Dynamic action of the Sec machinery during initiation, protein translocation and termination

Abstract

Protein translocation across cell membranes is a ubiquitous process required for protein secretion and membrane protein insertion. In bacteria, this is mostly mediated by the conserved SecYEG complex, driven through rounds of ATP hydrolysis by the cytoplasmic SecA, and the trans-membrane proton motive force. We have used single molecule techniques to explore SecY pore dynamics on multiple timescales in order to dissect the complex reaction pathway. The results show that SecA, both the signal sequence and mature components of the pre-protein, and ATP hydrolysis each have important and specific roles in channel unlocking, opening and priming for transport. After channel opening, translocation proceeds in two phases: a slow phase independent of substrate length, and a length-dependent transport phase with an intrinsic translocation rate of ~40 amino acids per second for the proOmpA substrate. Broad translocation rate distributions reflect the stochastic nature of polypeptide transport.

Data availability

Compressed data are available together with the relevant scripts as Supplementary Source Data and Code

Article and author information

Author details

  1. Tomas Fessl

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Daniel Watkins

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Peter Oatley

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. William John 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
  5. Robin Adam Corey

    School of Biochemistry, University of Bristol, Bristol, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Jim Horne

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Steve A Baldwin

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  8. Sheena E Radford

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-3079-8039
  9. 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
  10. Roman Tuma

    Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds, United Kingdom
    For correspondence
    r.tuma@leeds.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0047-0013

Funding

Biotechnology and Biological Sciences Research Council (BB/N017307/1)

  • Tomas Fessl
  • Sheena E Radford
  • Roman Tuma

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

  • Daniel Watkins

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

  • Robin Adam Corey

Wellcome (104632)

  • William John Allen
  • Ian Collinson

Seventh Framework Programme (32240)

  • Sheena E Radford

European Regional Development Fund (CZ.02.1.01/0.0/0.0/15_003/0000441)

  • Tomas Fessl
  • Roman Tuma

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

  • Daniel Watkins
  • Ian Collinson

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

  • Peter Oatley
  • Steve A Baldwin
  • Sheena E Radford
  • Roman Tuma

Biotechnology and Biological Sciences Research Council (BB/M011151/1)

  • Jim Horne

Biotechnology and Biological Sciences Research Council (BB/I006737/1)

  • William John Allen
  • Ian Collinson

Biotechnology and Biological Sciences Research Council (BBSRC South West Bioscience Doctoral Training Partnership)

  • Robin Adam Corey

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

Copyright

© 2018, Fessl 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.

Metrics

  • 3,045
    views
  • 512
    downloads
  • 58
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Tomas Fessl
  2. Daniel Watkins
  3. Peter Oatley
  4. William John Allen
  5. Robin Adam Corey
  6. Jim Horne
  7. Steve A Baldwin
  8. Sheena E Radford
  9. Ian Collinson
  10. Roman Tuma
(2018)
Dynamic action of the Sec machinery during initiation, protein translocation and termination
eLife 7:e35112.
https://doi.org/10.7554/eLife.35112

Share this article

https://doi.org/10.7554/eLife.35112

Further reading

    1. Immunology and Inflammation
    2. Structural Biology and Molecular Biophysics
    Douwe Schulte, Marta Šiborová ... Joost Snijder
    Research Article

    Antibodies are a major component of adaptive immunity against invading pathogens. Here, we explore possibilities for an analytical approach to characterize the antigen-specific antibody repertoire directly from the secreted proteins in convalescent serum. This approach aims to perform simultaneous antibody sequencing and epitope mapping using a combination of single particle cryo-electron microscopy (cryoEM) and bottom-up proteomics techniques based on mass spectrometry (LC-MS/MS). We evaluate the performance of the deep-learning tool ModelAngelo in determining de novo antibody sequences directly from reconstructed 3D volumes of antibody-antigen complexes. We demonstrate that while map quality is a critical bottleneck, it is possible to sequence antibody variable domains from cryoEM reconstructions with accuracies of up to 80–90%. While the rate of errors exceeds the typical levels of somatic hypermutation, we show that the ModelAngelo-derived sequences can be used to assign the used V-genes. This provides a functional guide to assemble de novo peptides from LC-MS/MS data more accurately and improves the tolerance to a background of polyclonal antibody sequences. Following this proof-of-principle, we discuss the feasibility and future directions of this approach to characterize antigen-specific antibody repertoires.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Yamato Niitani, Kohei Matsuzaki ... Michio Tomishige
    Research Article

    The two identical motor domains (heads) of dimeric kinesin-1 move in a hand-over-hand process along a microtubule, coordinating their ATPase cycles such that each ATP hydrolysis is tightly coupled to a step and enabling the motor to take many steps without dissociating. The neck linker, a structural element that connects the two heads, has been shown to be essential for head–head coordination; however, which kinetic step(s) in the chemomechanical cycle is ‘gated’ by the neck linker remains unresolved. Here, we employed pre-steady-state kinetics and single-molecule assays to investigate how the neck-linker conformation affects kinesin’s motility cycle. We show that the backward-pointing configuration of the neck linker in the front kinesin head confers higher affinity for microtubule, but does not change ATP binding and dissociation rates. In contrast, the forward-pointing configuration of the neck linker in the rear kinesin head decreases the ATP dissociation rate but has little effect on microtubule dissociation. In combination, these conformation-specific effects of the neck linker favor ATP hydrolysis and dissociation of the rear head prior to microtubule detachment of the front head, thereby providing a kinetic explanation for the coordinated walking mechanism of dimeric kinesin.