Figure 1: E3 ligase activity of RING1B–PCGF complexes.

Unless specified, RING1B is common to all E3 complexes, so only the identity of the PCGFs is shown (P1–P6). Reactions throughout were monitored by western blot (α-Ub) unless stated otherwise. (a) Human and Drosophila PCGF (green) and RING1 proteins (purple). Boundaries of the RING domain constructs used in this study are shown. (b) Auto-ubiquitination by the various GST–RING1B–PCGF complexes. (c) Discharge of Ub from the UbcH5c∼Ub conjugate (‘∼’ represents a covalent thiolester bond) to excess solution lysine in the presence of the indicated E3 complex. The discharge assay is a direct readout of intrinsic E3 catalytic activity. * represents a band that appears to be UbcH5c conjugated to diUb (note discharge in parallel with E2∼Ub). (d) Rate of ubiquitin discharge from the indicated PCGF–RING1B–UbcH5c∼Ub complex. Reaction progress was monitored by discharge-induced fluorescence polarization (FP) changes of BODIPY-FL-labelled ubiquitin (see Methods and Table 1) and is plotted as apparent, pseudo first-order rate constant (k′_app±s.d.) at the indicated lysine concentration. Rates were determined from duplicate measurements for PCGFs 1, 2 and 4. For PCGFs 3, 5 and 6, four replicates were averaged (except two replicates at 20 mM lysine). (e) Auto-ubiquitination by human, Drosophila and human–Drosophila chimeric GST–RING1–PCGF complexes.