RESUMO
The anaphase-promoting complex (APC/C) is a multimeric RING E3 ubiquitin ligase that controls chromosome segregation and mitotic exit. Its regulation by coactivator subunits, phosphorylation, the mitotic checkpoint complex and interphase early mitotic inhibitor 1 (Emi1) ensures the correct order and timing of distinct cell-cycle transitions. Here we use cryo-electron microscopy to determine atomic structures of APC/C-coactivator complexes with either Emi1 or a UbcH10-ubiquitin conjugate. These structures define the architecture of all APC/C subunits, the position of the catalytic module and explain how Emi1 mediates inhibition of the two E2s UbcH10 and Ube2S. Definition of Cdh1 interactions with the APC/C indicates how they are antagonized by Cdh1 phosphorylation. The structure of the APC/C with UbcH10-ubiquitin reveals insights into the initiating ubiquitination reaction. Our results provide a quantitative framework for the design of future experiments to investigate APC/C functions in vivo.
Assuntos
Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Ubiquitinação , Ciclossomo-Complexo Promotor de Anáfase/química , Antígenos CD , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc1 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc10 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc11 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc3 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/química , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/metabolismo , Subunidade Apc8 do Ciclossomo-Complexo Promotor de Anáfase/ultraestrutura , Caderinas/química , Caderinas/metabolismo , Caderinas/ultraestrutura , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/ultraestrutura , Microscopia Crioeletrônica , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/metabolismo , Proteínas F-Box/química , Proteínas F-Box/metabolismo , Proteínas F-Box/ultraestrutura , Humanos , Lisina/metabolismo , Modelos Moleculares , Fosforilação , Ligação Proteica , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Especificidade por Substrato , Ubiquitina/química , Ubiquitina/metabolismo , Ubiquitina/ultraestrutura , Enzimas de Conjugação de Ubiquitina/química , Enzimas de Conjugação de Ubiquitina/metabolismo , Enzimas de Conjugação de Ubiquitina/ultraestruturaRESUMO
The ubiquitination of cell cycle regulatory proteins by the anaphase-promoting complex/cyclosome (APC/C) controls sister chromatid segregation, cytokinesis and the establishment of the G1 phase of the cell cycle. The APC/C is an unusually large multimeric cullin-RING ligase. Its activity is strictly dependent on regulatory coactivator subunits that promote APC/C-substrate interactions and stimulate its catalytic reaction. Because the structures of many APC/C subunits and their organization within the assembly are unknown, the molecular basis for these processes is poorly understood. Here, from a cryo-electron microscopy reconstruction of a human APC/C-coactivator-substrate complex at 7.4 Å resolution, we have determined the complete secondary structural architecture of the complex. With this information we identified protein folds for structurally uncharacterized subunits, and the definitive location of all 20 APC/C subunits within the 1.2 MDa assembly. Comparison with apo APC/C shows that the coactivator promotes a profound allosteric transition involving displacement of the cullin-RING catalytic subunits relative to the degron-recognition module of coactivator and APC10. This transition is accompanied by increased flexibility of the cullin-RING subunits and enhanced affinity for UBCH10-ubiquitin, changes which may contribute to coactivator-mediated stimulation of APC/C E3 ligase activity.