The Parkinson's disease–linked proteins Fbxo7 and Parkin interact to mediate mitophagy

Abstract

Compelling evidence indicates that two autosomal recessive Parkinson's disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain–containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinson's disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinson's disease–causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinson's disease pathogenesis.

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Figure 1: The N-terminal Ubl domain of Fbxo7 interacts directly with Parkin.
Figure 2: Fbxo7 participates in CCCP-induced accumulation of Parkin at the mitochondria.
Figure 3: Expression of Fbxo7 rescues parkin mutant phenotypes.
Figure 4: PINK1 interacts directly with the amino terminus of Fbxo7.
Figure 5: Functional interaction of Fbxo7 with PINK1.
Figure 6: Fbxo7 promotes Mfn1 ubiquitination and restores Mfn levels and mitochondrial morphology in Parkin- but not PINK1-deficient cells.
Figure 7: Fbxo7 is important for mitophagy.

References

  1. 1

    Narendra, D., Tanaka, A., Suen, D.F. & Youle, R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy. J. Cell Biol. 183, 795–803 (2008).

  2. 2

    Vives-Bauza, C. et al. PINK1-dependent recruitment of Parkin to mitochondria in mitophagy. Proc. Natl. Acad. Sci. USA 107, 378–383 (2010).

  3. 3

    Narendra, D.P. et al. PINK1 is selectively stabilized on impaired mitochondria to activate Parkin. PLoS Biol. 8, e1000298 (2010).

  4. 4

    Geisler, S. et al. PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/SQSTM1. Nat. Cell Biol. 12, 119–131 (2010).

  5. 5

    Deas, E., Wood, N.W. & Plun-Favreau, H. Mitophagy and Parkinson's disease: the PINK1-parkin link. Biochim. Biophys. Acta 1813, 623–633 (2011).

  6. 6

    Jin, S.M. et al. Mitochondrial membrane potential regulates PINK1 import and proteolytic destabilization by PARL. J. Cell Biol. 191, 933–942 (2010).

  7. 7

    Whitworth, A.J. et al. Rhomboid-7 and HtrA2/Omi act in a common pathway with the Parkinson's disease factors Pink1 and Parkin. Dis. Model. Mech. 1, 168–174 discussion 173 (2008).

  8. 8

    Ziviani, E., Tao, R.N. & Whitworth, A.J. Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin. Proc. Natl. Acad. Sci. USA 107, 5018–5023 (2010).

  9. 9

    Poole, A.C., Thomas, R.E., Yu, S., Vincow, E.S. & Pallanck, L. The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway. PLoS ONE 5, e10054 (2010).

  10. 10

    Gegg, M.E. et al. Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy. Hum. Mol. Genet. 19, 4861–4870 (2010).

  11. 11

    Tanaka, A. Parkin-mediated selective mitochondrial autophagy, mitophagy: Parkin purges damaged organelles from the vital mitochondrial network. FEBS Lett. 584, 1386–1392 (2010).

  12. 12

    Rakovic, A. et al. Mutations in PINK1 and Parkin impair ubiquitination of Mitofusins in human fibroblasts. PLoS ONE 6, e16746 (2011).

  13. 13

    Sarraf, S.A. et al. Landscape of the PARKIN-dependent ubiquitylome in response to mitochondrial depolarization. Nature 496, 372–376 (2013).

  14. 14

    Tanaka, A. et al. Proteasome and p97 mediate mitophagy and degradation of mitofusins induced by Parkin. J. Cell Biol. 191, 1367–1380 (2010).

  15. 15

    Shojaee, S. et al. Genome-wide linkage analysis of a Parkinsonian-pyramidal syndrome pedigree by 500 K SNP arrays. Am. J. Hum. Genet. 82, 1375–1384 (2008).

  16. 16

    Di Fonzo, A. et al. FBXO7 mutations cause autosomal recessive, early-onset parkinsonian-pyramidal syndrome. Neurology 72, 240–245 (2009).

  17. 17

    Paisán-Ruiz, C. et al. Early-onset L-dopa-responsive parkinsonism with pyramidal signs due to ATP13A2, PLA2G6, FBXO7 and spatacsin mutations. Mov. Disord. 25, 1791–1800 (2010).

