A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins

Abstract

Signal-anchored (SA) proteins are anchored into the mitochondrial outer membrane (OM) via a single transmembrane segment at their N-terminus while the bulk of the proteins is facing the cytosol. These proteins are encoded by nuclear DNA, translated on cytosolic ribosomes, and are then targeted to the organelle and inserted into its OM by import factors. Recently, research on the insertion mechanisms of these proteins into the mitochondrial OM have gained a lot of attention. In contrast, the early cytosolic steps of their biogenesis are unresolved. Using various proteins from this category and a broad set of in vivo, in organello, and in vitro assays, we reconstituted the early steps of their biogenesis. We identified a subset of molecular (co)chaperones that interact with newly synthesized SA proteins, namely, Hsp70 and Hsp90 chaperones and co-chaperones from the Hsp40 family like Ydj1 and Sis1. These interactions were mediated by the hydrophobic transmembrane segments of the SA proteins. We further demonstrate that interfering with these interactions inhibits the biogenesis of SA proteins to a various extent. Finally, we could demonstrate direct interaction of peptides corresponding to the transmembrane segments of SA proteins with the (co)chaperones and reconstitute in vitro the transfer of such peptides from the Hsp70 chaperone to the mitochondrial Tom70 receptor. Collectively, this study unravels an array of cytosolic chaperones and mitochondrial import factors that facilitates the targeting and membrane integration of mitochondrial SA proteins.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031610.All data generated or analyzed during this study are included in the manuscript and supporting file; Source Data files have been provided for all relevant Figures.

The following data sets were generated

Article and author information

Author details

  1. Layla Drwesh

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  2. Benjamin Heim

    Department of Chemistry, Technische Universität München, Munich, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Max Graf

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  4. Linda Kehr

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  5. Lea Hansen-Palmus

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Mirita Franz-Wachtel

    Proteome Center Tübingen, Interfaculty Institute for Cell Biology,, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Boris Macek

    Proteome Center Tübingen, Interfaculty Institute for Cell Biology,, University of Tübingen, Tübingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  8. Hubert Kalbacher

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  9. Johannes Buchner

    Department of Chemistry, Technische Universität München, Garching, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1282-7737
  10. Doron Rapaport

    Interfaculty Institute of Biochemistry, University of Tübingen, Tuebingen, Germany
    For correspondence
    doron.rapaport@uni-tuebingen.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3136-1207

Funding

Deutsche Forschungsgemeinschaft (RA 1028/7-2)

  • Doron Rapaport

Deutsche Forschungsgemeinschaft (RA 1028/10-2)

  • Doron Rapaport

Minerva Foundation (PhD fellowship)

  • Layla Drwesh

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Reviewing Editor

  1. Nikolaus Pfanner, University of Freiburg, Germany

Version history

  1. Received: February 8, 2022
  2. Preprint posted: February 17, 2022 (view preprint)
  3. Accepted: July 25, 2022
  4. Accepted Manuscript published: July 25, 2022 (version 1)
  5. Version of Record published: August 5, 2022 (version 2)

Copyright

© 2022, Drwesh et al.

This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.

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  1. Layla Drwesh
  2. Benjamin Heim
  3. Max Graf
  4. Linda Kehr
  5. Lea Hansen-Palmus
  6. Mirita Franz-Wachtel
  7. Boris Macek
  8. Hubert Kalbacher
  9. Johannes Buchner
  10. Doron Rapaport
(2022)
A network of cytosolic (co)chaperones promotes the biogenesis of mitochondrial signal-anchored outer membrane proteins
eLife 11:e77706.
https://doi.org/10.7554/eLife.77706

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https://doi.org/10.7554/eLife.77706

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