Figure 6. USP8 hydrolyzes K6 linkages in parkin Ub conjugates.

A, B USP8 is unable to hydrolyze preassembled parkin Ub conjugates when K6 linkages are absent. GST-parkin bound to glutathione beads was left to ubiquitinate for 2 h alone at 37°C in the presence of wild-type Ub or Ub^K6R (A). After 2 h, the beads were washed to remove reaction components. Ubiquitinated GST-parkin was then incubated in the presence or absence of His-tagged full-length USP8, the USP8 catalytic domain, or the untagged USP2 catalytic domain for 1 h at 37°C. Reactions were immunoblotted for Ub and GST-parkin was stained with Ponceau S. The optical densities of the Ub conjugates relative to total GST-parkin were quantified using NIH ImageJ (B), and the data represent the mean ± SEM for three independent experiments. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, **P < 0.01; NS, not significant.

C, D Expression of HA-Ub^K6R rescues the delay in mitochondrial recruitment of parkin following USP8 siRNA. U2OS-GFP-parkin cells were co-transfected with USP8 siRNA (5 nM) and either HA-Ub^wild-type, HA-Ub^{K6 only}, or HA-Ub^K6R (1 μg) for 60 h (C). Cells were treated with CCCP for the indicated time periods and fixed. Immunofluorescence images of cells were acquired after staining for HA and TOM20. After 1-h treatment with CCCP, cells were analyzed for the recruitment of GFP-parkin onto TOM20-positive mitochondria in HA-positive cells (D). Experiments were blinded and performed in triplicate with 100 cells analyzed for each condition. The vertical bars represent SEM. For statistical analysis, a two-way ANOVA with Tukey post-test was performed, *P < 0.05, **P < 0.01; NS, not significant.

Source data are available online for this figure.