Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle

  1. Miriam Böhm
  2. Kerstin Killinger
  3. Alexander Dudziak
  4. Pradeep Pant
  5. Karolin Jänen
  6. Simone Hohoff
  7. Karl Mechtler
  8. Mihkel Örd
  9. Mart Loog
  10. Elsa Sanchez-Garcia
  11. Stefan Westermann  Is a corresponding author
  1. University of Duisburg-Essen, Germany
  2. Research Institute of Molecular Pathology, Austria
  3. University of Tartu, Estonia

Abstract

Kinetochores are multi-subunit protein assemblies that link chromosomes to microtubules of the mitotic and meiotic spindle. It is still poorly understood how efficient, centromere-dependent kinetochore assembly is accomplished from hundreds of individual protein building blocks in a cell cycle dependent manner. Here, by combining comprehensive phosphorylation analysis of native Ctf19CCAN subunits with biochemical and functional assays in the model system budding yeast, we demonstrate that Cdk1 phosphorylation activates phospho-degrons on the essential subunit Ame1CENP-U which are recognized by the E3 ubiquitin ligase complex SCF-Cdc4. Gradual phosphorylation of degron motifs culminates in M-Phase and targets the protein for degradation. Binding of the Mtw1Mis12 complex shields the proximal phospho-degron, protecting kinetochore-bound Ame1 from the degradation machinery. Artificially increasing degron strength partially suppresses the temperature-sensitivity of a cdc4 mutant, while overexpression of Ame1-Okp1 is toxic in SCF mutants, demonstrating the physiological importance of this mechanism. We propose that phospho-regulated clearance of excess CCAN subunits facilitates efficient centromere-dependent kinetochore assembly. Our results suggest a novel strategy for how phospho-degrons can be used to regulate the assembly of multi-subunit complexes.

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. Miriam Böhm

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-6054-1912
  2. Kerstin Killinger

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  3. Alexander Dudziak

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-5082-3468
  4. Pradeep Pant

    Computational Biochemistry, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-3890-1958
  5. Karolin Jänen

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  6. Simone Hohoff

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
  7. Karl Mechtler

    Research Institute of Molecular Pathology, Vienna, Austria
    Competing interests
    The authors declare that no competing interests exist.
  8. Mihkel Örd

    Institute of Technology, University of Tartu, Tartu, Estonia
    Competing interests
    The authors declare that no competing interests exist.
  9. Mart Loog

    Institute of Technology, University of Tartu, Tartu, Estonia
    Competing interests
    The authors declare that no competing interests exist.
  10. Elsa Sanchez-Garcia

    Computational Biochemisty, University of Duisburg-Essen, Essen, Germany
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-9211-5803
  11. Stefan Westermann

    Molecular Genetics I, University of Duisburg-Essen, Essen, Germany
    For correspondence
    Stefan.Westermann@uni-due.de
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6921-9113

Funding

Deutsche Forschungsgemeinschaft (WE-2886/2)

  • Miriam Böhm
  • Stefan Westermann

Deutsche Forschungsgemeinschaft (CRC1093)

  • Elsa Sanchez-Garcia
  • Stefan Westermann

Deutsche Forschungsgemeinschaft (CRC1430)

  • Elsa Sanchez-Garcia
  • Stefan Westermann

H2020 European Research Council (ERC consolidator grant 649124)

  • Mart Loog

Estonian Science Agency (Grant PRG550)

  • Mart Loog

Centre of Excellence for Molecular Cell Technologies (TK143)

  • Mart Loog

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

Reviewing Editor

  1. Silke Hauf, Virginia Tech, United States

Publication history

  1. Received: February 9, 2021
  2. Preprint posted: February 16, 2021 (view preprint)
  3. Accepted: July 24, 2021
  4. Accepted Manuscript published: July 26, 2021 (version 1)
  5. Version of Record published: August 5, 2021 (version 2)

Copyright

© 2021, Böhm 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. Miriam Böhm
  2. Kerstin Killinger
  3. Alexander Dudziak
  4. Pradeep Pant
  5. Karolin Jänen
  6. Simone Hohoff
  7. Karl Mechtler
  8. Mihkel Örd
  9. Mart Loog
  10. Elsa Sanchez-Garcia
  11. Stefan Westermann
(2021)
Cdc4 phospho-degrons allow differential regulation of Ame1CENP-U protein stability across the cell cycle
eLife 10:e67390.
https://doi.org/10.7554/eLife.67390
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