Abstract

Most of the mitochondrial proteome originates from nuclear genes and is transported into the mitochondria after synthesis in the cytosol. Complex machineries which maintain the specificity of protein import and sorting include the TIM23 translocase responsible for the transfer of precursor proteins into the matrix, and the mitochondrial intermembrane space import and assembly (MIA) machinery required for the biogenesis of intermembrane space proteins. Dysfunction of mitochondrial protein sorting pathways results in diminishing specific substrate proteins, followed by systemic pathology of the organelle and organismal death1,2,3,4. The cellular responses caused by accumulation of mitochondrial precursor proteins in the cytosol are mainly unknown. Here we present a comprehensive picture of the changes in the cellular transcriptome and proteome in response to a mitochondrial import defect and precursor over-accumulation stress. Pathways were identified that protect the cell against mitochondrial biogenesis defects by inhibiting protein synthesis and by activation of the proteasome, a major machine for cellular protein clearance. Proteasomal activity is modulated in proportion to the quantity of mislocalized mitochondrial precursor proteins in the cytosol. We propose that this type of unfolded protein response activated by mistargeting of proteins (UPRam) is beneficial for the cells. UPRam provides a means for buffering the consequences of physiological slowdown in mitochondrial protein import and for counteracting pathologies that are caused or contributed by mitochondrial dysfunction.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Accessions

Primary accessions

ArrayExpress

Data deposits

RNA-seq data have been submitted to the ArrayExpress database under accession number E-MTAB-3588. The mass spectrometry data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD001495.

References

  1. 1.

    , , , & Importing mitochondrial proteins: machineries and mechanisms. Cell 138, 628–644 (2009)

  2. 2.

    , & Structural insight into the mitochondrial protein import system. Biochim. Biophys. Acta 1808, 955–970 (2011)

  3. 3.

    & Translocation of proteins into mitochondria. Annu. Rev. Biochem. 76, 723–749 (2007)

  4. 4.

    , & On the mechanism of preprotein import by the mitochondrial presequence translocase. Biochim. Biophys. Acta 1803, 732–739 (2010)

  5. 5.

    et al. Essential role of Mia40 in import and assembly of mitochondrial intermembrane space proteins. EMBO J. 23, 3735–3746 (2004)

  6. 6.

    et al. Mitochondrial protein import: precursor oxidation in a ternary complex with disulfide carrier and sulfhydryl oxidase. J. Cell Biol. 183, 195–202 (2008)

  7. 7.

    et al. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol. Cell. Proteomics 1, 376–386 (2002)

  8. 8.

    , , & The ubiquitin-proteasome system regulates mitochondrial intermembrane space proteins. Mol. Cell. Biol. 33, 2136–2148 (2013)

  9. 9.

    Cleaning up: ER-associated degradation to the rescue. Cell 151, 1163–1167 (2012)

  10. 10.

    Intracellular protein degradation: from a vague idea through the lysosome and the ubiquitin-proteasome system and onto human diseases and drug targeting. Biochim. Biophys. Acta 1824, 3–13 (2012)

  11. 11.

    Recognition and processing of ubiquitin-protein conjugates by the proteasome. Annu. Rev. Biochem. 78, 477–513 (2009)

  12. 12.

    Protein degradation and protection against misfolded or damaged proteins. Nature 426, 895–899 (2003)

  13. 13.

    et al. Retro-translocation of mitochondrial intermembrane space proteins. Proc. Natl Acad. Sci. USA 112, 7713–7718 (2015)

  14. 14.

    , , & A multimeric assembly factor controls the formation of alternative 20S proteasomes. Nature Struct. Mol. Biol. 15, 237–244 (2008)

  15. 15.

    et al. 20S proteasome assembly is orchestrated by two distinct pairs of chaperones in yeast and in mammals. Mol. Cell 27, 660–674 (2007)

  16. 16.

    et al. Cooperation of multiple chaperones required for the assembly of mammalian 20S proteasomes. Mol. Cell 24, 977–984 (2006)

  17. 17.

    , , , & Ump1p is required for proper maturation of the 20S proteasome and becomes its substrate upon completion of the assembly. Cell 92, 489–499 (1998)

  18. 18.

    et al. An inducible chaperone adapts proteasome assembly to stress. Mol. Cell 55, 566–577 (2014)

  19. 19.

    et al. Rapid degradation of a large fraction of newly synthesized proteins by proteasomes. Nature 404, 770–774 (2000)

  20. 20.

    et al. Protein import and oxidative folding in the mitochondrial intermembrane space of intact mammalian cells. Mol. Biol. Cell 24, 2160–2170 (2013)

  21. 21.

    et al. Pam16 has an essential role in the mitochondrial protein import motor. Nature Struct. Mol. Biol. 11, 226–233 (2004)

  22. 22.

    et al. Mitochondrial presequence translocase: switching between TOM tethering and motor recruitment involves Tim21 and Tim17. Cell 120, 817–829 (2005)

  23. 23.

