ARL3 activation requires the co-GEF BART and effector-mediated turnover

  1. Yasmin ElMaghloob
  2. Begoña Sot
  3. Michael J McIlwraith
  4. Esther Garcia
  5. Tamas Yelland
  6. Shehab Ismail  Is a corresponding author
  1. Beatson Institute for Cancer Research, United Kingdom
  2. IMDEA Nanoscience, Spain

Abstract

The ADP-ribosylation factor-like 3 (ARL3) is a ciliopathy G-protein which regulates the ciliary trafficking of several lipid-modified proteins. ARL3 is activated by its guanine exchange factor (GEF) ARL13B via an unresolved mechanism. BART is described as an ARL3 effector which has also been implicated in ciliopathies, although the role of its ARL3 interaction is unknown. Here we show that, at physiological GTP:GDP levels, human ARL3GDP is weakly activated by ARL13B. However, BART interacts with nucleotide-free ARL3 and, in concert with ARL13B, efficiently activates ARL3. In addition, BART binds ARL3GTP and inhibits GTP dissociation, thereby stabilising the active G-protein; the binding of ARL3 effectors then releases BART. Finally, using live cell imaging, we show that BART accesses the primary cilium and colocalises with ARL13B. We propose a model wherein BART functions as a bona fide co-GEF for ARL3 and maintains the active ARL3GTP, until it is recycled by ARL3 effectors.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files. Source data files have been provided for: Figures 1A and 1B, Figures 2B, 2C, 2D, 2E, and 2F, Figures 3A,3B, 3C, 3D, 3F, Figures 4A, 4B, and 4C and Figure 5B, 5C

Article and author information

Author details

  1. Yasmin ElMaghloob

    Structural biology, Beatson Institute for Cancer Research, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  2. Begoña Sot

    Nanobiosystems, IMDEA Nanoscience, Madrid, Spain
    Competing interests
    The authors declare that no competing interests exist.
  3. Michael J McIlwraith

    Structural biology, Beatson Institute for Cancer Research, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Esther Garcia

    Structural biology, Beatson Institute for Cancer Research, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Tamas Yelland

    Structural biology, Beatson Institute for Cancer Research, Glasgow, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  6. Shehab Ismail

    Structural biology, Beatson Institute for Cancer Research, Glasgow, United Kingdom
    For correspondence
    shehab.ismail@glasgow.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4150-1077

Funding

Cancer Research UK (A17196)

  • Shehab Ismail

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

Copyright

© 2021, ElMaghloob 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

  • 1,288
    views
  • 200
    downloads
  • 19
    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. Yasmin ElMaghloob
  2. Begoña Sot
  3. Michael J McIlwraith
  4. Esther Garcia
  5. Tamas Yelland
  6. Shehab Ismail
(2021)
ARL3 activation requires the co-GEF BART and effector-mediated turnover
eLife 10:e64624.
https://doi.org/10.7554/eLife.64624

Share this article

https://doi.org/10.7554/eLife.64624

Further reading

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics
    Joar Esteban Pinto Torres, Mathieu Claes ... Yann G-J Sterckx
    Research Article

    African trypanosomes are the causative agents of neglected tropical diseases affecting both humans and livestock. Disease control is highly challenging due to an increasing number of drug treatment failures. African trypanosomes are extracellular, blood-borne parasites that mainly rely on glycolysis for their energy metabolism within the mammalian host. Trypanosomal glycolytic enzymes are therefore of interest for the development of trypanocidal drugs. Here, we report the serendipitous discovery of a camelid single-domain antibody (sdAb aka Nanobody) that selectively inhibits the enzymatic activity of trypanosomatid (but not host) pyruvate kinases through an allosteric mechanism. By combining enzyme kinetics, biophysics, structural biology, and transgenic parasite survival assays, we provide a proof-of-principle that the sdAb-mediated enzyme inhibition negatively impacts parasite fitness and growth.

    1. Biochemistry and Chemical Biology
    2. Microbiology and Infectious Disease
    Eva Herdering, Tristan Reif-Trauttmansdorff ... Ruth Anne Schmitz
    Research Article

    Glutamine synthetases (GS) are central enzymes essential for the nitrogen metabolism across all domains of life. Consequently, they have been extensively studied for more than half a century. Based on the ATP-dependent ammonium assimilation generating glutamine, GS expression and activity are strictly regulated in all organisms. In the methanogenic archaeon Methanosarcina mazei, it has been shown that the metabolite 2-oxoglutarate (2-OG) directly induces the GS activity. Besides, modulation of the activity by interaction with small proteins (GlnK1 and sP26) has been reported. Here, we show that the strong activation of M. mazei GS (GlnA1) by 2-OG is based on the 2-OG dependent dodecamer assembly of GlnA1 by using mass photometry (MP) and single particle cryo-electron microscopy (cryo-EM) analysis of purified strep-tagged GlnA1. The dodecamer assembly from dimers occurred without any detectable intermediate oligomeric state and was not affected in the presence of GlnK1. The 2.39 Å cryo-EM structure of the dodecameric complex in the presence of 12.5 mM 2-OG demonstrated that 2-OG is binding between two monomers. Thereby, 2-OG appears to induce the dodecameric assembly in a cooperative way. Furthermore, the active site is primed by an allosteric interaction cascade caused by 2-OG-binding towards an adaption of an open active state conformation. In the presence of additional glutamine, strong feedback inhibition of GS activity was observed. Since glutamine dependent disassembly of the dodecamer was excluded by MP, feedback inhibition most likely relies on the binding of glutamine to the catalytic site. Based on our findings, we propose that under nitrogen limitation the induction of M. mazei GS into a catalytically active dodecamer is not affected by GlnK1 and crucially depends on the presence of 2-OG.