Structure/function analysis of RIC-7. (A) 3D structure of RIC-7b predicted by Alphafold. Model confidence is color-coded. The highest confidence region (471–709 aa) is structurally similar to human OTU domain. (B) Quantification of the ability of different RIC-7 truncations to rescue mitochondria localization defects in ric-7 mutants. Transgenes were driven by itr-1 promoter (see Materials and methods). Each data point represents one animal. Data are pooled from four experiments. ***P < 0.001, ****P < 0.0001 (one-way ANOVA with multiple comparisons). (C) Localization of truncated proteins. White arrows highlight the distal end localization of 1–470 aa::EGFP; White arrowheads indicate the non-distal localization of truncated proteins; Magenta arrowheads indicate mitochondria that are not associated with the truncated proteins; Scale bar = 5 μm. (D) Quantification of the percentage of mitochondria associated with the GFP transgenic fusions. Number of mitochondria counted are indicated at the top of each bar. Error bars represent 95% confidence intervals. Total number of animals: 1–470 aa = 8; 1–99 aa = 11. **P < 0.01, ****P < 0.0001 (Fisher’s exact test). (E) Quantification of the percentage of mitochondria based on the polarization status of the GFP transgenic fusions. Same dataset as in D. *P < 0.05 (Fisher’s exact test of Non-distal versus Distal).