Fig. 3.

Analysis of Ddi1 domains required for proteolytic activity. (A) Scheme of Ddi1 domain organization. The domains are shown in different colors with the amino acid residue number at the beginning and end of a domain indicated above. (B) Wild-type or mutant Ddi1 was purified as a His14–SUMO fusion. The N-terminal SUMO tag was cleaved off with SUMO protease, and the proteins were further purified by gel filtration. They were then incubated with DyLight800-labeled substrate bearing long polyubiquitin chains purified by gel filtration. Samples were taken at the indicated time points and subjected to SDS-PAGE and fluorescence scanning. FL refers to the full-length construct. ΔUBL, ΔUBA, and ΔHDD are mutants lacking the indicated domains. RVP and HDD–RVP are constructs containing only the indicated domains. The arrowheads indicate a generated proteolytic fragment. (C) As in B, but with constructs containing the D220N mutation in the active site of the RVP domain.