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.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Amino Acid Motifs
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Anaphase-Promoting Complex-Cyclosome
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Animals
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Apc2 Subunit, Anaphase-Promoting Complex-Cyclosome
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Apc5 Subunit, Anaphase-Promoting Complex-Cyclosome
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Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome
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Biocatalysis
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Cell Line
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Holoenzymes / chemistry
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Holoenzymes / metabolism
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Holoenzymes / ultrastructure
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Mass Spectrometry
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Microscopy, Electron
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Models, Molecular
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Molecular Weight
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Protein Binding
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Protein Conformation
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Protein Subunits / chemistry
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Protein Subunits / isolation & purification
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Protein Subunits / metabolism
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Recombinant Proteins / chemistry
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Recombinant Proteins / metabolism
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Recombinant Proteins / ultrastructure
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Saccharomyces cerevisiae / chemistry
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / isolation & purification
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Saccharomyces cerevisiae Proteins / metabolism
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Saccharomyces cerevisiae Proteins / ultrastructure
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Scattering, Radiation
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Schizosaccharomyces / chemistry
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Structure-Activity Relationship
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Substrate Specificity
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Ubiquitin-Protein Ligase Complexes / chemistry*
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Ubiquitin-Protein Ligase Complexes / metabolism*
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Ubiquitin-Protein Ligase Complexes / ultrastructure
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Ubiquitination
Substances
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APC5 protein, S cerevisiae
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Apc5 Subunit, Anaphase-Promoting Complex-Cyclosome
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Apc8 Subunit, Anaphase-Promoting Complex-Cyclosome
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CDC23 protein, S cerevisiae
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Holoenzymes
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Protein Subunits
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Ubiquitin-Protein Ligase Complexes
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APC2 protein, S cerevisiae
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Anaphase-Promoting Complex-Cyclosome
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Apc2 Subunit, Anaphase-Promoting Complex-Cyclosome