FIGURE 5.

Substrate ubiquitination activity of the HUWE1 HECT domains. a and b, the Mcl-1 ubiquitination activity of HUWE1 HECT domain was tested using 5 μm ³²P-labeled Mcl-1 as substrate and 100 nm WT Δ α1 (a) or 300 nm WT + α1 (b) HECT domains incubated with UBE1, UBE2L3, Ub, and an ATP regenerating system. HECT domain was omitted from the lane marked N. Concentrations of HECT domain were chosen to obtain initial rate conditions. c, ligation activity of the HECT domains in the Mcl-1 ubiquitination assay is shown. Activity is given as the ratio between initial velocity (pmol of total ³²P-labeled Ub product/min) and total enzyme concentration E (pmol). Errors are the S.D. calculated from three independent experiments. d and e, shown is a graph of the percent ubiquitinated Mcl-1 as a function of time in the reactions shown in panels a and b catalyzed by HUWE1 Δ α1 (d) or HUWE1 + α1 (e) HECT domains.