Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion

  1. William J Allen
  2. Robin A Corey
  3. Daniel W Watkins
  4. A Sofia F Oliveira
  5. Kiel Hards
  6. Gregory M Cook
  7. Ian Collinson  Is a corresponding author
  1. University of Bristol, United Kingdom
  2. University of Oxford, United Kingdom
  3. University of Otago, New Zealand

Abstract

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism through which the PMF can aid transport by enabling a 'proton ratchet', wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

Data availability

All raw data generated during this study are included as supplementary files, and annotated with the figure they were used in.

Article and author information

Author details

  1. 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
  2. Robin A Corey

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1820-7993
  3. Daniel W Watkins

    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:" 0000-0003-3825-5036
  4. A Sofia F Oliveira

    School of Chemistry, 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:" 0000-0001-8753-4950
  5. Kiel Hards

    Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  6. Gregory M Cook

    Department of Microbiology and Immunology, University of Otago, Duneding, New Zealand
    Competing interests
    The authors declare that no competing interests exist.
  7. 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)

  • William J Allen
  • Ian Collinson

Biotechnology and Biological Sciences Research Council (BB/S008349/1)

  • Daniel W Watkins
  • Ian Collinson

Biotechnology and Biological Sciences Research Council (BB/N015126/1)

  • Daniel W Watkins
  • Ian Collinson

Biotechnology and Biological Sciences Research Council (BB/M003604/1)

  • Robin A Corey
  • Ian Collinson

Biotechnology and Biological Sciences Research Council (BB/I008675/1)

  • William J Allen
  • 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. Lejla Zubcevic, University of Kansas Medical Center, United States

Version history

  1. Preprint posted: March 29, 2019 (view preprint)
  2. Received: February 4, 2022
  3. Accepted: April 29, 2022
  4. Accepted Manuscript published: April 29, 2022 (version 1)
  5. Version of Record published: May 16, 2022 (version 2)

Copyright

© 2022, Allen 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. William J Allen
  2. Robin A Corey
  3. Daniel W Watkins
  4. A Sofia F Oliveira
  5. Kiel Hards
  6. Gregory M Cook
  7. Ian Collinson
(2022)
Rate-limiting transport of positively charged arginine residues through the Sec-machinery is integral to the mechanism of protein secretion
eLife 11:e77586.
https://doi.org/10.7554/eLife.77586

Share this article

https://doi.org/10.7554/eLife.77586

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