Figure 1: Activating Parkin mutations.
From: Structure-guided mutagenesis reveals a hierarchical mechanism of Parkin activation
(a) Cartoon representation adapted from ref. 26 of the auto-inhibited structure of Parkin with activating mutations highlighted in red: F146A (RING0:RING2 interface); N273K (Ubl:RING1 interface); and W403A (REP:RING1 interface). (b) Time-lapse microscopy showing Parkin recruitment to mitochondria upon membrane depolarization with CCCP in U2OS cells stably expressing GFP-Parkin WT, F146A, N273K or W403A. 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. (c) Quantification of GFP-Parkin recruitment to the mitochondria. 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). (d) Western blot of in organello ubiquitination reactions showing WT, F146A, N273K or W403A ubiquitination of Mfn2. All in organello ubiquitination reactions are performed with CCCP-treated mitochondria.
