Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase

  1. Ryo Fujisawa
  2. Cristian Polo Rivera
  3. Karim Labib  Is a corresponding author
  1. University of Dundee, United Kingdom

Abstract

The p97 / Cdc48 ATPase and its ubiquitin receptors Ufd1-Npl4 are essential to unfold ubiquitylated proteins in many areas of eukaryotic cell biology. In yeast, Cdc48-Ufd1-Npl4 is controlled by a quality control mechanism, whereby substrates must be conjugated to at least five ubiquitins. Here we show that mammalian p97-UFD1-NPL4 is governed by a complex interplay between additional p97 cofactors and the number of conjugated ubiquitins. Using reconstituted assays for the disassembly of ubiquitylated CMG (Cdc45-MCM-GINS) helicase by human p97-UFD1-NPL4, we show that the unfoldase has a high ubiquitin threshold for substrate unfolding, which can be reduced by the UBX proteins UBXN7, FAF1 or FAF2. Our data indicate that the UBX proteins function by binding to p97-UFD1-NPL4 and stabilising productive interactions between UFD1-NPL4 and K48-linked chains of at least five ubiquitins. Stimulation by UBXN7 is dependent upon known ubiquitin binding motifs, whereas FAF1 and FAF2 use a previously uncharacterised coiled-coil domain to reduce the ubiquitin threshold of p97-UFD1-NPL4. We show that deleting the Ubnx7 and Faf1 genes impairs CMG disassembly during S-phase and mitosis and sensitises cells to reduced ubiquitin ligase activity. These findings indicate that multiple UBX proteins are important for the efficient unfolding of ubiquitylated proteins by p97-UFD1-NPL4 in mammalian cells.

Data availability

All data generated or analysed during this study are included in the manuscript and supporting files; Source Data files have been provided .

Article and author information

Author details

  1. Ryo Fujisawa

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-1985-1668
  2. Cristian Polo Rivera

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    Competing interests
    The authors declare that no competing interests exist.
  3. Karim Labib

    MRC Protein Phosphorylation and Ubiquitylation Unit, University of Dundee, Dundee, United Kingdom
    For correspondence
    kpmlabib@dundee.ac.uk
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-8861-379X

Funding

Medical Research Council (MC_UU_12016/13)

  • Ryo Fujisawa
  • Cristian Polo Rivera
  • Karim Labib

Cancer Research UK (C578/A24558; C578/A25671)

  • Ryo Fujisawa
  • Cristian Polo Rivera
  • Karim Labib

Japan Society for the Promotion of Science (202160572)

  • Ryo Fujisawa

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

Reviewing Editor

  1. Wade Harper, Harvard Medical School, United States

Publication history

  1. Received: January 4, 2022
  2. Preprint posted: January 14, 2022 (view preprint)
  3. Accepted: August 2, 2022
  4. Accepted Manuscript published: August 3, 2022 (version 1)
  5. Version of Record published: August 15, 2022 (version 2)

Copyright

© 2022, Fujisawa 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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  1. Ryo Fujisawa
  2. Cristian Polo Rivera
  3. Karim Labib
(2022)
Multiple UBX proteins reduce the ubiquitin threshold of the mammalian p97-UFD1-NPL4 unfoldase
eLife 11:e76763.
https://doi.org/10.7554/eLife.76763

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