Figure 4: Deletion of the Ubl impairs recruitment and mitochondrial substrate ubiquitination.

(a) Time-lapse microscopy of Parkin recruitment to mitochondria upon treatment with CCCP in U2OS cells stably expressing WT, ΔUbl (deletion of residues 1–76), ΔUbl/F146A or ΔUbl/N273K Parkin double mutants. Recruitment can be visualized by the appearance of punctate GFP fluorescence. Scale bar located at the top left of the first pictograph represents 20 μm. (b) Quantification of GFP-Parkin recruitment to the mitochondria for Parkin double mutant-expressing cells. The percentage of cells showing recruitment of GFP-Parkin to mitochondria was determined every 5 min over a period of 90 min. The vertical bars represent s.e.m. from three independent experiments. *P<0.05 (two-way ANOVA with Bonferroni post test). Data from WT and hyperactive mutants from experiments performed in parallel from Fig. 1 are shown for comparison in each graph. (c) Western blot of in organello ubiquitination assays for the rescue of ΔUbl (ref. 2; deletion of residues 1–140). (d) Quantification of Mfn2 ubiquitination signals from three independent experiments. The vertical bars represent s.e.m. *P<0.05, **P<0.01; n.s., nonsignificant (one-way ANOVA with Dunnett’s test). All in organello ubiquitination reactions are performed with CCCP-treated mitochondria unless stated differently.