FIGURE 1.

The UBA domain does not contribute to acceptor ubiquitin binding or RING enhancement of E2 catalysis. A, Ubc1 (wild type; 0.5 μm) was charged with ³²P-radiolabeled K48R ubiquitin and incubated with either GST-Apc11ΔN (80 μm) or buffer. Wild-type ubiquitin was added at the indicated concentrations, and reactions were carried out at 4 °C for 5 s (+GST-Apc11ΔN) or 10 min (buffer alone). Reaction products were analyzed by SDS-PAGE and autoradiography with a phosphorimaging system. The right panel displays the quantification of diubiquitin synthesis assays, showing the dependence of catalytic rate (k_obs) on ubiquitin concentration. Autoradiographs were quantified using ImageQuant, and data were fit to the Michaelis-Menten equation in Prism software. The average of three experiments is shown. Error bars represent S.E. B, Ubc1 (wild type) and Ubc4 (each at 0.5 μm) were charged with ³²P-radiolabeled K48R ubiquitin and incubated with buffer or increasing concentrations of GST-Apc11ΔN. Unlabeled CycB^N (200 μm; Ubc4 reactions) or ubiquitin (100 μm; Ubc1 reactions) was added, and reactions were carried out at 4 °C for 5 s. Reactions were analyzed as in A. Results are representative of three independent experiments. C, Ubc1 (wild type or ΔUBA at 0.5 μm) reactions were carried out as in A except that a subsaturating concentration of GST-Apc11ΔN (6.5 μm) was used. Reactions were carried out at room temperature for 3 (wild-type Ubc1) or 5 min (Ubc1ΔUBA). The average of three experiments is shown. Error bars represent S.E. D, Ubc1 and Ubc1ΔUBA were charged as in A and incubated with buffer or increasing concentrations of GST-Apc11ΔN. Unlabeled wild-type ubiquitin (100 μm) was added, and reactions were carried out, visualized, and quantified as in A. Results are representative of three independent experiments. Ub, ubiquitin.