model organisms7, 8. reactive oxygen species metabolism and the mitochondrial unfolded protein response. Our results demonstrate that a respiratory enzyme acts in the nucleus to control mitochondrial stress responses and longevity. Introduction Mitochondria function as cellular energy generators producing the fuel, predominantly in the form of adenosine triphosphate (ATP), required to drive biological processes. They act as a hub for many essential biochemical pathways, the metabolites of which are closely monitored by the cell1-3. The majority of the enzymes that are required for these pathways are encoded by the nuclear genome compared with a few that are encoded directly by the mitochondrial genome. Therefore, coordinated regulation of nuclear and mitochondrial gene expression is usually essential4, 5. Mitochondrial activity is usually monitored through a variety of mitochondrial readouts that include the amount of reactive oxygen species (ROS) produced during oxidative metabolism, the rate of ATP production, and the level of misfolded proteins. These readouts activate or inhibit cytosolic signalling pathways that make sure the appropriate changes in nuclear gene expression occur to maintain mitochondrial homeostasis2-6. Mitochondrial dysfunction is usually a hallmark of ageing and alterations in mitochondrial activity affect the lifespan of model organisms7, 8. Indeed, increased mitochondrial ROS modulates stress responses and promotes longevity3, 8-10. In addition, the initiation of a distinct mitochondrial to nuclear retrograde signaling pathway, the mitochondrial unfolded protein response (UPRmt), has been proposed to extend lifespan11-13. These findings suggest a direct link between mitochondrial stress and longevity. In mutants of components of the electron transport chain display reduced oxidative phosphorylation and increased longevity14. The mitochondrial diiron made up of monooxygenase CLK-1 catalyzes the hydroxylation of 5-demethoxyubiquinone, a critical step in the Xanthone (Genicide) biosynthesis of ubiquinone, an essential cofactor of the electron transport chain15, 16. However, null mutants and heterozygous mice display altered mitochondrial metabolism and extended lifespans through a pathway that appears to be impartial of ubiquinone biosynthesis and ATP production17-21. This indicates that additional functions for CLK-1 may exist. CLK-1, and its human homologue COQ7, contain an N-terminal mitochondrial targeting sequence (MTS) and are assumed to reside exclusively within mitochondria22, 23. However, we have observed CLK-1/COQ7 present in the nuclei of both and cultured human cells. Furthermore, we have uncovered a specific role for the nuclear pool of CLK-1/COQ7 in the regulation of ROS metabolism, mitochondrial stress responses and longevity. Results CLK-1 and its human homologue Rabbit Polyclonal to MPRA COQ7 localise to mitochondria and nuclei We found that endogenous and exogenously expressed COQ7 display both mitochondrial and nuclear immunostaining in HeLa cells (Fig. 1a, b and Supplementary Fig. 1a), while adult transgenic worms expressing CLK-1 fused to green fluorescent protein (GFP) also display fluorescence in both compartments (Fig. 1c). We identified a sequence in COQ7 between amino acids 11 and 29 that is required for nuclear localisation (Supplementary Fig. 1b). This nuclear Xanthone (Genicide) targeting sequence (NTS) is usually adjacent to the MTS, but within the N-terminal region that is cleaved and Xanthone (Genicide) degraded by the mitochondrial processing peptidase (MPP) following mitochondrial import23 (Fig. 2b). This suggested that a pool of COQ7, rather than being imported into mitochondria and cleaved, remains uncleaved and localises to the nucleus. This scenario is supported by nuclear-specific immunostaining of endogenous COQ7 with antibodies that specifically recognise the N-terminal region (Fig. 1d and Supplementary Fig. 1c). It was also observed that two forms of COQ7 were visible on immunoblots of cell lysates and that both were decreased by transcripts decrease levels of both cleaved and uncleaved COQ7 protein. Immunoblots of lysates from.