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APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Nature Cell Biology volume 13, pages 1234–1243 (2011)Cite this article

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Abstract

Faithful chromosome segregation during mitosis depends on the spindle assembly checkpoint (SAC), which monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the anaphase-promoting complex, or cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important for the understanding of how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator CDC20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated CDC20 remain ‘locked’ onto the APC/C, which prevents the ubiquitylation and degradation of cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of CDC20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores.

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Figure 1: APC15 is a subunit of the human APC/C.
Figure 2: APC15 is an APC/C subunit required for timely entry into anaphase.
Figure 3: APC15 depletion causes a SAC-dependent delay in mitosis but does not affect APC/C activity.
Figure 4: APC15 is required for the turnover of MAD2, BUBR1, BUB3 and CDC20 on the APC/C during prometaphase.
Figure 5: APC15 is required for the release of MAD2, BUBR1 and BUB3 from the APC/C after the SAC had been satisfied.
Figure 6: Ubiquitylation of CDC20 is not required to release MCCs from the APC/C.
Figure 7: Ubiquitylation contributes to the release of MCCs from the APC/C.

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Acknowledgements

We thank S. Taylor for the HeLa FRT cell line, A. Musacchio for the anti-MAD2 and anti-p31comet antibodies and for communicating results before publication, T. Hunt for the anti-APC3 antibody, members of our laboratory for discussions and O. Nashchekina, F. Walton, E. Russe and S. Wieser for help with somatic cell knock-ins, mass spectrometry, statistical and flow cytometry analyses, respectively. J.M. was supported by FEBS and EMBO fellowships. This work was supported by a project grant from the BBSRC and by a Programme grant from Cancer Research UK to J.P. and core grant support to the Sanger Institute (M.O.C. and J.S.C.) from The Wellcome Trust (grant number 079643/Z/06/Z).

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  1. Philippe Collin and Mark O. Collins: These authors contributed equally

Authors and Affiliations

  1. The Gurdon Institute and Department of Zoology, Tennis Court Road, Cambridge CB2 1QN, UK

    Jörg Mansfeld, Philippe Collin & Jonathon Pines

  2. Proteomic Mass Spectrometry Laboratory, The Wellcome Trust Sanger Institute, Hinxton, Cambridge CB10 1SA, UK

    Mark O. Collins & Jyoti S. Choudhary

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Contributions

J.M. carried out all of the experiments, P.C. generated the RPE1 cyclin-A2– and B1–Venus knock-in cell lines, M.O.C. and J.S.C. carried out the mass spectrometry that identified APC15 and the CDC20 ubiquitylation site. J.M. and J.P. designed the experiments and wrote the paper. All authors contributed to the interpretation of the results.

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Correspondence to Jonathon Pines.

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Mansfeld, J., Collin, P., Collins, M. et al. APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment. Nat Cell Biol 13, 1234–1243 (2011). https://doi.org/10.1038/ncb2347

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