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APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex

Nature Structural & Molecular Biology volume 19, pages 1116–1123 (2012)Cite this article

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Abstract

The anaphase-promoting complex/cyclosome (APC/C) bound to CDC20 (APC/CCDC20) initiates anaphase by ubiquitylating B-type cyclins and securin. During chromosome bi-orientation, CDC20 assembles with MAD2, BUBR1 and BUB3 into a mitotic checkpoint complex (MCC) that inhibits substrate recruitment to the APC/C. APC/C activation depends on MCC disassembly, which was proposed to require CDC20 autoubiquitylation. Here we characterize APC15, a human APC/C subunit related to yeast Mnd2. APC15 is located near APC/C's MCC binding site; it is required for APC/C-bound MCC (APC/CMCC)-dependent CDC20 autoubiquitylation and degradation and for timely anaphase initiation but is dispensable for substrate ubiquitylation by APC/CCDC20 and APC/CCDH1. Our results support the model wherein MCC is continuously assembled and disassembled to enable rapid activation of APC/CCDC20 and CDC20 autoubiquitylation promotes MCC disassembly. We propose that APC15 and Mnd2 negatively regulate APC/C coactivators and report generation of recombinant human APC/C.

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Figure 1: C11ORF51 (APC15) is a subunit of APC/C's platform domain.
Figure 2: APC15 depletion causes delay in mitosis.
Figure 3: APC15 is required for the turnover of BUBR1, CDC20 and MAD2 proteins on APC/C during prometaphase in the presence of an active checkpoint.
Figure 4: APC15 is required for MCC disassembly in vivo and in vitro.
Figure 5: CDC20 turnover in prometaphase depends on APC15.
Figure 6: APC15 is not essential for cyclin-B1 and CDC20 ubiquitylation by APC/CCDH1.
Figure 7: APC15 is required for CDC20 autoubiquitylation by APC/CMCC.

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Acknowledgements

Research in the laboratory of J.-M.P is supported by Boehringer Ingelheim, the Vienna Science and Technology Fund (WWTF LS09-13), the Laura Bassi Center for Optimized Structural Studies (FFG 822736) and the Austrian Science Fund (FWF special research program SFB F34 'Chromosome Dynamics', grant W1221 'DK: Structure and Interaction of Biological Macromolecules' and Wittgenstein award Z196-B20). Research in the laboratories of J.-M.P. and A.A.H. is supported by the European Community's Seventh Framework Programme (FP7/2007-2013) under grant agreement no. 241548 (MitoSys). Research in the laboratory of B.A.S. is supported by the American Lebanese Syrian Associated Charities/St. Jude and the Howard Hughes Medical Institute. Research in the laboratory of H. Stark is supported by the German Research Foundation (DFG) under grant agreement SFB860. Research in the laboratory of K.M. is supported by European Community's Seventh Framework Programme under grant agreement no. 262067 (PRIME-XS). Y.T. was supported by the Japan Society for the Promotion of Science (Postdoctoral Fellowship for Research Abroad). N.G.B. is supported as a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research.

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  1. Kristina Uzunova, Billy T Dye and Hannelore Schutz: These authors contributed equally to this work.

Authors and Affiliations

  1. Research Institute of Molecular Pathology, Vienna, Austria

    Kristina Uzunova, Hannelore Schutz, Rene Ladurner, Georg Petzold, Marc A Jarvis, Maria Novatchkova, Karl Mechtler & Jan-Michael Peters

  2. Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Billy T Dye, Nicholas G Brown & Brenda A Schulman

  3. Howard Hughes Medical Institute, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Billy T Dye & Brenda A Schulman

  4. Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany

    Yusuke Toyoda, Ina Poser & Anthony A Hyman

  5. Max Planck Institute for Biophysical Chemistry, Göttingen, Germany

    Holger Stark

  6. Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universität, Göttingen, Germany

    Holger Stark

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Contributions

B.A.S. and J.-M.P. planned and supervised the project. K.U., B.T.D., H. Schutz, R.L., G.P., Y.T., M.A.J., N.G.B. and I.P. designed the experiments. K.U. performed experiments on APC15 function. B.T.D. and N.G.B. performed experiments on recombinant human APC/C. M.A.J. and G.P. established protocols for coactivator protein purification. H. Schutz performed experiments on APC15 being an APC/C core subunit. R.L. performed IFM experiments. G.P. performed antibody labeling and contributed to EM experiments. Y.T. performed time-lapse microscopy. I.P. and A.A.H. created LAP-tagged APC15. M.N. performed APC15 homology searches. K.M. performed mass spectrometry. H. Stark performed EM experiments, calculated 3D EM structures and analyzed APC15 antibody labeling. K.U., R.L., G.P., B.T.D., N.G.B., B.A.S. and J.-M.P. wrote the paper.

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Correspondence to Brenda A Schulman or Jan-Michael Peters.

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Uzunova, K., Dye, B., Schutz, H. et al. APC15 mediates CDC20 autoubiquitylation by APC/CMCC and disassembly of the mitotic checkpoint complex. Nat Struct Mol Biol 19, 1116–1123 (2012). https://doi.org/10.1038/nsmb.2412

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