The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication

  1. Agnieszka Fatalska  Is a corresponding author
  2. Emma Stepinac
  3. Magdalena Richter
  4. Levente Kovacs
  5. Zbigniew Pietras
  6. Martin Puchinger
  7. Gang Dong
  8. Michal Dadlez
  9. David M Glover  Is a corresponding author
  1. University of Cambridge, United Kingdom
  2. Medical University of Vienna, Austria
  3. Institute of Biochemistry and Biophysics PAS, Poland
  4. University of Vienna, Austria

Abstract

The duplication and 9-fold symmetry of the Drosophila centriole requires that the cartwheel molecule, Sas6, physically associates with Gorab, a trans-Golgi component. How Gorab achieves these disparate associations is unclear. Here we use hydrogen-deuterium exchange mass spectrometry to define Gorab's interacting surfaces that mediate its sub-cellular localization. We identify a core stabilization sequence within Gorab's C-terminal coiled-coil domain that enables homodimerization, binding to Rab6, and thereby trans-Golgi localization. By contrast, part of the Gorab monomer's coiled-coil domain undergoes an anti-parallel interaction with a segment of the parallel coiled-coil dimer of Sas6. This stable hetero-trimeric complex can be visualized by electron microscopy. Mutation of a single leucine residue in Sas6's Gorab-binding domain generates a Sas6 variant with a 16-fold reduced binding affinity for Gorab that can not support centriole duplication. Thus Gorab dimers at the Golgi exist in equilibrium with Sas-6 associated monomers at the centriole to balance Gorab's dual role.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files.

Article and author information

Author details

  1. Agnieszka Fatalska

    Department of Genetics, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    af589@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-1720-4742
  2. Emma Stepinac

    Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  3. Magdalena Richter

    Department of Genetics, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  4. Levente Kovacs

    Department of Genetics, University of Cambridge, Cambridge, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  5. Zbigniew Pietras

    Laboratory of RNA Biology and Functional Genomics, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland
    Competing interests
    The authors declare that no competing interests exist.
  6. Martin Puchinger

    Department of Structural and Computational Biology, University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  7. Gang Dong

    Department of Medical Biochemistry, Medical University of Vienna, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Michal Dadlez

    Biophysics, Mass Spectrometry Laboratory, Institute of Biochemistry and Biophysics PAS, Warsaw, Poland
    Competing interests
    The authors declare that no competing interests exist.
  9. David M Glover

    Genetics, University of Cambridge, Cambridge, United Kingdom
    For correspondence
    dmg25@cam.ac.uk
    Competing interests
    The authors declare that no competing interests exist.

Funding

Wellcome Trust (Investigator Award)

  • David M Glover

National Institute of Neurological Disorders and Stroke (R01NS113930)

  • David M Glover

National Science Centre (MAESTRO project UMO-2014/14/A/NZ1/00306)

  • Agnieszka Fatalska
  • Michal Dadlez

Austrian Science Fund (P28231-B28)

  • Gang Dong

Austrian Science Fund (W-1258 Doktoratskollegs)

  • Emma Stepinac

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

Copyright

© 2021, Fatalska 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

  • 1,703
    views
  • 278
    downloads
  • 6
    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. Agnieszka Fatalska
  2. Emma Stepinac
  3. Magdalena Richter
  4. Levente Kovacs
  5. Zbigniew Pietras
  6. Martin Puchinger
  7. Gang Dong
  8. Michal Dadlez
  9. David M Glover
(2021)
The dimeric Golgi protein Gorab binds to Sas6 as a monomer to mediate centriole duplication
eLife 10:e57241.
https://doi.org/10.7554/eLife.57241

Share this article

https://doi.org/10.7554/eLife.57241

Further reading

    1. Cell Biology
    2. Neuroscience
    Vibhavari Aysha Bansal, Jia Min Tan ... Toh Hean Ch'ng
    Research Article

    The emergence of Aβ pathology is one of the hallmarks of Alzheimer’s disease (AD), but the mechanisms and impact of Aβ in progression of the disease is unclear. The nuclear pore complex (NPC) is a multi-protein assembly in mammalian cells that regulates movement of macromolecules across the nuclear envelope; its function is shown to undergo age-dependent decline during normal aging and is also impaired in multiple neurodegenerative disorders. Yet not much is known about the impact of Aβ on NPC function in neurons. Here, we examined NPC and nucleoporin (NUP) distribution and nucleocytoplasmic transport using a mouse model of AD (AppNL-G-F/NL-G-F) that expresses Aβ in young animals. Our studies revealed that a time-dependent accumulation of intracellular Aβ corresponded with a reduction of NPCs and NUPs in the nuclear envelope which resulted in the degradation of the permeability barrier and inefficient segregation of nucleocytoplasmic proteins, and active transport. As a result of the NPC dysfunction App KI neurons become more vulnerable to inflammation-induced necroptosis – a programmed cell death pathway where the core components are activated via phosphorylation through nucleocytoplasmic shutting. Collectively, our data implicates Aβ in progressive impairment of nuclear pore function and further confirms that the protein complex is vulnerable to disruption in various neurodegenerative diseases and is a potential therapeutic target.

    1. Cell Biology
    Qi Zeng, Chen Yao ... Shuai Chen
    Research Article

    Mounting evidence has demonstrated the genetic association of ORMDL sphingolipid biosynthesis regulator 3 (ORMDL3) gene polymorphisms with bronchial asthma and a diverse set of inflammatory disorders. However, its role in type I interferon (type I IFN) signaling remains poorly defined. Herein, we report that ORMDL3 is a negative modulator of the type I IFN signaling by interacting with mitochondrial antiviral signaling protein (MAVS) and subsequently promoting the proteasome-mediated degradation of retinoic acid-inducible gene I (RIG-I). Immunoprecipitation coupled with mass spectrometry (IP-MS) assays uncovered that ORMDL3 binds to ubiquitin-specific protease 10 (USP10), which forms a complex with and stabilizes RIG-I through decreasing its K48-linked ubiquitination. ORMDL3 thus disrupts the interaction between USP10 and RIG-I, thereby promoting RIG-I degradation. Additionally, subcutaneous syngeneic tumor models in C57BL/6 mice revealed that inhibition of ORMDL3 enhances anti-tumor efficacy by augmenting the proportion of cytotoxic CD8 positive T cells and IFN production in the tumor microenvironment (TME). Collectively, our findings reveal the pivotal roles of ORMDL3 in maintaining antiviral innate immune responses and anti-tumor immunity.