Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structure of the mitotic checkpoint complex

Abstract

In mitosis, the spindle assembly checkpoint (SAC) ensures genome stability by delaying chromosome segregation until all sister chromatids have achieved bipolar attachment to the mitotic spindle. The SAC is imposed by the mitotic checkpoint complex (MCC), whose assembly is catalysed by unattached chromosomes and which binds and inhibits the anaphase-promoting complex/cyclosome (APC/C), the E3 ubiquitin ligase that initiates chromosome segregation. Here, using the crystal structure of Schizosaccharomyces pombe MCC (a complex of mitotic spindle assembly checkpoint proteins Mad2, Mad3 and APC/C co-activator protein Cdc20), we reveal the molecular basis of MCC-mediated APC/C inhibition and the regulation of MCC assembly. The MCC inhibits the APC/C by obstructing degron recognition sites on Cdc20 (the substrate recruitment subunit of the APC/C) and displacing Cdc20 to disrupt formation of a bipartite D-box receptor with the APC/C subunit Apc10. Mad2, in the closed conformation (C-Mad2), stabilizes the complex by optimally positioning the Mad3 KEN-box degron to bind Cdc20. Mad3 and p31comet (also known as MAD2L1-binding protein) compete for the same C-Mad2 interface, which explains how p31comet disrupts MCC assembly to antagonize the SAC. This study shows how APC/C inhibition is coupled to degron recognition by co-activators.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.

$32.00

All prices are NET prices.

Figure 1: Structure of S. pombe MCC trimer.
Figure 2: Details of Cdc20–Mad2–Mad3 interactions.
Figure 3: The KEN box binds to a conserved surface at the centre of the top side of the WD40 domain.
Figure 4: The D box binds in an extended conformation to a conserved inter-blade channel on the Cdc20 WD40 domain.
Figure 5: Pseudo-atomic structure of human APC/CMCC.

Accession codes

Primary accessions

Protein Data Bank

Data deposits

The MCC coordinates have been deposited in the Protein Data Bank under accession number 4aez.

References

  1. Musacchio, A. & Salmon, E. D. The spindle-assembly checkpoint in space and time. Nature Rev. Mol. Cell Biol. 8, 379–393 (2007)

    CAS  Google Scholar 

  2. Kim, S. & Yu, H. Mutual regulation between the spindle checkpoint and APC/C. Semin. Cell Dev. Biol. (2011)

  3. Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517 (1991)

    CAS  Google Scholar 

  4. Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519–531 (1991)

    CAS  Google Scholar 

  5. Li, Y., Gorbea, C., Mahaffey, D., Rechsteiner, M. & Benezra, R. MAD2 associates with the cyclosome/anaphase-promoting complex and inhibits its activity. Proc. Natl Acad. Sci. USA 94, 12431–12436 (1997)

    CAS  Google Scholar 

  6. Fang, G., Yu, H. & Kirschner, M. W. The checkpoint protein MAD2 and the mitotic regulator CDC20 form a ternary complex with the anaphase-promoting complex to control anaphase initiation. Genes Dev. 12, 1871–1883 (1998)

    CAS  Google Scholar 

  7. Hwang, L. H. et al. Budding yeast Cdc20: a target of the spindle checkpoint. Science 279, 1041–1044 (1998)

    CAS  Google Scholar 

  8. Kim, S. H., Lin, D. P., Matsumoto, S., Kitazono, A. & Matsumoto, T. Fission yeast Slp1: an effector of the Mad2-dependent spindle checkpoint. Science 279, 1045–1047 (1998)

    CAS  Google Scholar 

  9. Kallio, M., Weinstein, J., Daum, J. R., Burke, D. J. & Gorbsky, G. J. Mammalian p55CDC mediates association of the spindle checkpoint protein Mad2 with the cyclosome/anaphase-promoting complex, and is involved in regulating anaphase onset and late mitotic events. J. Cell Biol. 141, 1393–1406 (1998)

    CAS  Google Scholar 

  10. Hardwick, K. G., Johnston, R. C., Smith, D. L. & Murray, A. W. MAD3 encodes a novel component of the spindle checkpoint which interacts with Bub3p, Cdc20p, and Mad2p. J. Cell Biol. 148, 871–882 (2000)

