Assembling the Tat protein translocase

  1. Felicity Alcock
  2. Phillip J Stansfeld  Is a corresponding author
  3. Hajra Basit
  4. Johann Habersetzer
  5. Matthew AB Baker
  6. Tracy Palmer
  7. Mark I Wallace
  8. Ben C Berks  Is a corresponding author
  1. University of Oxford, United Kingdom
  2. Kings College London, United Kingdom
  3. University of Dundee, United Kingdom
  4. University of New South Wales, Australia

Abstract

The twin-arginine protein translocation system (Tat) transports folded proteins across the bacterial cytoplasmic membrane and the thylakoid membranes of plant chloroplasts. The Tat transporter is assembled from multiple copies of the membrane proteins TatA, TatB, and TatC. We combine sequence co-evolution analysis, molecular simulations, and experimentation to define the interactions between the Tat proteins of Escherichia coli at molecular-level resolution. In the TatBC receptor complex the transmembrane helix of each TatB molecule is sandwiched between two TatC molecules, with one of the inter-subunit interfaces incorporating a functionally important cluster of interacting polar residues. Unexpectedly, we find that TatA also associates with TatC at the polar cluster site. Our data provide a structural model for assembly of the active Tat translocase in which substrate binding triggers replacement of TatB by TatA at the polar cluster site. Our work demonstrates the power of co-evolution analysis to predict protein interfaces in multi-subunit complexes.

Article and author information

Author details

  1. Felicity Alcock

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Phillip J Stansfeld

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    For correspondence
    phillip.stansfeld@bioch.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
  3. Hajra Basit

    Department of Chemistry, Kings College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Johann Habersetzer

    Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Matthew AB Baker

    EMBL Australia Node for Single Molecule Science, University of New South Wales, Kensington, Australia
    Competing interests
    The authors declare that no competing interests exist.
  6. Tracy Palmer

    Division of Molecular Microbiology, College of Life Sciences, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  7. Mark I Wallace

    Department of Chemistry, Kings College London, London, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5692-8313
  8. Ben C Berks

    Department of Biochemistry, University of Oxford, Oxford, United Kingdom
    For correspondence
    ben.berks@bioch.ox.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9685-4067

Funding

Biotechnology and Biological Sciences Research Council (BB/L002531/1)

  • Tracy Palmer
  • Ben C Berks

Wellcome (Investigator Award 107929/Z/15/Z)

  • Ben C Berks

Medical Research Council (G1001640)

  • Tracy Palmer
  • Ben C Berks

European Commission (Marie Curie Fellowship Programme: GP7-PEOPLE-2013-IEF 626436)

  • Hajra Basit
  • Mark I Wallace

Biotechnology and Biological Sciences Research Council (BB/I019855/1)

  • Phillip J Stansfeld

Wellcome (Investigator Award 110183/Z/15/Z)

  • Tracy Palmer

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

Reviewing Editor

  1. Nir Ben-Tal, Tel Aviv University, Israel

Publication history

  1. Received: August 22, 2016
  2. Accepted: November 29, 2016
  3. Accepted Manuscript published: December 3, 2016 (version 1)
  4. Accepted Manuscript updated: December 16, 2016 (version 2)
  5. Accepted Manuscript updated: December 20, 2016 (version 3)
  6. Version of Record published: December 30, 2016 (version 4)

Copyright

© 2016, Alcock 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

  • 2,888
    Page views
  • 770
    Downloads
  • 41
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

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. Felicity Alcock
  2. Phillip J Stansfeld
  3. Hajra Basit
  4. Johann Habersetzer
  5. Matthew AB Baker
  6. Tracy Palmer
  7. Mark I Wallace
  8. Ben C Berks
(2016)
Assembling the Tat protein translocase
eLife 5:e20718.
https://doi.org/10.7554/eLife.20718

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Edmundo G Vides, Ayan Adhikari ... Suzanne R Pfeffer
    Research Advance

    Activating mutations in the Leucine Rich Repeat Kinase 2 (LRRK2) cause Parkinson's disease and previously we showed that activated LRRK2 phosphorylates a subset of Rab GTPases (Steger et al., 2017). Moreover, Golgi-associated Rab29 can recruit LRRK2 to the surface of the Golgi and activate it there for both auto- and Rab substrate phosphorylation. Here we define the precise Rab29 binding region of the LRRK2 Armadillo domain between residues 360-450 and show that this domain, termed 'Site #1', can also bind additional LRRK2 substrates, Rab8A and Rab10. Moreover, we identify a distinct, N-terminal, higher affinity interaction interface between LRRK2 phosphorylated Rab8 and Rab10 termed 'Site #2', that can retain LRRK2 on membranes in cells to catalyze multiple, subsequent phosphorylation events. Kinase inhibitor washout experiments demonstrate that rapid recovery of kinase activity in cells depends on the ability of LRRK2 to associate with phosphorylated Rab proteins, and phosphorylated Rab8A stimulates LRRK2 phosphorylation of Rab10 in vitro. Reconstitution of purified LRRK2 recruitment onto planar lipid bilayers decorated with Rab10 protein demonstrates cooperative association of only active LRRK2 with phospho-Rab10-containing membrane surfaces. These experiments reveal a feed-forward pathway that provides spatial control and membrane activation of LRRK2 kinase activity.

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Andrea Volante, Juan Carlos Alonso, Kiyoshi Mizuuchi
    Research Article Updated

    Three-component ParABS partition systems ensure stable inheritance of many bacterial chromosomes and low-copy-number plasmids. ParA localizes to the nucleoid through its ATP-dependent nonspecific DNA-binding activity, whereas centromere-like parS-DNA and ParB form partition complexes that activate ParA-ATPase to drive the system dynamics. The essential parS sequence arrangements vary among ParABS systems, reflecting the architectural diversity of their partition complexes. Here, we focus on the pSM19035 plasmid partition system that uses a ParBpSM of the ribbon-helix-helix (RHH) family. We show that parSpSM with four or more contiguous ParBpSM-binding sequence repeats is required to assemble a stable ParApSM-ParBpSM complex and efficiently activate the ParApSM-ATPase, stimulating complex disassembly. Disruption of the contiguity of the parSpSM sequence array destabilizes the ParApSM-ParBpSM complex and prevents efficient ATPase activation. Our findings reveal the unique architecture of the pSM19035 partition complex and how it interacts with nucleoid-bound ParApSM-ATP.