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A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity

Nature Cell Biology volume 17, pages 782–792 (2015)Cite this article

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Abstract

The coordinated regulation of mitochondrial and nuclear activities is essential for cellular respiration and its disruption leads to mitochondrial dysfunction, a hallmark of ageing. Mitochondria communicate with nuclei through retrograde signalling pathways that modulate nuclear gene expression to maintain mitochondrial homeostasis. The monooxygenase CLK-1 (human homologue COQ7) was previously reported to be mitochondrial, with a role in respiration and longevity. We have uncovered a distinct nuclear form of CLK-1 that independently regulates lifespan. Nuclear CLK-1 mediates a retrograde signalling pathway that is conserved from Caenorhabditis elegans to humans and is responsive to mitochondrial reactive oxygen species, thus acting as a barometer of oxidative metabolism. We show that, through modulation of gene expression, the pathway regulates both mitochondrial 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.

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Figure 1: CLK-1 and its human homologue COQ7 localize to mitochondria and nuclei.
Figure 2: Nuclear COQ7 functions independently of the mitochondrial form.
Figure 3: A truncated form of C. elegans CLK-1 with impaired mitochondrial targeting is predominantly nuclear and does not rescue ubiquinone biosynthesis.
Figure 4: Nuclear CLK-1 and COQ7 regulate ROS metabolism.
Figure 5: Mitochondrial and nuclear CLK-1 independently contribute to longevity.
Figure 6: Nuclear CLK-1 and COQ7 suppress the expression of a subset of UPRmt genes.
Figure 7: COQ7 associates with chromatin.
Figure 8: Model for the regulation of ROS metabolism, the UPRmt and lifespan by nuclear CLK-1.

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Acknowledgements

We thank I. Donaldson and A. Hayes of the Bioinformatics and Genomic Technologies Core Facilities at the University of Manchester for providing support with regard to ChIP–chip tiling arrays. We thank M. Howard for assistance with RP-HPLC. This work was supported by the Biotechnology and Biological Sciences Research Council (BB/J014834/1 to A.J.W. and G.B.P.) and the Wellcome Trust (093176/Z/10/Z to A.J.W. and 097820/Z/11/Z to A.J.W. and G.B.P.). Some strains were provided by the Caenorhabditis Genetics Center, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank A. Sharrocks, P. Shore, S. H. Yang and A. Gilmore for helpful comments on the manuscript.

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  1. Tereza Andreou & Nicholas Rooney

    Present address: Present addresses: Institute of Cancer and Pathology, University of Leeds, St James’s University Hospital, Leeds LS9 7TF, UK (T.A.); The Beatson Institute for Cancer Research, Switchback Road, Bearsden, Glasgow G61 1BD, UK (N.R.).,

Authors and Affiliations

  1. Faculty of Life Sciences, University of Manchester, Michael Smith Building, Oxford Road Manchester M13 9PT, UK,

    Richard M. Monaghan, Robert G. Barnes, Kate Fisher, Tereza Andreou, Nicholas Rooney, Gino B. Poulin & Alan J. Whitmarsh

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Contributions

R.M.M. conceived and designed the study, performed most of the experiments, analysed the data and wrote the paper; R.G.B. generated worm strains, imaged worms and conducted lifespan experiments; K.F. generated worm strains; T.A. and N.R. screened non-nuclear COQ7 mutants; G.B.P. conceived and designed the study; A.J.W. conceived and designed the study, analysed the data and wrote the paper.

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Correspondence to Gino B. Poulin or Alan J. Whitmarsh.

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Integrated supplementary information

Supplementary Figure 1 A distinct pool of COQ7 localises to nuclei.

(a) HeLa cells expressing COQ7-Myc were scored for mitochondrial, nuclear, mitochondrial and nuclear, or disperse COQ7 immunostaining (40 cells assessed in each of n = 3 independent experiments; error bars, s.e.m. ∗∗P < 0.005 compared to other localisations). Representative image of cells is shown in Fig. 1b. (b) The region of COQ7 required for specific nuclear localisation resides between amino acids 11 and 29. GFP fluorescence of COS7 cells expressing GFP-COQ7 and the deletion mutants GFP-COQ7(11-217), lacking amino acids 1-10, and GFP-COQ7(30-217), lacking amino acids 1-29. Orientating the GFP tag on the N-terminus of COQ7 abolished mitochondrial localisation and promoted nuclear localisation, probably due to disruption of the interaction between the N-terminal MTS and the mitochondrial import machinery. Mitochondria are stained with MitoTracker (MT) and nuclei with DAPI. Schematic depicts the GFP-COQ7 deletion mutants used and summarises their localisation. (c) Endogenous uncleaved COQ7 is nuclear. HeLa cells immunostained with a second antibody specific to the N-terminus of COQ7 (COQ7N−term2). Nuclei are stained with DAPI. (d) Immunoblot demonstrating that uncleaved wild type COQ7 (WT) migrates at the same position as COQ7 S36A (containing a point mutation in the predicted mitochondrial processing peptidase cleavage site) and that cleaved COQ7 migrates at the same position as COQ7(37-217) that lacks the N-terminal region cleaved by MPP. ∗ denotes partial cleavage of COQ7 S36A at a site upstream of the predicted MPP site. (e) The intensity of nuclear anti-Myc immunostaining in HeLa cells expressing COQ7-Myc or COQ7-R11/14/16D-Myc was quantified. Cells were treated with H2O2 (150 μM, 4 h) or NAC (10 mM, 6 h). 50 cells assessed in each of n = 3 independent experiments; error bars, s.e.m. n.s., no significant difference; ∗P < 0.05 relative to untreated.