    , , & Global impairment of the ubiquitin–proteasome system by nuclear or cytoplasmic protein aggregates precedes inclusion body formation. Mol. Cell 17, 351–365 (2005)

  24. 24.

    , & Spatial sequestration of misfolded proteins by a dynamic chaperone pathway enhances cellular fitness during stress. Nature Cell Biol. 15, 1231–1243 (2013)

  25. 25.

    & The mitochondrial UPR — protecting organelle protein homeostasis. J. Cell Sci. 123, 3849–3855 (2010)

  26. 26.

    et al. A mitochondrial specific stress response in mammalian cells. EMBO J. 21, 4411–4419 (2002)

  27. 27.

    , , , & Mitochondrial import efficiency of ATFS-1 regulates mitochondrial UPR activation. Science 337, 587–590 (2012)

  28. 28.

    et al. RPN-6 determines C. elegans longevity under proteotoxic stress conditions. Nature 489, 263–268 (2012)

  29. 29.

    , & The cell-non-autonomous nature of electron transport chain-mediated longevity. Cell 144, 79–91 (2011)

  30. 30.

    et al. Mitonuclear protein imbalance as a conserved longevity mechanism. Nature 497, 451–457 (2013)

  31. 31.

    et al. A J-protein is an essential subunit of the presequence translocase-associated protein import motor of mitochondria. J. Cell Biol. 163, 707–713 (2003)

  32. 32.

    et al. In vivo evidence for cooperation of Mia40 and Erv1 in the oxidation of mitochondrial proteins. Mol. Biol. Cell 23, 3957–3969 (2012)

  33. 33.

    et al. Uniform nomenclature for the mitochondrial contact site and cristae organizing system. J. Cell Biol. 204, 1083–1086 (2014)

  34. 34.

    & Point mutations destabilizing a precursor protein enhance its post-translational import into mitochondria. EMBO J. 7, 1147–1151 (1988)

  35. 35.

    et al. Role of the AAA protease Yme1 in folding of proteins in the intermembrane space of mitochondria. Mol. Biol. Cell 23, 4335–4346 (2012)

  36. 36.

    et al. TopHat2: accurate alignment of transcriptomes in the presence of insertions, deletions and gene fusions. Genome Biol. 14, R36 (2013)

  37. 37.

    & Fast gapped-read alignment with Bowtie 2. Nature Methods 9, 357–359 (2012)

  38. 38.

    et al. The Sequence Alignment/Map format and SAMtools. Bioinformatics 25, 2078–2079 (2009)

  39. 39.

    et al. The INA complex facilitates assembly of the peripheral stalk of the mitochondrial F1Fo-ATP synthase. EMBO J. 33, 1624–1638 (2014)

  40. 40.

    et al. Proteomics characterization of mouse kidney peroxisomes by tandem mass spectrometry and protein correlation profiling. Mol. Cell. Proteomics 6, 2045–2057 (2007)

  41. 41.

    & MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nature Biotechnol. 26, 1367–1372 (2008)

  42. 42.

    et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011)

  43. 43.

    et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nature Biotechnol. 32, 223–226 (2014)

  44. 44.

    et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 41, D1063–D1069 (2013)

  45. 45.

    , & BiNGO: a Cytoscape plugin to assess overrepresentation of gene ontology categories in biological networks. Bioinformatics 21, 3448–3449 (2005)

  46. 46.

    et al. Cytoscape: a software environment for integrated models of biomolecular interaction networks. Genome Res. 13, 2498–2504 (2003)

  47. 47.

    et al. Elevated proteasome capacity extends replicative lifespan in Saccharomyces cerevisiae. PLoS Genet. 7, e1002253 (2011)

  48. 48.

    , & Isolation of yeast mitochondria. Methods Mol. Biol. 313, 33–39 (2006)

  49. 49.

    & Fungal small nuclear ribonucleoproteins share properties with plant and vertebrate U-snRNPs. EMBO J. 6, 469–476 (1987)

  50. 50.

    et al. Accurate normalization of real-time quantitative RT–PCR data by geometric averaging of multiple internal control genes. Genome Biol. 3, research0034 (2002)

  51. 51.

    , , & Validation of reference genes for quantitative expression analysis by real-time RT–PCR in Saccharomyces cerevisiae. BMC Mol. Biol. 10, 99 (2009)

  52. 52.

    et al. A novel strategy for selection and validation of reference genes in dynamic multidimensional experimental design in yeast. PLoS ONE 7, e38351 (2012)

  53. 53.

