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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.

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.

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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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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.

Corresponding author

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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