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Mitochondrial protein translocation-associated degradation

Abstract

Mitochondrial biogenesis and functions depend on the import of precursor proteins via the ‘translocase of the outer membrane’ (TOM complex). Defects in protein import lead to an accumulation of mitochondrial precursor proteins that induces a range of cellular stress responses. However, constitutive quality-control mechanisms that clear trapped precursor proteins from the TOM channel under non-stress conditions have remained unknown. Here we report that in Saccharomyces cerevisiae Ubx2, which functions in endoplasmic reticulum-associated degradation, is crucial for this quality-control process. A pool of Ubx2 binds to the TOM complex to recruit the AAA ATPase Cdc48 for removal of arrested precursor proteins from the TOM channel. This mitochondrial protein translocation-associated degradation (mitoTAD) pathway continuously monitors the TOM complex under non-stress conditions to prevent clogging of the TOM channel with precursor proteins. The mitoTAD pathway ensures that mitochondria maintain their full protein-import capacity, and protects cells against proteotoxic stress induced by impaired transport of proteins into mitochondria.

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Fig. 1: Mitochondria-localized Ubx2 interacts with the TOM complex.
Fig. 2: Tom70 and Mim1 promote import of Ubx2 into mitochondria.
Fig. 3: Ubx2 recruits Cdc48 to the TOM complex in mitochondrial quality control.
Fig. 4: Ubx2 promotes clearance of arrested precursor proteins from the TOM channel.

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Data availability

All data of this study are available in the Article, its Extended Data and Supplementary Information. The uncropped blots and gels are provided in Supplementary Fig. 1.

References

  1. Pfanner, N., Warscheid, B. & Wiedemann, N. Mitochondrial proteins: from biogenesis to functional networks. Nat. Rev. Mol. Cell Biol. 20, 267–284 (2019).

    Article  CAS  Google Scholar 

  2. Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007).

    Article  CAS  Google Scholar 

  3. Endo, T. & Yamano, K. Multiple pathways for mitochondrial protein traffic. Biol. Chem. 390, 723–730 (2009).

    Article  CAS  Google Scholar 

  4. Boos, F. et al. Mitochondrial protein-induced stress triggers a global adaptive transcriptional programme. Nat. Cell Biol. 21, 442–451 (2019).

    Article  CAS  Google Scholar 

  5. Pickles, S., Vigié, P. & Youle, R. J. Mitophagy and quality control mechanisms in mitochondrial maintenance. Curr. Biol. 28, R170–R185 (2018).

    Article  CAS  Google Scholar 

  6. Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing. Nat. Rev. Mol. Cell Biol. 19, 109–120 (2018).

    Article  CAS  Google Scholar 

  7. Wrobel, L. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488 (2015).

    Article  ADS  CAS  Google Scholar 

  8. Wang, X. & Chen, X. J. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484 (2015).

    Article  ADS  CAS  Google Scholar 

  9. Weidberg, H. & Amon, A. MitoCPR – a surveillance pathway that protects mitochondria in response to protein import stress. Science 360, eaan4146 (2018).

    Article  Google Scholar 

  10. Bausewein, T. et al. Cryo-EM structure of the TOM core complex from Neurospora crassa. Cell 170, 693–700.e7 (2017).

    Article  CAS  Google Scholar 

  11. Neuber, O., Jarosch, E., Volkwein, C., Walter, J. & Sommer, T. Ubx2 links the Cdc48 complex to ER-associated protein degradation. Nat. Cell Biol. 7, 993–998 (2005).

    Article  CAS  Google Scholar 

  12. Schuberth, C. & Buchberger, A. Membrane-bound Ubx2 recruits Cdc48 to ubiquitin ligases and their substrates to ensure efficient ER-associated protein degradation. Nat. Cell Biol. 7, 999–1006 (2005).

    Article  CAS  Google Scholar 

  13. Zahedi, R. P. et al. Proteomic analysis of the yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins. Mol. Biol. Cell 17, 1436–1450 (2006).

    Article  CAS  Google Scholar 

  14. Wang, C.-W. & Lee, S. C. The ubiquitin-like (UBX)-domain-containing protein Ubx2/Ubxd8 regulates lipid droplet homeostasis. J. Cell Sci. 125, 2930–2939 (2012).

    Article  CAS  Google Scholar 

  15. Becker, T. et al. The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins. J. Cell Biol. 194, 387–395 (2011).

    Article  CAS  Google Scholar 

  16. Papić, D., Krumpe, K., Dukanovic, J., Dimmer, K. S. & Rapaport, D. Multispan mitochondrial outer membrane protein Ugo1 follows a unique Mim1-dependent import pathway. J. Cell Biol. 194, 397–405 (2011).

