Fig. 1.

Cleavage of ATP at the β–γ bond is required for the release of APC/C from the checkpoint-inhibited state and for the disassembly of MCC. (A) Effects of incubation of extracts with ATP or its analogues on the release of APC/C from inhibition by the mitotic checkpoint. Extracts from nocodazole-arrested cells were incubated with the indicated additions as described under Methods. APC/C was isolated by immunoprecipitation with anti-Cdc27 and its activity in the ligation of ¹²⁵I-cyclin to ubiquitin was determined. (B) Effects of ATP or its analogues on the phosphorylation state of Cdc27 and on the degradation of endogenous cyclin B. Samples of extracts (30 μg of protein) from the incubations described in A were blotted with antibodies to the indicated proteins. (C) Influence of ATP and its analogues on the release of MCC components from APC/C. Extracts were incubated as described in A and were subjected to immunoprecipitation with anti-Cdc27. Samples were resolved by SDS-PAGE and were blotted with the antibodies indicated. Blots were also probed for Cdc27 as the loading control, following phosphatase treatment of samples as described under Methods. (D) Quantitation of data from C. (E) Effects of ATP and its analogues on the dissociation of free MCC. Extracts were incubated as in A and then were subjected to sequential immunoprecipitations as described in Methods. Samples from anti-Cdc20 (Upper) and anti-BubR1 (Lower) immunoprecipitations were immunoblotted as indicated.