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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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.
Pfanner, N., Warscheid, B. & Wiedemann, N. Mitochondrial proteins: from biogenesis to functional networks. Nat. Rev. Mol. Cell Biol. 20, 267–284 (2019).
Neupert, W. & Herrmann, J. M. Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007).
Endo, T. & Yamano, K. Multiple pathways for mitochondrial protein traffic. Biol. Chem. 390, 723–730 (2009).
Boos, F. et al. Mitochondrial protein-induced stress triggers a global adaptive transcriptional programme. Nat. Cell Biol. 21, 442–451 (2019).
Pickles, S., Vigié, P. & Youle, R. J. Mitophagy and quality control mechanisms in mitochondrial maintenance. Curr. Biol. 28, R170–R185 (2018).
Shpilka, T. & Haynes, C. M. The mitochondrial UPR: mechanisms, physiological functions and implications in ageing. Nat. Rev. Mol. Cell Biol. 19, 109–120 (2018).
Wrobel, L. et al. Mistargeted mitochondrial proteins activate a proteostatic response in the cytosol. Nature 524, 485–488 (2015).
Wang, X. & Chen, X. J. A cytosolic network suppressing mitochondria-mediated proteostatic stress and cell death. Nature 524, 481–484 (2015).
Weidberg, H. & Amon, A. MitoCPR – a surveillance pathway that protects mitochondria in response to protein import stress. Science 360, eaan4146 (2018).
Bausewein, T. et al. Cryo-EM structure of the TOM core complex from Neurospora crassa. Cell 170, 693–700.e7 (2017).
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).
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).
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).
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).
Becker, T. et al. The mitochondrial import protein Mim1 promotes biogenesis of multispanning outer membrane proteins. J. Cell Biol. 194, 387–395 (2011).
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).
Backes, S. et al. Tom70 enhances mitochondrial preprotein import efficiency by binding to internal targeting sequences. J. Cell Biol. 217, 1369–1382 (2018).
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).
Heo, J.-M. et al. A stress-responsive system for mitochondrial protein degradation. Mol. Cell 40, 465–480 (2010).
Wu, X., Li, L. & Jiang, H. Doa1 targets ubiquitinated substrates for mitochondria-associated degradation. J. Cell Biol. 213, 49–63 (2016).
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).
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).
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).
Tran, J. R. & Brodsky, J. L. The Cdc48–Vms1 complex maintains 26S proteasome architecture. Biochem. J. 458, 459–467 (2014).
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).
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).
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).
Verma, R. et al. Vms1 and ANKZF1 peptidyl-tRNA hydrolases release nascent chains from stalled ribosomes. Nature 557, 446–451 (2018).
Zurita Rendón, O. et al. Vms1p is a release factor for the ribosome-associated quality control complex. Nat. Commun. 9, 2197 (2018).
Ast, T., Michaelis, S. & Schuldiner, M. The protease Ste24 clears clogged translocons. Cell 164, 103–114 (2016).
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).
Ellenrieder, L. et al. Separating mitochondrial protein assembly and endoplasmic reticulum tethering by selective coupling of Mdm10. Nat. Commun. 7, 13021 (2016).
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).
Morgenstern, M. et al. Definition of a high-confidence mitochondrial proteome at quantitative scale. Cell Reports 19, 2836–2852 (2017).
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).
Kushnirov, V. V. Rapid and reliable protein extraction from yeast. Yeast 16, 857–860 (2000).
Opaliński, Ł. et al. Recruitment of cytosolic J-proteins by TOM receptors promotes mitochondrial protein biogenesis. Cell Reports 25, 2036–2043.e5 (2018).
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).
Wittig, I., Braun, H. P. & Schägger, H. Blue native PAGE. Nat. Protocols 1, 418–428 (2006).
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).
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).
Neal, S. et al. The Dfm1 derlin is required for ERAD retrotranslocation of integral membrane proteins. Mol. Cell 69, 306–320.e4 (2018).
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).
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.
The authors declare no competing interests.
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Extended data figures and tables
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%.
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.
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).
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%.
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.
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.
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. c–e, 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.
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).
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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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