FIGURE 3.

E3 ubiquitin ligase activity of HUWE1 HECT domain. a and b, the autoubiquitination activity of HUWE1 HECT domain was tested using 60 μm ³²P-labeled Ub as substrate and 2 μm WT Δ α1 (a) or 2.9 μm WT + α1 (b) HECT domains incubated with UBE1, UBE2L3, and an ATP regenerating system (note the different time scale for the two variants of the HECT domain). The HECT domain is omitted in the lane marked N. The asterisk denotes a likely ubiquitin polymer; the double asterisk denotes likely mono-ubiquitinated UBE2L3. Concentrations of HECT domain were chosen to obtain initial rate conditions. c, ligation activity of the WT Δ α1 and WT + α1 HECT domains in the autoubiquitination 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, shown is a single turnover assay monitoring transfer of Ub from the UBE2L3∼Ub thioester to a lysine in the WT Δ α1 and WT + α1 HECT domains. The UBE2L3∼Ub thioester is generated in a pulse reaction containing E1, UBE2L3, ATP regenerating system, and a Ub mutant in which all lysines are mutated to arginine (K0 Ub). Ub is chased from the E2 enzyme to the HECT domain added to the reaction. Ub-conjugated HECT domain is visualized by anti-Ub immunoblot. Samples were terminated in reducing or non-reducing sample buffer as indicated. The panel marked N is a chase reaction performed in the absence of HECT domain and terminated in non-reducing sample buffer.