Figure 5.

The BubR1 Loop Is Required for APC/C Inhibition In Vitro

(A) BubR1^wt:Bub3 as well as the two loop mutants BubR1^B1-LL:Bub3 and BubR1^ΔLL:Bub3 interact in size exclusion chromatography with the other two MCC components Cdc20 and Mad2. In the chromatogram, the height of elution curves for the three different MCC complexes were rescaled to match that of MCC containing BubR1^ΔLL, which emphasizes the remarkable similarity of the elution profiles, an indication that the different MCC complexes are structurally stable and virtually identical. Vertical dashed lines indicate the elution volumes of the individual constituents of the three MCC complexes. The corresponding elution profiles and SDS-PAGE analyses are shown in Figure S7B.

(B) Ubiquitination reactions in the presence of recombinant APC/C-pE (carrying 68 phospho-mimicking mutations) [70] and the fluorescently labeled N-terminal domain of cyclin B were analyzed by SDS-PAGE and fluorescence scanning. MCC containing BubR1^ΔLL:Bub3 is less efficient in inhibiting APC/C cyclin B ubiquitination activity in comparison to MCC containing BubR1^wt:Bub3. Omitting Bub3 from MCC also reduces APC/C inhibition. NTD, N-terminal domain; Ub, ubiquitin.

See also Figures S6 and S7.