Localization of Cdc20 and MCC bound to APC/C, analyzed by negative-staining EM. (A) Distinct conformers of apo-APC/C isolated from cells with an active checkpoint. (B) Localization of recombinant Cdc20 (purple) bound to apo-APC/C. A model of Cdc20’s WD40 propeller is projected onto APC/C. (C) 3D structure of APC/C^MCC. MCC is labeled in red. (D) Superposition of the MCC (red) and Cdc20 (purple) densities shown from orthogonal directions.

MCC inhibits the binding of substrates to APC/C. (A) A tandem fragment of wild-type (wt) or D-box mutant (dm) fission yeast cyclin B was immobilized on glutathione beads at the indicated concentrations, and binding of APC/C from cells with checkpoints on or off was analyzed with SDS-PAGE immunoblotting. (B) APC/C from cells with checkpoints on or off was bound to beads coupled with antibody to Cdc27, and binding of soluble wild-type or D-box mutant cyclin B was analyzed in the same manner as in (A). (C) The binding of fulllength human securin to APC/C was analyzed in the same manner as in (B).

APC/C subunit topology and structural changes upon MCC binding. (A) APC/C subunits were localized with antibody labeling. Antibody epitopes are marked on the surface of the APC/C 3D model, and the labeling accuracy is indicated by the size of the differently colored areas. (B)Models of Apc1, Apc2, Apc4, Apc5, and Cdc27 were docked into the 3D density of the APC/C reconstruction. (C)The apo-APC/Cstructure in its most open conformation. (D)TheAPC/C^MCC structure with a closed conformation. (E) Conformational changes induced by MCC binding to APC/C.

Biochemical characterization of APC/C^MCC and apo-APC/C. (A) SDS-PAGE–silver-staining analysis of APC/C and MCC from cells in which the checkpoint was active (SC on) or inhibited (SC off). (B) Semiquantitative immunoblot analysis of APC/C from cells in which the checkpoint was on or off. (C and D) Cyclin B ubiquitylation activity of APC/C isolated from cells in which the checkpoint was on or off, analyzed with SDS-PAGE and autoradiography (C) and quantified (D). (E)Checkpoint activation reduces APC/C activity similarly toward prometaphase (cyclin A and Nek2A) and metaphase substrates (cyclin B and securin). Ratios of ubiquitiylation by the two APC/C samples were calculated for each substrate in three independent experiments. Error bars indicate SD of the mean. (F) Schematic representation of APC/C^MCC and apo-APC/C isolation from cells with an active checkpoint (reIP, reimmunoprecipitation). (G and H) Immunoblot analyses of complexes obtained in (F). (I) Cyclin B ubiquitylation activities of APC/C^Cdc20 from cells with an inactive checkpoint and of APC/C^MCC and apo-APC/C from cells with an active checkpoint. APC/C levels were normalized to Apc2. APC/C^MCC and apo-APC/C were preincubated with various concentrations of recombinant Cdc20 or Cdh1. Cyclin B ubiquitylation was quantified after 4- and 8-min reaction times. Data represent three independent experiments and error bars indicate SD of the mean.