Structure of the Anaphase-Promoting Complex/Cyclosome Interacting with a Mitotic Checkpoint Complex

Abstract

Once all chromosomes are connected to the mitotic spindle (bioriented), anaphase is initiated by the protein ubiquitylation activity of the anaphase-promoting complex/cyclosome (APC/C) and its coactivator Cdc20 (APC/C(Cdc20)). Before chromosome biorientation, anaphase is delayed by a mitotic checkpoint complex (MCC) that inhibits APC/C(Cdc20). We used single-particle electron microscopy to obtain three-dimensional models of human APC/C in various functional states: bound to MCC, to Cdc20, or to neither (apo-APC/C). These experiments revealed that MCC associates with the Cdc20 binding site on APC/C, locks the otherwise flexible APC/C in a "closed" state, and prevents binding and ubiquitylation of a wide range of different APC/C substrates. These observations clarify the structural basis for the inhibition of APC/C by spindle checkpoint proteins.

biGBac enables rapid gene assembly for the expression of large multisubunit protein complexes

“…For example, by combining four of the five pBIG1 constructs that we had used for the APC/C-CDH1-EMI1-SKP1 construct (all except pBIG1e FW_I-5, which contains cDNAs encoding CDH1, EMI1, and SKP1; Table S3), we assembled a construct (pBIG2abcd FW_II-2) encoding the 14 core subunits of APC/C. By combining pBIG1a FW_I-1 and pBIG1b FW_I-2, we generated a construct encoding the six subunits of an APC/C subcomplex known as the "platform" (17)(18)(19). By combining pBIG1c and pBIG1d constructs FW_I-3 and FW_I-4 with "empty" pBIG1a and pBIG1b vectors, we assembled a construct encoding APC/C's "arc lamp" subcomplex (17)(18)(19).…”

“…By combining pBIG1a FW_I-1 and pBIG1b FW_I-2, we generated a construct encoding the six subunits of an APC/C subcomplex known as the "platform" (17)(18)(19). By combining pBIG1c and pBIG1d constructs FW_I-3 and FW_I-4 with "empty" pBIG1a and pBIG1b vectors, we assembled a construct encoding APC/C's "arc lamp" subcomplex (17)(18)(19). To generate mutated forms of the APC/C that are predicted to have deficiencies in coactivator and substrate binding (20,21), we generated pLIB vectors encoding APC3 N575A,L606A , APC8 N339A , and APC10 N144A,H145A,R149A,D150A and generated pBIG1d and pBIG2abcd constructs with combinations of these mutated subunits, as well as a version lacking the cDNA for APC10.…”

Systems Biology Modeling of Five Pathways for Regulation and Potent Inhibition of the Anaphase-Promoting Complex (APC/C): Pivotal Roles for MCC and BubR1

“…The APC can be inhibited in multiple ways, and complexes of APC together with either Cdc20:C-Mad2, Bub3:BubR1, Bub3:BubR1:Cdc20, MCC, or MCF2 have been found to be inactive (Eytan et al, 2008;Herzog et al, 2009;Kulukian et al, 2009a;Malureanu et al, 2009;Medema, 2009;Sudakin et al, 2001). However, the demand mechanisms for binding the inhibitory complexes to the APC are subject to current research.…”

“…The MCC may form more stably at unattached kinetochores and recruit APC from the cytoplasm (Herzog et al, 2009). The MCC subcomplexes Bub3:BubR1:Cdc20 and Cdc20:C-Mad2 can bind to the APC independently from unattached kinetochores, although their binding may be facilitated in a kinetochore-dependent manner (Kulukian et al, 2009a;Malureanu et al, 2009;Medema 2009).…”

Understanding the structural basis for controlling chromosome division

“…One role of Mad2 is to order the N-terminal region of Mad3, incorporating the KEN motif, and to correctly position Mad3 onto Cdc20. To understand how the MCC regulates the APC/C through blocking D box and KEN box recognition, we docked the MCC structure [34] and pseudo-atomic model of the APC/C [18] into the cryo-EM map of the human APC/C-MCC complex [54]. This map is at around 25-20 Å resolution.…”