Fig. 1. Bypassing System (ByS). (A) C-terminal ubiquitin thioester Ub–MES can form a catalytically active HECT E3∼Ub thioester adduct and conjugate ubiquitin to protein substrates, bypassing ATP, E1, and E2. (B) A time course of Sic60-GFP ubiquitination by Rsp5 and Tagged-Ub–MES. Reaction mixtures were incubated at room temperature for indicated times, quenched with Laemmli buffer, resolved by SDS-PAGE and imaged with in-gel fluorescence scanning and coomassie staining.
