Figure 3. Maintaining the association of BubR1^N with Cdc20 does not require the Mad2-Cdc20 interaction.

(A) Schematic of a Mad2 variant modified to contain a HRV 3C cleavage site. (B) Immunoblot showing HRV 3C-mediated cleavage of Mad2^HRV in vitro. Mad2^HRV was incubated at room temperature for 20 min in the presence or absence of HRV 3C protease. (C) After incubation with BubR1^N, Bub3, Mad2^HRV and Cdc20, APC/C was affinity-purified to remove unbound components. Following incubation with or without HRV 3C protease, dissociated components were washed away and APC/C was peptide-eluted from beads and analyzed for associated components by immunoblotting. The supernatant from the protease digestion mixture was collected and analyzed for release of the digested Mad2^HRV fragments. (D) The amount of APC/C-bound BubR1^N or Mad2^HRV in (C) was measured. Values are plotted as the molar ratio of APC/C-bound Mad2^HRV compared with APC/C-bound BubR1^N. The level of APC/C-bound BubR1^N without protease treatment was set to 1. Bars represent the mean. Error bars represent SEM (n=3). (E) Schematic of an HRV 3C cleavable variant of BubR1^N. (F) HRV 3C-mediated digestion of the cleavable BubR1^N in vitro. Cleavable BubR1^N was incubated at room temperature for 20 min in the presence or absence of HRV 3C prior to analysis. (G–H) Effect of forced BubR1^N release on the association of Mad2 with APC/C^Cdc20. (G) Experimental procedure is the same as in (C). (H) Values were plotted as the molar ratio compared with APC/C-bound BubR1^N-HRV. The level of APC/C-bound cleavable BubR1^N without the protease treatment was set to 1. Bars represent the mean. Error bars represent SEM (n=3). ***=P<0.0001. (I) Assay of Mad2 binding to APC/C in the presence and absence of Cdc20 (see also Fig. S3).