FIGURE 3.

CDC34-Ub^Cys is an excellent functional mimic of the E2∼ubiquitin thiol ester intermediate. A, dose-dependent increases in the polyubiquitylation of ³²P-labeled IκBα-ubiquitin by CDC34 are shown. B and C, CDC34-Ub^Cys competitively inhibits CDC34/SCF-dependent ubiquitylation. All reactions contained CDC34 (1.3 μm) with increasing concentrations of CDC34-Ub^Cys and show decreased ubiquitin conjugation to ³²P-labeled IκBα-ubiquitin, demonstrating that CDC34-Ub^Cys competes for the same site as CDC34∼ubiquitin. Reaction mixtures were prepared as described under “Experimental Procedures” visualized by autoradiogram, and the amount of remaining unreacted ³²P-labeled IκBα-ubiquitin was quantified by phosphorimaging analysis.