    CAS  Google Scholar 

  11. Tang, Z., Bharadwaj, R., Li, B. & Yu, H. Mad2-Independent inhibition of APCCdc20 by the mitotic checkpoint protein BubR1. Dev. Cell 1, 227–237 (2001)

    CAS  Google Scholar 

  12. Fang, G. Checkpoint protein BubR1 acts synergistically with Mad2 to inhibit anaphase-promoting complex. Mol. Biol. Cell 13, 755–766 (2002)

    CAS  Google Scholar 

  13. Sudakin, V., Chan, G. K. & Yen, T. J. Checkpoint inhibition of the APC/C in HeLa cells is mediated by a complex of BUBR1, BUB3, CDC20, and MAD2. J. Cell Biol. 154, 925–936 (2001)

    CAS  Google Scholar 

  14. Herzog, F. et al. Structure of the anaphase-promoting complex/cyclosome interacting with a mitotic checkpoint complex. Science 323, 1477–1481 (2009)

    CAS  Google Scholar 

  15. Burton, J. L. & Solomon, M. J. Mad3p, a pseudosubstrate inhibitor of APCCdc20 in the spindle assembly checkpoint. Genes Dev. 21, 655–667 (2007)

    CAS  Google Scholar 

  16. Davenport, J., Harris, L. D. & Goorha, R. Spindle checkpoint function requires Mad2-dependent Cdc20 binding to the Mad3 homology domain of BubR1. Exp. Cell Res. 312, 1831–1842 (2006)

    CAS  Google Scholar 

  17. Kulukian, A., Han, J. S. & Cleveland, D. W. Unattached kinetochores catalyze production of an anaphase inhibitor that requires a Mad2 template to prime Cdc20 for BubR1 binding. Dev. Cell 16, 105–117 (2009)

    CAS  Google Scholar 

  18. Nilsson, J., Yekezare, M., Minshull, J. & Pines, J. The APC/C maintains the spindle assembly checkpoint by targeting Cdc20 for destruction. Nature Cell Biol. 10, 1411–1420 (2008)

    CAS  Google Scholar 

  19. Luo, X. & Yu, H. Protein metamorphosis: the two-state behavior of Mad2. Structure 16, 1616–1625 (2008)

    CAS  Google Scholar 

  20. Luo, X. et al. Structure of the Mad2 spindle assembly checkpoint protein and its interaction with Cdc20. Nature Struct. Biol. 7, 224–229 (2000)

    CAS  Google Scholar 

  21. Luo, X., Tang, Z., Rizo, J. & Yu, H. The Mad2 spindle checkpoint protein undergoes similar major conformational changes upon binding to either Mad1 or Cdc20. Mol. Cell 9, 59–71 (2002)

    Google Scholar 

  22. Sironi, L. et al. Crystal structure of the tetrameric Mad1–Mad2 core complex: implications of a ‘safety belt’ binding mechanism for the spindle checkpoint. EMBO J. 21, 2496–2506 (2002)

    CAS  Google Scholar 

  23. De Antoni, A. et al. The Mad1/Mad2 complex as a template for Mad2 activation in the spindle assembly checkpoint. Curr. Biol. 15, 214–225 (2005)

    CAS  Google Scholar 

  24. Barford, D. Structure, function and mechanism of the anaphase promoting complex (APC/C). Q. Rev. Biophys. 44, 153–190 (2011)

    CAS  Google Scholar 

  25. Glotzer, M., Murray, A. W. & Kirschner, M. W. Cyclin is degraded by the ubiquitin pathway. Nature 349, 132–138 (1991)

    CAS  Google Scholar 

  26. Pfleger, C. M. & Kirschner, M. W. The KEN box: an APC recognition signal distinct from the D box targeted by Cdh1. Genes Dev. 14, 655–665 (2000)

    CAS  Google Scholar 

  27. King, E. M., van der Sar, S. J. & Hardwick, K. G. Mad3 KEN boxes mediate both Cdc20 and Mad3 turnover, and are critical for the spindle checkpoint. PLoS ONE 2, e342 (2007)

    Google Scholar 

  28. Sczaniecka, M. et al. The spindle checkpoint functions of Mad3 and Mad2 depend on a Mad3 KEN box-mediated interaction with Cdc20-anaphase-promoting complex (APC/C). J. Biol. Chem. 283, 23039–23047 (2008)

