FIGURE 8.
A, competition with β-catenin-Ub1 and β-catenin-Ub4 as substrates. β-cat-Ub1 or β-cat-Ub4 were prepared as described under “Experimental Procedures.” These preformed β-cat-Ub1 (∼2 pmol) or β-cat-Ub4 (∼2 pmol) were mixed with increasing amounts of β-catenin peptide (1-, 3-, 10-, and 30-fold excess) in the presence of SCFβTrCP2, and the resulting mixture was incubated for 10 min at 25 °C. A separate mixture containing Cdc34∼Ub (17 μm E2) was added, and the reaction was incubated at 37 °C for 10 min. For quantification, the difference in substrate utilization between the reaction with or without competitor was calculated. The calculated difference was expressed as % inhibition. This graph integrates the results of three independent experiments, with error bars for the calculated S.D. B, substrate-independent di-Ub synthesis by Cdc34 with Ub or Lys-48-Ub4 as the receptor. The detailed procedure is described under “Experimental Procedures.” The concentrations of Ub or Lys-48-Ub4 are 0.85, 2.55, and 8.5 μm. This graph integrates the results of three independent experiments, with error bars for the calculated standard deviation. C, comparison of Cdc34-catalyzed di-Ub synthesis with Lys-48-Ub4 or Lys-63-Ub4 as the receptor. The concentrations of Lys-48-Ub4 or Lys-63-Ub4 are 4.25, 8.5, and 12.75 μm. D, an overall model for SCF-directed ubiquitination. See “Discussion” for detail.