Supplementary Figure 2 Identifying a non-nuclear mutant of COQ7.

(a) Alignment of mammalian COQ7 N-terminal protein sequences (amino acids 1 to 42) using Clone Manager (Sci-Ed Software); HS, Homo sapiens; CL, Canis lupus familiaris; MM, Mus musculus; RN, Rattus norvegicus. Conserved residues are in red and R28 is in blue. MPP marks the predicted mitochondrial processing peptidase cleavage site. Residues mutated and assessed in the fluorescence studies in panel b are denoted with asterisks. (b) Point mutations in the COQ7 N-terminus cause reduced nuclear localisation. GFP fluorescence in COS7 cells expressing COQ7 fused at the C-terminus to GFP and harboring the point mutations R21A, Y26F and R28A were analysed. Quantification of the percent of cells displaying nuclear staining is shown (100 cells assessed in each of n = 3 independent experiments; error bars, s.e.m. ∗∗P < 0.005). The most severe loss of nuclear staining was observed with the R28A mutation. (c) COQ7 (R28A) mutant displays reduced levels of the uncleaved form. Lysates from HEK293 cells expressing OLLAS and FLAG tagged COQ7 (COQ7O/F) or COQ7(R28A)O/F (Fig. 2b) were immunoblotted with anti-COQ7 antibody. (d) Parent HEK293 cells (Ctrl) or cells stably expressing untagged (WT) or non-nuclear COQ7 (R28A) were transfected with siCTRL or siCOQ7that specifically targets endogenous COQ7 mRNA. Transcript levels of endogenous COQ7 mRNA (5′UTR amplicon) were analysed (mean values from 3 reactions per condition in n = 4 independent experiments; error bars, s.e.m. ∗∗P < 0.005). (e) Reverse phase HPLC chromatograms of quinones. Purified ubiquinone-10 (UQ10) was used as a standard. Levels of UQ10 and demethoxyubiquinone-10 (DMQ10) were measured in HEK293 cells treated with the COQ7 inhibitor clioquinol (CQ; 10 μM, 24 h), or from WT or R28A expressing HEK293 cells. CQ caused the appearance of DMQ10. UQ10 peak at 8.63 min, DMQ10 peak at 8.39 min. (f) Levels of lactate dehydrogenase (LDH) in media from cultured WT and R28A cells are similar, indicating that cell survival under basal conditions is not changed (mean values from 4 wells of cells per condition in n = 3 independent experiments; error bars, s.e.m. ∗∗P < 0.005). Treatment with 0.1% (v/v) Triton X-100 (TX) for 30 min was used as a positive control.

Supplementary Figure 3 Nuclear CLK-1/COQ7 regulates ROS metabolic gene expression.

(a) Nuclear CLK-1 regulates the expression of genes involved in ROS metabolism. qPCR analysis of transcripts from ROS-sensitive retrograde genes (mean values from 3 reactions per condition in n = 3 independent experiments; error bars, s.e.m. ∗P < 0.05 for clk-1(−) compared to other strains). (b) CLK-1nuc(+) rescues the increased transcript levels of genes known to be responsive to ROS (sod-2 and skn-1) in clk-1 null worms (mean values from 3 reactions per condition in n = 3 independent experiments; error bars, s.e.m. ∗P < 0.05 for clk-1(−) compared to other strains). (c) Human homologues of these genes, SOD2 and NRF2, and the NRF2 target gene HMOX1 are increased in cells that have lost nuclear COQ7 (R28A) (mean values from 3 reactions per condition for n = 4 independent experiments; error bars, s.e.m. ∗P < 0.05). (d) Quantification of immunoblots for GLS2, WWOX and HTRA2 proteins (mean values from n = 3 independent experiments; error bars, s.e.m. ∗P < 0.05,∗P < 0.005). Representative blots are shown in Fig. 4i.

Supplementary Figure 4 Nuclear CLK-1/COQ7 suppresses the expression of UPRmt genes.

(a) qPCR analysis of UPRmt genes in CLK-1 transgenic worm strains relative to N2 (mean values from 3 reactions per condition in n = 3 independent experiments; error bars, s.e.m. n.s., no significant difference; ∗P < 0.05). The increase in expression of hsp-6, hsp-60 and spg-7 in clk-1(−) worms was abrogated by expression of either CLK-1wt or CLK-1nuc(+). mRNA levels of the endoplasmic reticulum UPR (UPRER)-regulated gene hsp-4 were not changed in any of the worm strains. (b) qPCR of analysis of transcripts of UPRmt genes and UPRER genes in WT and R28A expressing HEK293 cells (mean values from 3 reactions per condition in n = 4 independent experiments; error bars, s.e.m. ∗P < 0.05.∗∗P < 0.005). Heatmap of this data is shown in Fig. 6c. (c) The ratio of COXIV to MTCO1 protein levels in WT and R28A expressing cells quantified from n = 3 independent immunoblots (error bars, s.e.m. n.s., no significant difference). See Fig. 6d for representative immunoblot.

Supplementary Figure 5 Uncropped scans of western blots.

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Monaghan, R., Barnes, R., Fisher, K. et al. A nuclear role for the respiratory enzyme CLK-1 in regulating mitochondrial stress responses and longevity. Nat Cell Biol 17, 782–792 (2015). https://doi.org/10.1038/ncb3170

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