    CAS  Google Scholar 

  29. Pan, J. & Chen, R. H. Spindle checkpoint regulates Cdc20p stability in Saccharomyces cerevisiae. Genes Dev. 18, 1439–1451 (2004)

    CAS  Google Scholar 

  30. Habu, T., Kim, S. H., Weinstein, J. & Matsumoto, T. Identification of a MAD2-binding protein, CMT2, and its role in mitosis. EMBO J. 21, 6419–6428 (2002)

    CAS  Google Scholar 

  31. Yang, M. et al. p31comet blocks Mad2 activation through structural mimicry. Cell 131, 744–755 (2007)

    CAS  Google Scholar 

  32. Reddy, S. K., Rape, M., Margansky, W. A. & Kirschner, M. W. Ubiquitination by the anaphase-promoting complex drives spindle checkpoint inactivation. Nature 446, 921–925 (2007)

    CAS  Google Scholar 

  33. D’Arcy, S., Davies, O. R., Blundell, T. L. & Bolanos-Garcia, V. M. Defining the molecular basis of BubR1 kinetochore interactions and APC/C-CDC20 inhibition. J. Biol. Chem. 285, 14764–14776 (2010)

    Google Scholar 

  34. Malureanu, L. A. et al. BubR1 N terminus acts as a soluble inhibitor of cyclin B degradation by APC/C(Cdc20) in interphase. Dev. Cell 16, 118–131 (2009)

    CAS  Google Scholar 

  35. Schwab, M., Neutzner, M., Mocker, D. & Seufert, W. Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC. EMBO J. 20, 5165–5175 (2001)

    CAS  Google Scholar 

  36. Vodermaier, H. C., Gieffers, C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J. M. TPR subunits of the anaphase-promoting complex mediate binding to the activator protein CDH1. Curr. Biol. 13, 1459–1468 (2003)

    CAS  Google Scholar 

  37. Mapelli, M., Massimiliano, L., Santaguida, S. & Musacchio, A. The Mad2 conformational dimer: structure and implications for the spindle assembly checkpoint. Cell 131, 730–743 (2007)

    CAS  Google Scholar 

  38. Tipton, A. R. et al. BUBR1 and closed MAD2 (C-MAD2) interact directly to assemble a functional mitotic checkpoint complex. J. Biol. Chem. 286, 21173–21179 (2011)

    CAS  Google Scholar 

  39. Kraft, C., Vodermaier, H. C., Maurer-Stroh, S., Eisenhaber, F. & Peters, J. M. The WD40 propeller domain of Cdh1 functions as a destruction box receptor for APC/C substrates. Mol. Cell 18, 543–553 (2005)

    CAS  Google Scholar 

  40. Carroll, C. W., Enquist-Newman, M. & Morgan, D. O. The APC subunit Doc1 promotes recognition of the substrate destruction box. Curr. Biol. 15, 11–18 (2005)

    CAS  Google Scholar 

  41. da Fonseca, P. C. et al. Structures of APC/C(Cdh1) with substrates identify Cdh1 and Apc10 as the D-box co-receptor. Nature 470, 274–278 (2011)

    CAS  Google Scholar 

  42. King, R. W., Glotzer, M. & Kirschner, M. W. Mutagenic analysis of the destruction signal of mitotic cyclins and structural characterization of ubiquitinated intermediates. Mol. Biol. Cell 7, 1343–1357 (1996)

    CAS  Google Scholar 

  43. Choi, E., Dial, J. M., Jeong, D. E. & Hall, M. C. Unique D box and KEN box sequences limit ubiquitination of Acm1 and promote pseudosubstrate inhibition of the anaphase-promoting complex. J. Biol. Chem. 283, 23701–23710 (2008)

    CAS  Google Scholar 

  44. Ostapenko, D., Burton, J. L., Wang, R. & Solomon, M. J. Pseudosubstrate inhibition of the anaphase-promoting complex by Acm1: regulation by proteolysis and Cdc28 phosphorylation. Mol. Cell. Biol. 28, 4653–4664 (2008)

    CAS  Google Scholar 

  45. Kimata, Y. et al. A mutual inhibition between APC/C and its substrate Mes1 required for meiotic progression in fission yeast. Dev. Cell 14, 446–454 (2008)

    CAS  Google Scholar 

  46. Schreiber, A. et al. Structural basis for the subunit assembly of the anaphase-promoting complex. Nature 470, 227–232 (2011)