    Article  Google Scholar 

  17. Backes, S. et al. Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences. J. Cell Biol. 217, 1369–1382 (2018).

    Article  CAS  Google Scholar 

  18. Bodnar, N. O. et al. Structure of the Cdc48 ATPase with its ubiquitin-binding cofactor Ufd1–Npl4. Nat. Struct. Mol. Biol. 25, 616–622 (2018).

    Article  CAS  Google Scholar 

  19. Heo, J.-M. et al. A stress-responsive system for mitochondrial protein degradation. Mol. Cell 40, 465–480 (2010).

    Article  CAS  Google Scholar 

  20. Wu, X., Li, L. & Jiang, H. Doa1 targets ubiquitinated substrates for mitochondria-associated degradation. J. Cell Biol. 213, 49–63 (2016).

    Article  CAS  Google Scholar 

  21. Okreglak, V. & Walter, P. The conserved AAA-ATPase Msp1 confers organelle specificity to tail-anchored proteins. Proc. Natl Acad. Sci. USA 111, 8019–8024 (2014).

    Article  ADS  CAS  Google Scholar 

  22. Chen, Y. C. et al. Msp1/ATAD1 maintains mitochondrial function by facilitating the degradation of mislocalized tail-anchored proteins. EMBO J. 33, 1548–1564 (2014).

    Article  CAS  Google Scholar 

  23. Izawa, T., Park, S.-H., Zhao, L., Hartl, F. U. & Neupert, W. Cytosolic protein Vms1 links ribosome quality control to mitochondrial and cellular homeostasis. Cell 171, 890–903.e18 (2017).

    Article  CAS  Google Scholar 

  24. Tran, J. R. & Brodsky, J. L. The Cdc48–Vms1 complex maintains 26S proteasome architecture. Biochem. J. 458, 459–467 (2014).

    Article  CAS  Google Scholar 

  25. Schuberth, C., Richly, H., Rumpf, S. & Buchberger, A. Shp1 and Ubx2 are adaptors of Cdc48 involved in ubiquitin-dependent protein degradation. EMBO Rep. 5, 818–824 (2004).

    Article  CAS  Google Scholar 

  26. van der Laan, M. et al. Pam17 is required for architecture and translocation activity of the mitochondrial protein import motor. Mol. Cell. Biol. 25, 7449–7458 (2005).

    Article  Google Scholar 

  27. Bömer, U. et al. The sorting route of cytochrome b 2 branches from the general mitochondrial import pathway at the preprotein translocase of the inner membrane. J. Biol. Chem. 272, 30439–30446 (1997).

    Article  Google Scholar 

  28. Verma, R. et al. Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes. Nature 557, 446–451 (2018).

    Article  ADS  CAS  Google Scholar 

  29. Zurita Rendón, O. et al. Vms1p is a release factor for the ribosome-associated quality control complex. Nat. Commun. 9, 2197 (2018).

    Article  ADS  Google Scholar 

  30. Ast, T., Michaelis, S. & Schuldiner, M. The protease Ste24 clears clogged translocons. Cell 164, 103–114 (2016).

    Article  CAS  Google Scholar 

  31. Wenz, L. S. et al. Sam37 is crucial for formation of the mitochondrial TOM–SAM supercomplex, thereby promoting β-barrel biogenesis. J. Cell Biol. 210, 1047–1054 (2015).

    Article  CAS  Google Scholar 

  32. Ellenrieder, L. et al. Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10. Nat. Commun. 7, 13021 (2016).

    Article  ADS  CAS  Google Scholar 

  33. Longtine, M. S. et al. Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14, 953–961 (1998).

    Article  CAS  Google Scholar 

  34. Morgenstern, M. et al. Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Reports 19, 2836–2852 (2017).

    Article  CAS  Google Scholar 

  35. Janke, C. et al. A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes. Yeast 21, 947–962 (2004).

    Article  CAS  Google Scholar 

  36. Kushnirov, V. V. Rapid and reliable protein extraction from yeast. Yeast 16, 857–860 (2000).

    Article  CAS  Google Scholar 

  37. Opaliński, Ł. et al. Recruitment of cytosolic J-proteins by TOM receptors promotes mitochondrial protein biogenesis. Cell Reports 25, 2036–2043.e5 (2018).

    Article  Google Scholar 

  38. Schuler, M. H., Di Bartolomeo, F., Mårtensson, C. U., Daum, G. & Becker, T. Phosphatidylcholine affects inner membrane protein translocases of mitochondria. J. Biol. Chem. 291, 18718–18729 (2016).

