FIGURE 3.

A, reaction scheme for in vitro labeling experiments performed in B to E using the active site-directed probe Ub-VS. B, Ub-VS conjugates to Parkin and IBR-RING2 proteins. C and D, the Ub-VS adduct was inhibited by preincubation of IBR-RING2 with NEM (C) or the C431S mutation (D). E, C431S is the lone free cysteine mutation to specifically inhibit Ub-VS conjugation. F, E3 activity of Parkin lacking the Ubl and RING1 domains in cells. HeLa cells expressing GFP-Parkin or GFP-IBR-RING2 with the C431A or T415N mutation were treated with CCCP and subjected to immunoblotting. G, GFP-IBR-RING2 catalyzes autoubiquitylation in cells irrespective of a decrease in ΔΨm. H, cytosolic localization of GFP-IBR-RING2 following CCCP treatment. The mitochondrial localization of GFP-Parkin following CCCP treatment is shown as a control.