Fig. 4.

Functional interplay between the catalytic activity of the JD of ataxin-3 and its UIMs during catalysis. (A) Pull-down of synthetic K48-linked polyubiquitin chains onto recombinant His-tagged wild-type and mutant ataxin-3 (Atx) and isolated JD. Bound polyubiquitin and the bait proteins were detected by Western blotting (WB). Note the preference of ataxin-3, primarily mediated by the first two UIMs, for the longest chains. Ctr, control. (B) Deubiquitination assays carried out by incubating tetraubiquitin with recombinant His-tagged ataxin-3 and JD and their mutants. At the end of the reaction, Ub and ataxin-3 proteins were detected by Western blotting. Activity is indicated by the conversion of tetraubiquitin to triubiquitin. (C) In vivo substrate trapping by catalytically inactive ataxin-3. HeLa cells were transfected with FLAG-tagged wild-type ataxin-3 or mutant ataxin-3 constructs or with the empty vector (Ctr lane). FLAG-tagged proteins were then lysed and immunoprecipitated in JS buffer (Left) or radioimmunoprecipitation assay buffer (to disrupt Ub–UIM interactions) (Right), and the immunoprecipitates were analyzed by Western blotting for Ub or for ataxin-3 with an antibody that recognizes its JD. Arrows indicates IgG heavy chains.