Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes

  1. Holly C Ford
  2. William J Allen
  3. Gonçalo C Pereira
  4. Xia Liu
  5. Mark Simon Dillingham
  6. Ian Collinson  Is a corresponding author
  1. University of Bristol, United Kingdom
  2. University of Cambridge, United Kingdom

Abstract

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how ∆ψ and ATP hydrolysis, the two main driving forces for import into the matrix, contribute to the transport of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the outer membrane and initiation of inner membrane transport by the pre-sequence - the rates of which are influenced by PCP properties such as size and net charge. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel – presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.

Data availability

We present secondary analysis of raw optical readout data. All raw data is included in the manuscript, supplementary information and source data.

Article and author information

Author details

  1. Holly C Ford

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. William J Allen

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9513-4786
  3. Gonçalo C Pereira

    MRC - Mitochondrial Biology Unit, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Xia Liu

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Mark Simon Dillingham

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4612-7141
  6. Ian Collinson

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    For correspondence
    ian.collinson@bristol.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3931-0503

Funding

Wellcome Trust (104632)

  • Holly C Ford

Wellcome Trust (104632)

  • William J Allen

Wellcome Trust (104632)

  • Gonçalo C Pereira

Wellcome Trust (104632)

  • Xia Liu

Wellcome Trust (104632)

  • Ian Collinson

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

Publication history

  1. Received: November 9, 2021
  2. Accepted: June 7, 2022
  3. Accepted Manuscript published: June 8, 2022 (version 1)

Copyright

© 2022, Ford 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. Holly C Ford
  2. William J Allen
  3. Gonçalo C Pereira
  4. Xia Liu
  5. Mark Simon Dillingham
  6. Ian Collinson
(2022)
Towards a molecular mechanism underlying mitochondrial protein import through the TOM and TIM23 complexes
eLife 11:e75426.
https://doi.org/10.7554/eLife.75426

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