FIGURE 2.
The UBA domain is independent of ubiquitin on substrate. APC/C purified from yeast cells was mixed with Cdh1 and 125I-CycB 1K. Ubc1 (either wild type or ΔUBA) charged with K48R mutant ubiquitin was added at the indicated concentrations, and reactions were carried out at room temperature for 15 min. Reaction products were analyzed by SDS-PAGE and autoradiography with a phosphorimaging system. The bottom panel displays quantification of results, showing the dependence of APC/C activity on concentration of Ubc1 or Ubc1ΔUBA. Autoradiographs were quantified using ImageQuant, and data were fit to the Michaelis-Menten equation in Prism software. The average of four experiments is shown. Error bars represent S.E. PIU/s, phosphorimaging units/s.