    CAS  Google Scholar 

  47. Zhang, Y. & Lees, E. Identification of an overlapping binding domain on Cdc20 for Mad2 and anaphase-promoting complex: model for spindle checkpoint regulation. Mol. Cell. Biol. 21, 5190–5199 (2001)

    CAS  Google Scholar 

  48. Kimata, Y., Baxter, J. E., Fry, A. M. & Yamano, H. A role for the Fizzy/Cdc20 family of proteins in activation of the APC/C distinct from substrate recruitment. Mol. Cell 32, 576–583 (2008)

    CAS  Google Scholar 

  49. Xia, G. et al. Conformation-specific binding of p31(comet) antagonizes the function of Mad2 in the spindle checkpoint. EMBO J. 23, 3133–3143 (2004)

    CAS  Google Scholar 

  50. Westhorpe, F. G., Tighe, A., Lara-Gonzalez, P. & Taylor, S. S. p31comet-mediated extraction of Mad2 from the MCC promotes efficient mitotic exit. J. Cell Sci. 124, 3905–3916 (2011)

    CAS  Google Scholar 

  51. Berger, I., Fitzgerald, D. J. & Richmond, T. J. Baculovirus expression system for heterologous multiprotein complexes. Nature Biotechnol. 22, 1583–1587 (2004)

    CAS  Google Scholar 

  52. Sironi, L. et al. Mad2 binding to Mad1 and Cdc20, rather than oligomerization, is required for the spindle checkpoint. EMBO J. 20, 6371–6382 (2001)

    CAS  Google Scholar 

  53. Evans, P. Scaling and assessment of data quality. Acta Crystallogr. D 62, 72–82 (2006)

    Google Scholar 

  54. Navaza, J. Implementation of molecular replacement in AMoRe. Acta Crystallogr. D 57, 1367–1372 (2001)

    CAS  Google Scholar 

  55. Yang, M. et al. Insights into mad2 regulation in the spindle checkpoint revealed by the crystal structure of the symmetric mad2 dimer. PLoS Biol. 6, e50 (2008)

    Google Scholar 

  56. Song, J. J. & Kingston, R. E. WDR5 interacts with mixed lineage leukemia (MLL) protein via the histone H3-binding pocket. J. Biol. Chem. 283, 35258–35264 (2008)

    CAS  Google Scholar 

  57. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Google Scholar 

  58. Adams, P. D. et al. PHENIX: building new software for automated crystallographic structure determination. Acta Crystallogr. D 58, 1948–1954 (2002)

    Google Scholar 

  59. Painter, J. & Merritt, E. A. Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D 62, 439–450 (2006)

    Google Scholar 

  60. Davis, I. W. et al. MolProbity: all-atom contacts and structure validation for proteins and nucleic acids. Nucleic Acids Res. 35, W375–W383 (2007)

    Google Scholar 

  61. Passmore, L. A., Barford, D. & Harper, J. W. Purification and assay of the budding yeast anaphase-promoting complex. Methods Enzymol. 398, 195–219 (2005)

    CAS  Google Scholar 

Download references

Acknowledgements

This work was funded by a Cancer Research UK grant to D.B. and an ICR studentship to W.C.H.C. We thank J. Yang and J. He for advice, and staff at the Diamond Light Source beamline I02 for help with data collection.

Author information

Authors and Affiliations

Authors

Contributions

W.C.H.C. cloned, purified and crystallized the S. pombe MCC, and performed the biochemistry and mutagenesis experiments. W.C.H.C., K.K. and D.B. collected the X-ray diffraction data. K.K. determined the complex structure and modelled the D-box and KEN–D peptides. Z.Z. advised on cloning strategies of the MCC. E.H.K. provided purified endogenous APC/C. W.C.H.C., K.H.K. and D.B. docked crystal coordinates into the human APC/CMCC electron-microscope map. W.C.H.C. and D.B. wrote the manuscript and the others authors provided editorial input.

Corresponding author

Correspondence to David Barford.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

This file contains Supplementary Table 1 and Supplementary Figures 1-10. (PDF 1792 kb)

PowerPoint slides

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Chao, W., Kulkarni, K., Zhang, Z. et al. Structure of the mitotic checkpoint complex. Nature 484, 208–213 (2012). https://doi.org/10.1038/nature10896

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nature10896

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing