FIGURE 4.

ROC1-CUL1-Nedd8 acts catalytically, converting Cdc34∼Ub to di-Ub. Multiple (panel A) and single (panel C) round di-Ub synthesis reactions were performed as described under “Experimental Procedures.” RING complex refers to ROC1-CUL1-(411–776). The inset graph in panel A reveals the effects of Nedd8 at low concentrations of the RING complex. Panel B shows that apyrase blocks the formation of E1∼Ub and Cdc34∼Ub. The Cdc34∼S∼³²P-Ub (5 μl) was prepared with 1.7 μm ³²P-PK-Ub, 0.05 μm E1, and 0.5 μm Cdc34. Increasing amounts of apyrase, as indicated, was added to the mix before ³²P-PK-Ub, and the incubation was at 37 °C for 1min. The inset graph in panel B reveals the effects of apyrase at low concentrations. mU, milliunits.