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In vivo identification of GTPase interactors by mitochondrial relocalization and proximity biotinylation

  1. Alison K Gillingham  Is a corresponding author
  2. Jessie Bertram
  3. Farida Begum
  4. Sean Munro  Is a corresponding author
  1. MRC Laboratory of Molecular Biology, United Kingdom
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Cite this article as: eLife 2019;8:e45916 doi: 10.7554/eLife.45916

Abstract

The GTPases of the Ras superfamily regulate cell growth, membrane traffic and the cytoskeleton, and a wide range of diseases are caused by mutations in particular members. They function as switchable landmarks with the active GTP-bound form recruiting to the membrane a specific set of effector proteins. The GTPases are precisely controlled by regulators that promote acquisition of GTP (GEFs) or its hydrolysis to GDP (GAPs). We report here MitoID, a method for identifying effectors and regulators by performing in vivo proximity biotinylation with mitochondrially-localized forms of the GTPases. Applying this to 11 human Rab GTPases identified many known effectors and GAPs, as well as putative novel effectors, with examples of the latter validated for Rab2, Rab5, Rab9 and Rab11. MitoID can also efficiently identify effectors and GAPs of Rho and Ras family GTPases such as Cdc42, RhoA, Rheb, and N-Ras, and can identify GEFs by use of GDP-bound forms.

Data availability

The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD013668 Apart from this, all data generated or analysed during this study are included in the manuscript and supporting files.

The following data sets were generated

Article and author information

Author details

  1. Alison K Gillingham

    Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    ag@mrc-lmb.cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
  2. Jessie Bertram

    Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Farida Begum

    Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Sean Munro

    Division of Cell Biology, MRC Laboratory of Molecular Biology, Cambridge, United Kingdom
    For correspondence
    sean@mrc-lmb.cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6160-5773

Funding

Medical Research Council (MC_U105178783)

  • Alison K Gillingham
  • Jessie Bertram
  • Farida Begum
  • Sean Munro

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

Reviewing Editor

  1. Reinhard Jahn, Max Planck Institute for Biophysical Chemistry, Germany

Publication history

  1. Received: February 8, 2019
  2. Accepted: July 10, 2019
  3. Accepted Manuscript published: July 11, 2019 (version 1)
  4. Version of Record published: July 18, 2019 (version 2)

Copyright

© 2019, Gillingham 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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