1. Structural Biology and Molecular Biophysics

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

  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  Is a corresponding author
  10. Roman Tuma  Is a corresponding author
  1. University of Leeds, United Kingdom
  2. University of Bristol, United Kingdom
https://doi.org/10.7554/eLife.35112
Share this article
Cite this article
  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
  2. Download BibTeX
  3. Download .RIS

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.

Reviewing Editor

  1. Taekjip Ha, Johns Hopkins University School of Medicine, United States

Version history

  1. Received: January 16, 2018
  2. Accepted: June 5, 2018
  3. Accepted Manuscript published: June 7, 2018 (version 1)
  4. Version of Record published: June 27, 2018 (version 2)

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

  • 2,859
    views
  • 496
    downloads
  • 45
    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

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    X-ray structure and enzymatic study of a bacterial NADPH oxidase highlight the activation mechanism of eukaryotic NOX

    Isabelle Petit-Hartlein, Annelise Vermot ... Franck Fieschi
    Research Article

    NADPH oxidases (NOX) are transmembrane proteins, widely spread in eukaryotes and prokaryotes, that produce reactive oxygen species (ROS). Eukaryotes use the ROS products for innate immune defense and signaling in critical (patho)physiological processes. Despite the recent structures of human NOX isoforms, the activation of electron transfer remains incompletely understood. SpNOX, a homolog from Streptococcus pneumoniae, can serves as a robust model for exploring electron transfers in the NOX family thanks to its constitutive activity. Crystal structures of SpNOX full-length and dehydrogenase (DH) domain constructs are revealed here. The isolated DH domain acts as a flavin reductase, and both constructs use either NADPH or NADH as substrate. Our findings suggest that hydride transfer from NAD(P)H to FAD is the rate-limiting step in electron transfer. We identify significance of F397 in nicotinamide access to flavin isoalloxazine and confirm flavin binding contributions from both DH and Transmembrane (TM) domains. Comparison with related enzymes suggests that distal access to heme may influence the final electron acceptor, while the relative position of DH and TM does not necessarily correlate with activity, contrary to previous suggestions. It rather suggests requirement of an internal rearrangement, within the DH domain, to switch from a resting to an active state. Thus, SpNOX appears to be a good model of active NOX2, which allows us to propose an explanation for NOX2’s requirement for activation.

    1. Cell Biology
    2. Structural Biology and Molecular Biophysics

    Effect of α-tubulin acetylation on the doublet microtubule structure

    Shun Kai Yang, Shintaroh Kubo ... Khanh Huy Bui
    Research Article

    Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.

    1. Structural Biology and Molecular Biophysics

    Structure-guided mutagenesis of OSCAs reveals differential activation to mechanical stimuli

    Sebastian Jojoa-Cruz, Adrienne E Dubin ... Andrew B Ward
    Research Advance

    The dimeric two-pore OSCA/TMEM63 family has recently been identified as mechanically activated ion channels. Previously, based on the unique features of the structure of OSCA1.2, we postulated the potential involvement of several structural elements in sensing membrane tension (Jojoa-Cruz et al., 2018). Interestingly, while OSCA1, 2, and 3 clades are activated by membrane stretch in cell-attached patches (i.e. they are stretch-activated channels), they differ in their ability to transduce membrane deformation induced by a blunt probe (poking). Here, in an effort to understand the domains contributing to mechanical signal transduction, we used cryo-electron microscopy to solve the structure of Arabidopsis thaliana (At) OSCA3.1, which, unlike AtOSCA1.2, only produced stretch- but not poke-activated currents in our initial characterization (Murthy et al., 2018). Mutagenesis and electrophysiological assessment of conserved and divergent putative mechanosensitive features of OSCA1.2 reveal a selective disruption of the macroscopic currents elicited by poking without considerable effects on stretch-activated currents (SAC). Our results support the involvement of the amphipathic helix and lipid-interacting residues in the membrane fenestration in the response to poking. Our findings position these two structural elements as potential sources of functional diversity within the family.