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

Download references

Acknowledgements

We thank A. Fergin, B. Knapp, B. Guiard, W. Voos, M. Glickman, A. Gornicka, A. Loniewska-Lwowska, and T. Wegierski for materials, experimental assistance and discussions. Deposition of the data to the ProteomeXchange Consortium is supported by PRIDE Team, EBI. Research in the B.W. laboratory is supported by the Deutsche Forschungsgemeinschaft and the Excellence Initiative of the German Federal & State Governments (EXC 294 BIOSS). Research in the A.C. laboratory was supported by Foundation for Polish Science – Welcome Programme co-financed by the EU within the European Regional Development Fund (L.W., M.E.S. and E.J.), National Science Centre grants 2011/02/B/NZ2/01402 (L.W., U.T. and A.V.) and 2013/11/B/NZ3/00974 (P.C.) and Ministerial Ideas Plus schema 000263 (E.J.). L.W. and U.T. were also supported by National Science Centre grant 2013/08/T/NZ1/00770 and Swiss National Science Foundation postdoctoral fellowship (PP300P3-147899), respectively. P.B. was supported by the National Science Centre grant 2013/11/D/NZ1/02294.

Author information

Author notes

    • Sebastian Wiese

    Present address: Core Unit Mass Spectrometry and Proteomics, Medical Faculty, Ulm University, D-89081 Ulm, Germany.

    • Lidia Wrobel
    •  & Ulrike Topf

    These authors contributed equally to this work.

Affiliations

  1. International Institute of Molecular and Cell Biology, 02-109 Warsaw, Poland

    • Lidia Wrobel
    • , Ulrike Topf
    • , Piotr Bragoszewski
    • , Malgorzata E. Sztolsztener
    • , Aksana Varabyova
    • , Piotr Chroscicki
    • , Elzbieta Januszewicz
    •  & Agnieszka Chacinska
  2. Department of Biochemistry and Functional Proteomics, Faculty of Biology and BIOSS Centre for Biological Signalling Studies, University of Freiburg, D-79104 Freiburg, Germany

    • Sebastian Wiese
    • , Silke Oeljeklaus
    •  & Bettina Warscheid
  3. ZBSA Centre for Biological Systems Analysis, University of Freiburg, D-79104 Freiburg, Germany

    • Sebastian Wiese
    •  & Bettina Warscheid
  4. Institute of Biochemistry and Biophysics Polish Academy of Sciences, 02-106 Warsaw, Poland

    • Maciej Lirski
    • , Seweryn Mroczek
    • , Andrzej Dziembowski
    •  & Marta Koblowska
  5. Department of Genetics and Biotechnology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland

    • Seweryn Mroczek
    •  & Andrzej Dziembowski
  6. Laboratory of Systems Biology, Faculty of Biology, University of Warsaw, 02-106 Warsaw, Poland

    • Marta Koblowska

Authors

  1. Search for Lidia Wrobel in:

  2. Search for Ulrike Topf in:

  3. Search for Piotr Bragoszewski in:

  4. Search for Sebastian Wiese in:

  5. Search for Malgorzata E. Sztolsztener in:

  6. Search for Silke Oeljeklaus in:

  7. Search for Aksana Varabyova in:

  8. Search for Maciej Lirski in:

  9. Search for Piotr Chroscicki in:

  10. Search for Seweryn Mroczek in:

  11. Search for Elzbieta Januszewicz in:

  12. Search for Andrzej Dziembowski in:

  13. Search for Marta Koblowska in:

  14. Search for Bettina Warscheid in:

  15. Search for Agnieszka Chacinska in:

Contributions

P.B. and S.W. are joint second authors. L.W., U.T., P.B., M.E.S., A.V., P.C., S.M. and E.J. performed and analysed biochemical experiments. P.B. and M.L. performed RNA-seq and analyses. S.W. and S.O. performed the mass spectrometric measurements and analyses. A.C., B.W., M.K. and A.D. analysed and supervised the study. A.C and B.W. conceived the project. All authors interpreted the experiments. A.C. wrote the manuscript with the input of other authors.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Bettina Warscheid or Agnieszka Chacinska.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains full legends for Supplementary Tables 1-3 and Supplementary images.

Excel files

  1. 1.

    Supplementary Table 1

    This file contains RNA-Seq analysis of the mia40-4int mutant versus wild-type strain – see Supplementary Information file for full legend.

  2. 2.

    Supplementary Table 2

    This file contains SILAC-based proteomics analysis of mia40-4intS mutant versus wild-type - see Supplementary Information file for full legend.

  3. 3.

    Supplementary Table 3

    This file contains proteins quantified in SILAC-based proteomics in at least two biological replicates - see Supplementary Information file for full legend.

About this article

Publication history

Received

Accepted

Published

DOI

https://doi.org/10.1038/nature14951

Further reading

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.