    Article  CAS  Google Scholar 

  39. Wittig, I., Braun, H. P. & Schägger, H. Blue native PAGE. Nat. Protocols 1, 418–428 (2006).

    Article  CAS  Google Scholar 

  40. Haan, C. & Behrmann, I. A cost effective non-commercial ECL-solution for western blot detections yielding strong signals and low background. J. Immunol. Methods 318, 11–19 (2007).

    Article  CAS  Google Scholar 

  41. Emanuelsson, O., Brunak, S., von Heijne, G. & Nielsen, H. Locating proteins in the cell using TargetP, SignalP and related tools. Nat. Protocols 2, 953–971 (2007).

    Article  CAS  Google Scholar 

  42. Neal, S. et al. The Dfm1 derlin is required for ERAD retrotranslocation of integral membrane proteins. Mol. Cell 69, 306–320.e4 (2018).

    Article  CAS  Google Scholar 

  43. Chacinska, A. et al. Mitochondrial translocation contact sites: separation of dynamic and stabilizing elements in formation of a TOM–TIM-preprotein supercomplex. EMBO J. 22, 5370–5381 (2003).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank N. Pfanner and J. Herrmann for comments on the manuscript, B. Guiard, H. Meyer, B. Kravic, T. Sommer, E. Jarosch, N. Wiedemann, L.-S. Wenz and N. Gebert for discussions and materials. The work was supported by grants of the Deutsche Forschungsgemeinschaft (BE 4679/2-2), Research Training Group 278002225/RTG 2202 and Germany’s Excellence Strategy (CIBSS- EXC-2189 - Project ID 390939984 and BIOSS-EXC-294) and by the Joachim Hertz Stiftung (to F.B.). Work included in this study has also been performed in partial fulfilment of the requirements for the doctoral thesis of C.U.M., C.P. and K.N.D. at the University of Freiburg.

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Authors and Affiliations

Authors

Contributions

C.U.M., C.P., J.S., A.F. and N.Z. performed functional studies of Ubx2 and analysed data together with T.B. C.U.M., L.E., K.N.D. and C.P. analysed cellular localization of Ubx2. F.B. analysed the internal matrix-targeting signal of Ubx2. C.U.M. and S.O. analysed partner proteins of the TOM complex together with B.W. and T.B. T.B. designed and supervised the project. C.U.M., C.P., J.S. and T.B. prepared the figures. T.B. and C.U.M. wrote the manuscript. All authors discussed results from the experiments and commented on the manuscript.

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Correspondence to Thomas Becker.

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Extended data figures and tables

Extended Data Fig. 1 Partner proteins and stability of Ubx2.

a, c, Affinity purifications of lysed wild-type, HA–Tom5, HA–Tom6, HA–Tom7 (a) and Por1–HA (c) mitochondria. The data are representative of three independent experiments. Load 4%; elution 100%. b, Co-immunoprecipitation with Tom22 antiserum of lysed wild-type mitochondria. The data are representative of two independent experiments. Load 2%; elution 100%. d, Wild-type and ubx2∆ cell extracts and mitochondria were analysed by SDS–PAGE, and immunodetection with Ubx2-specific antiserum. The data are representative of two independent experiments. Asterisk marks an unspecific band recognized by the Ubx2 antiserum. e, Ubx2 stability in intact cells or ruptured cells incubated for the indicated time points. The data are representative of three independent experiments. f, Affinity purifications of lysed wild-type, Hrd1–HA, Ubx2–HA and Doa10ProtA cell extracts. The data are representative of three (Hrd1–HA) or two (Doa10ProtA) independent experiments. Load 1%; elution 100%.

Extended Data Fig. 2 Localization of Ubx2.

a, Fluorescence microscopy of cells expressing Ubx2–GFP. The green fluorescence of Ubx2–GFP, the red fluorescence of MitoTracker and the merged image are shown. Cell borders are marked in blue. The data are representative of three independent experiments. b, Wild-type mitochondria and mitoplasts (+ swelling) were treated with or without proteinase K as indicated. The data are representative of two independent experiments.