  18. 18

    Skowyra, D., Craig, K.L., Tyers, M., Elledge, S.J. & Harper, J.W. F-box proteins are receptors that recruit phosphorylated substrates to the SCF ubiquitin-ligase complex. Cell 91, 209–219 (1997).

  19. 19

    Hsu, J.M., Lee, Y.C., Yu, C.T. & Huang, C.Y. Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region. J. Biol. Chem. 279, 32592–32602 (2004).

  20. 20

    Laman, H. et al. Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6. EMBO J. 24, 3104–3116 (2005).

  21. 21

    Kuiken, H.J. et al. Identification of F-box only protein 7 as a negative regulator of NF-κB signalling. J. Cell Mol. Med. 16, 2140–2149 (2012).

  22. 22

    Kirk, R. et al. Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor. J. Biol. Chem. 283, 22325–22335 (2008).

  23. 23

    Chang, Y.F., Cheng, C.M., Chang, L.K., Jong, Y.J. & Yuo, C.Y. The F-box protein Fbxo7 interacts with human inhibitor of apoptosis protein cIAP1 and promotes cIAP1 ubiquitination. Biochem. Biophys. Res. Commun. 342, 1022–1026 (2006).

  24. 24

    Greene, J.C. et al. Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants. Proc. Natl. Acad. Sci. USA 100, 4078–4083 (2003).

  25. 25

    Whitworth, A.J. et al. Increased glutathione S-transferase activity rescues dopaminergic neuron loss in a Drosophila model of Parkinson's disease. Proc. Natl. Acad. Sci. USA 102, 8024–8029 (2005).

  26. 26

    Park, J. et al. Mitochondrial dysfunction in Drosophila PINK1 mutants is complemented by parkin. Nature 441, 1157–1161 (2006).

  27. 27

    Clark, I.E. et al. Drosophila pink1 is required for mitochondrial function and interacts genetically with parkin. Nature 441, 1162–1166 (2006).

  28. 28

    Bader, M. et al. A conserved F box regulatory complex controls proteasome activity in Drosophila. Cell 145, 371–382 (2011).

  29. 29

    Bader, M., Arama, E. & Steller, H. A novel F-box protein is required for caspase activation during cellular remodeling in Drosophila. Development 137, 1679–1688 (2010).

  30. 30

    Yang, Y. et al. Mitochondrial pathology and muscle and dopaminergic neuron degeneration caused by inactivation of Drosophila Pink1 is rescued by Parkin. Proc. Natl. Acad. Sci. USA 103, 10793–10798 (2006).

  31. 31

    Exner, N. et al. Loss-of-function of human PINK1 results in mitochondrial pathology and can be rescued by parkin. J. Neurosci. 27, 12413–12418 (2007).

  32. 32

    Song, S. et al. Characterization of PINK1 (PTEN-induced Putative Kinase 1) mutations associated with Parkinson disease in mammalian cells and Drosophila. J. Biol. Chem. 288, 5660–5672 (2013).

  33. 33

    Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy 4, 151–175 (2008).

  34. 34

    Ding, W.X. et al. Nix is critical to two distinct phases of mitophagy, reactive oxygen species-mediated autophagy induction and Parkin-ubiquitin-p62-mediated mitochondrial priming. J. Biol. Chem. 285, 27879–27890 (2010).

  35. 35

    Lee, S.J. et al. A functional role for the p62–ERK1 axis in the control of energy homeostasis and adipogenesis. EMBO Rep. 11, 226–232 (2010).

  36. 36

    Klionsky, D.J. et al. Guidelines for the use and interpretation of assays for monitoring autophagy. Autophagy 8, 445–544 (2012).

  37. 37

    Nelson, D.E. & Laman, H. A competitive binding mechanism between SKP1 and exportin 1 (CRM1) controls the localization of a subset of F-box proteins. J. Biol. Chem. 286, 19804–19815 (2011).

  38. 38

    Kondapalli, C. et al. PINK1 is activated by mitochondrial membrane potential depolarization and stimulates Parkin E3 ligase activity by phosphorylating serine 65. Open Biol. 2, 120080 (2012).

  39. 39

    Nakai, K. & Kanehisa, M. A knowledge base for predicting protein localization sites in eukaryotic cells. Genomics 14, 897–911 (1992).

  40. 40

    Claros, M.G. & Vincens, P. Computational method to predict mitochondrially imported proteins and their targeting sequences. Eur. J. Biochem. 241, 779–786 (1996).

  41. 41

    Small, I., Peeters, N., Legeai, F. & Lurin, C. Predotar: a tool for rapidly screening proteomes for N-terminal targeting sequences. Proteomics 4, 1581–1590 (2004).

  42. 42

    Dickins, R.A. et al. Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat. Genet. 37, 1289–1295 (2005).

  43. 43

    Ardley, H.C. et al. Inhibition of proteasomal activity causes inclusion formation in neuronal and non-neuronal cells overexpressing Parkin. Mol. Biol. Cell 14, 4541–4556 (2003).

  44. 44

    Plun-Favreau, H. et al. The mitochondrial protease HtrA2 is regulated by Parkinson's disease–associated kinase PINK1. Nat. Cell Biol. 9, 1243–1252 (2007).

  45. 45

    Samali, A., Cai, J., Zhivotovsky, B., Jones, D.P. & Orrenius, S. Presence of a pre-apoptotic complex of pro-caspase-3, Hsp60 and Hsp10 in the mitochondrial fraction of Jurkat cells. EMBO J. 18, 2040–2048 (1999).

  46. 46

    Tain, L.S. et al. Drosophila HtrA2 is dispensable for apoptosis but acts downstream of PINK1 independently from Parkin. Cell Death Differ. 16, 1118–1125 (2009).

  47. 47

    Sullivan, W., Ashburner, M. & Hawley, R.S. Drosophila Protocols Ch.13, 240–241 (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, USA, 2000).

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Acknowledgements

We would like to thank M. Mehrabian and E. Elahi for obtaining patient biopsies, H. Ardley (Leeds Institute of Molecular Medicine) for donating the Flag-Parkin construct and cell line, K. Holmström and A. Isaacs for discussions and M. Gegg for technical advice. We thank C. Hill for technical assistance within the University of Sheffield Electron Microscopy Facility. We thank the H. Steller (Rockefeller University) laboratory for kind provision of the ntc mutant lines. The work was funded by a Wellcome/Medical Research Council (MRC) Parkinson's Disease Consortium grant to the University College London Institute of Neurology, the University of Sheffield and the MRC Protein Phosphorylation Unit at the University of Dundee (grant number WT089698), an MRC Career Development Award (G0700183; H.P.-F.), the MEFOPA project funded through the European Union FP7 research program (A.J.W.), and an ERC Starting Grant (no. 309742; A.J.W.). This research was supported by the UK National Institute for Health Research University College London Hospitals Biomedical Research Centre. V.S.B. is funded by the MRC Doctoral Training Account. D.E.N. is funded by the Biotechnology and Biological Sciences Research Council (BB/F012764/1; BB/J007846/1). The Wellcome Trust is acknowledged for support of the Sheffield Light Microscopy Facility (GR077544AIA). The MRC Centre for Developmental and Biomedical Genetics is supported by grant G070091.

Author information

H.P.-F., H.L. and A.J.W. conceived the study. V.S.B., D.E.N., A.S.-M., A.J.W., H.L. and H.P.-F. designed and performed experiments. M.D.-C., R.M.I., J.H.P. and S.J.R. performed experiments. H.H., P.A.L. and S.W. obtained and cultured the patient fibroblasts. P.A.L., A.Y.A., J.H. and N.W.W. contributed to the design of the study. V.S.B., D.E.N., A.S.-M., H.P.-F., A.J.W. and H.L. wrote the manuscript.

Correspondence to Alexander J Whitworth or Heike Laman or Helene Plun-Favreau.

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Burchell, V., Nelson, D., Sanchez-Martinez, A. et al. The Parkinson's disease–linked proteins Fbxo7 and Parkin interact to mediate mitophagy. Nat Neurosci 16, 1257–1265 (2013). https://doi.org/10.1038/nn.3489

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