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Structural basis for the subunit assembly of the anaphase-promoting complex

Nature volume 470, pages 227–232 (2011)Cite this article

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Abstract

The anaphase-promoting complex or cyclosome (APC/C) is an unusually large E3 ubiquitin ligase responsible for regulating defined cell cycle transitions. Information on how its 13 constituent proteins are assembled, and how they interact with co-activators, substrates and regulatory proteins is limited. Here, we describe a recombinant expression system that allows the reconstitution of holo APC/C and its sub-complexes that, when combined with electron microscopy, mass spectrometry and docking of crystallographic and homology-derived coordinates, provides a precise definition of the organization and structure of all essential APC/C subunits, resulting in a pseudo-atomic model for 70% of the APC/C. A lattice-like appearance of the APC/C is generated by multiple repeat motifs of most APC/C subunits. Three conserved tetratricopeptide repeat (TPR) subunits (Cdc16, Cdc23 and Cdc27) share related superhelical homo-dimeric architectures that assemble to generate a quasi-symmetrical structure. Our structure explains how this TPR sub-complex, together with additional scaffolding subunits (Apc1, Apc4 and Apc5), coordinate the juxtaposition of the catalytic and substrate recognition module (Apc2, Apc11 and Apc10 (also known as Doc1)), and TPR-phosphorylation sites, relative to co-activator, regulatory proteins and substrates.

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Figure 1: Recombinant APC/C and sub-complex assembly and purification and nano-electrospray mass spectrometry of SC8.
Figure 2: Three-dimensional EM structure comparisons of recombinant APC/C and APC/C sub-complexes.
Figure 3: Three-dimensional localization of TPR subunits and atomic coordinate docking.
Figure 4: Subunit organization and pseudo-atomic model of APC/C.

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Primary accessions

EMBL/GenBank/DDBJ

Data deposits

EM maps have been deposited in EMDB with accession numbers EMD-1815 (endogenous cryo-EM APC/CCdh1·D-box), EMD-1816 (endogenous apo APC/C), EMD-1841 (TPR6), EMD-1842 (APC/CΔCde27ΔApc9), EMD-1843 (Apc4–Apc5), EMD-1844 (recombinant apo APC/C), and EMD-1845 (SC8).

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Acknowledgements

This work was funded by a Cancer Research UK grant to D.B. and EU FP7 PROSPECTS (Proteomics Specification in Space and Time) Grant HEALTH-F4-2008-201648 to C.V.R. We thank F. Beuron for help with electron microscopy and Jing Yang for help with insect cell expression.

Author information

Authors and Affiliations

  1. Section of Structural Biology, Institute of Cancer Research, Chester Beatty Laboratories, 237 Fulham Road, London, SW3 6JB, UK

    Anne Schreiber, Ziguo Zhang, Radoslav I. Enchev, Eric H. Kong, Edward P. Morris, Paula C. A. da Fonseca & David Barford

  2. Department of Chemistry, University of Oxford, South Parks Road, Oxford, OX1 3TA, UK

    Florian Stengel & Carol V. Robinson

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  1. Anne Schreiber
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Contributions

All authors contributed to experimental design, data analysis and manuscript preparation. A.S. generated recombinant APC/C, APC/CΔCdc27ΔApc9, SC8 and Apc4–Apc5 complexes and (with R.I.E.) determined their EM structures. Z.Z. generated recombinant TPR6 and Cdc23–Apc13 and performed MALS. P.C.A.F. and E.H.K. collected and analysed TPR6 EM data and P.C.A.F. determined the TPR6 EM structure. A.S. performed ubiquitylation assays. A.S., R.I.E. and P.C.A.F. fitted coordinates. A.S. and F.S. performed mass spectrometry experiments and A.S., F.S. and C.V.R. analysed mass spectrometry data. E.P.M. helped analyse EM data. D.B. directed the project.

Corresponding author

Correspondence to David Barford.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Figures 1-20 with legends, Supplementary Tables 1-7 and additional references. (PDF 22487 kb)

Supplementary Movie 1

The movie shows the atomic coordinates of Cdc16-Cdc26, Cdc23, Cdc27, Apc2, (N terminal b-strand of Apc11), Apc10 and Cdh1 were fitted to the 11 Å cryo-EM map of the APC/CCdh1•D-box ternary complex represented in grey mesh. Symmetry related monomers of the Cdc23, Cdc16 and Cdc27 homo-dimers are represented in light and dark orange, red and green, respectively. Other subunits are: Cdh1 (purple), Apc2 (yellow), Apc10 (blue), Cdc26 (cyan), N-terminal b-strand of Apc11 (orange). Red spheres indicate the C-termini of Cdc16, Cdc23 and Apc10, whereas red and blue spheres in Cdc27 denote the N- and C-termini of the inter-TPR insert. The surface molecular boundaries of Apc1 (salmon) and Apc4-Apc5 (green) are indicated. (MOV 30504 kb)

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Schreiber, A., Stengel, F., Zhang, Z. et al. Structural basis for the subunit assembly of the anaphase-promoting complex. Nature 470, 227–232 (2011). https://doi.org/10.1038/nature09756

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