Extended Data Fig. 3 Analysis of the import pathway of Ubx2 into mitochondria.

a, Cellular fractionation of wild-type, tom70∆ and mim1∆ cells. The data are representative for two (tom70∆) or three (mim1∆) independent experiments. b, Ubx2 levels in microsomal fraction of the indicated yeast strains. The data are representative of two independent experiments. c, [35S]Ubx2 was imported into wild-type and tom22∆ mitochondria and analysed by BN–PAGE and autoradiography. The data are representative of four independent experiments. d, Import of the indicated [35S]Ubx2 constructs into wild-type mitochondria on BN–PAGE. The data are representative of five independent experiments. TMD, transmembrane domain. Quantification of the Ubx2–TOM assembly. The assembly of Ubx2 wild type was set to 100% (control). Mean ± s.e.m. (n = 5).

Extended Data Fig. 4 Analysis of the association of Cdc48 with mitochondria.

a, Crude and purified wild-type and ubx2∆ mitochondria were analysed by SDS–PAGE and immunodetection with the indicated antisera. The data are representative of four independent experiments. b, Cdc48 levels in purified mitochondria from mutant strains of Cdc48-binding proteins19,20,25,42 were analysed by SDS–PAGE and immunodetection. The data are representative of five independent experiments. c, Affinity purifications of Tom40–HA, Tom40–HA Ubx2 ∆UBA and Tom40–HA Ubx2∆UBX cell extracts. The data are representative of three independent experiments. Load 2% (0.1% for Cdc48); elution 100%.

Extended Data Fig. 5 Characterization of ubx2∆ and vms1∆ mutant mitochondria.

a, Membrane potential of the wild-type, ubx2∆, vms1∆ and ubx2∆ vms1∆ mitochondria. Mean values of three independent measurements are shown. b, Respiratory chain complexes in wild-type, ubx2∆, vms1∆ and ubx2∆ vms1∆ mitochondria on BN–PAGE. The data are representative of two independent experiments. c, In-gel activity stain of complex IV in wild-type, ubx2∆, vms1∆ and ubx2∆ vms1∆ mitochondria on BN–PAGE. The data are representative of two independent experiments. III, cytochrome bc1 complex; IV, cytochrome c oxidase; V, ATP synthase.

Extended Data Fig. 6 Characterization of the role of Ubx2 in CCCP-induced precursor accumulation.

a, b, Wild-type, ubx2∆, ubx2∆ + UBX2 (a) and shp1∆ (b) cells were either left untreated or treated with CCCP for the indicated time periods. Cell extracts were analysed by SDS–PAGE and immunodetection. The data are representative of three independent experiments.

Extended Data Fig. 7 Analysis of the accumulation of the Mdj1 precursor.

a, Left, Mdj1 precursor in post-nuclear supernatant and mitochondrial fraction. The data are representative of two independent experiments. Right, degradation of the Mdj1 precursors by proteinase K (prot. K) in pam17∆ ubx2∆ cell extracts. The data are representative of three independent experiments. b, Mdj1 precursor levels in cell extracts from wild-type, pam17∆, pam17∆ ubx2∆ and pam17∆ ubx2∆ + UBX2 strains expressing Ubx2. The data are representative of three independent experiments. ce, Degradation kinetics of the Mdj1 precursor were analysed in cell extracts from the indicated mutant strains. The data are representative of three (c, d) or two (e) independent experiments.

Extended Data Fig, 8 Characterization of b2-DHFR–HB-construct overexpressing mutants.

a, Degradation of the b2–DHFR–HB precursor by proteinase K in ubx2∆ mitochondria. The data are representative of seven independent experiments. b, The TOM–TIM23–preprotein supercomplex43 on BN–PAGE in cell extracts from wild-type and ubx2∆ strains that express b2∆–DHFR–HB. The data are representative of three independent experiments. Quantification of the TOM–TIM23–preprotein supercomplex. The amount of the TOM–TIM23–preprotein supercomplex in wild-type cells was set to 100% (control). Mean ± s.e.m. (n = 3). c, Binding of Ubx2 to the TOM complex on BN–PAGE in wild-type and ubx2∆ mitochondria from strains that express b2–DHFR–HB or b2∆–DHFR–HB. The data are representative of three (b2–DHFR–HB) or five (b2∆–DHFR–HB) independent experiments. Quantification of the Ubx2–TOM complex in cell extracts. The amount of Ubx2–TOM complex in wild-type cells was set to 100% (control). Mean ± s.e.m. (n = 3 for b2–DHFR–HB; n = 5 for b2∆–DHFR–HB).

Supplementary information

Supplementary Figure 1

This file contains the uncropped versions of all the blots and gels in this study.

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Mårtensson, C.U., Priesnitz, C., Song, J. et al. Mitochondrial protein translocation-associated degradation. Nature 569, 679–683 (2019). https://doi.org/10.1038/s41586-019-1227-y

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