Time course of activation of APC/C and disassembly of MCC during exit from mitotic checkpoint. (A) Kinetics of activation of APC/C. Checkpoint extracts were incubated with ATP as described in Methods for the preparation of activated extracts. After incubation for the time periods indicated, APC/C was isolated on anti-Cdc27 beads, and the ligation of ¹²⁵I-cyclin to ubiquitin was determined in samples of 1-μl beads incubated at 23°C for 20 min without the addition of exogenous Cdc20. The first lane on the left is a sample incubated without a source of APC/C. Numbers on the right indicate the position of molecular mass marker proteins (kDa). (B) Quantitation of results from A. (C) Kinetics of decay of MCC components in salt eluates of APC/C immunoprecipitates. Extracts were incubated for the time periods indicated and then adsorbed to anti-cdc27 beads as in A. Subsequently, beads were subjected to elution with 0.3 M KCl as described in ref. 12. Salt eluates were concentrated by ultrafiltration to one-half of the volume of extracts. Samples of 5 μl were subjected to immunoblotting. (D) Quantitation of results from C. Results were expressed as the percentage of the value at time 0. (E) Kinetics of dissociation of Mad2 and BubR1 from Cdc20. Checkpoint extracts were incubated with ATP for the indicated times as in A and then were subjected to immunoprecipitation with a polyclonal anti-Cdc20 antibody. Immunoprecipitates were analyzed by quantitative immunoblotting.

High concentrations of Cdc20 overcome the effect of inhibitors on APC/C activity. (A) Effect of Cdc20 on the degradation of securin in checkpoint extracts. The degradation of [³⁵S]-securin in extracts from nocodazole-arrested cells was followed as described in ref. 12, except that 10 μM staurosporine was added to all incubations. Where indicated, 0.1 μM recombinant Cdc20 was supplemented. (B) Influence of high-salt wash of immunoprecipitated APC/C on its sensitivity to stimulation by Cdc20. APC/C from checkpoint extracts was adsorbed to anti-Cdc27 beads. Part of beads was washed with 0.3 M KCl, as described in ref. 12. Another portion (Untreated) was subjected to similar washes without KCl. The ligation of ¹²⁵I-cyclin to ubiquitin by both preparations was determined in the absence or presence of 50 nM Cdc20, after incubation (23°C with shaking at 1,000 rpm) for the time periods indicated. (C) MCF2 and MCC inhibit APC/C in a manner antagonistic to Cdc20. Ligation of ¹²⁵I-cyclin to ubiquitin was determined as described in Methods, in the presence of 0.7 μl of highly purified APC/C (including purification on MonoQ) and Cdc20 at the concentrations indicated. Where indicated, samples of 1.5 μl of MCC or MCF2 (Fig. 2A) were added. (D) Results from C were calculated as inhibition of APC/C activity relative to the corresponding sample without inhibitor.

Resolution of inhibitory factors associated with APC/C in the checkpoint-arrested state. (A) Separation of MCC and MCF2 by chromatography on MonoQ. Salt eluate of APC/C immunoprecipitate from checkpoint-arrested extract was subjected to chromatography on MonoQ as described under Methods. Samples of 2 μl of column fractions were tested for inhibition of APC/C^Cdc20 activity (see Methods). Results are expressed as percentage decrease in activity relative to that without inhibitor. Samples of 3 μl of column fractions were used for quantitative immunoblotting for BubR1 and Cdc20, as described in Methods. MCC eluted at 260 mM NaCl and MCF2 at 210 mM NaCl. The indicated column fractions of MCC and MCF2 were pooled and used for further experiments. (B) Chromatography on MonoQ of salt eluate of APC/C immunoprecipitate from activated extracts. Samples of column fractions were analyzed as in A. The regions where MCC and MCF2 eluted in A are marked by dotted lines. (C) Immunoprecipitation of preparations of MCC and MCF2 with antibodies against Bubr1 and Cdc20. Samples of 30 μl of pooled MCC and MCF2 fractions (from a MonoQ separation similar to that shown in A) were subjected to immunoprecipitation with 2 μg of affinity-purified polyclonal antibodies against BubR1 or Cdc20 adsorbed to 10 μl of Affi-Prep protein A beads (Bio-Rad). The control was a similar amount of nonimmune rabbit IgG. Equal samples of precipitates (P) and supernatants (S) were subjected to immunoblotting with the indicated monoclonal antibodies. The blot on the right was subjected to longer exposure than that on the left, to show more clearly residual BubR1 in MCF2. (D) Effects of immunodepletion on APC/C-inhibitory activities of MCC and MCF2. Samples of 3 μl of the supernatants of the immunoprecipitations shown in C were tested for the inhibition of APC/C^Cdc20, as described under Methods.