FIGURE 5.
The UBA domain is important for E2 competition. A, [35S]SecurinN was translated in vitro in rabbit reticulocyte lysate, purified, and mixed with purified APC/C and Cdh1. The indicated E2s (each at 3 μm final concentration) were charged with wild-type ubiquitin (Ub) and added. Reactions were carried out at room temperature for 20 min. Reaction products were analyzed by SDS-PAGE and autoradiography with a phosphorimaging system. The numbers below show quantification of APC/C activity (i.e. total modified substrate) in each lane with Ubc4 activity normalized to 100. Results are representative of three independent experiments. B, APC/C assays were performed as in A except that [35S]SecurinN 1K was used as the substrate. Results are representative of three independent experiments.