Fig. 1: mitoUFD is a model substrate for monitoring protein turnover at the OMM.
a, The mitoUFD model substrate is located at the OMM and a substrate for the UPS. IMM, inner mitochondrial membrane; Ub, ubiquitin. b, Confocal images showing that mitoUFD colocalizes with a previously established OMM marker TOMM-20::mKate2 (top row), surrounding the mitoTracker-stained mitochondrial matrix (middle row), and cytoUFD does not colocalize with TOMM-20::mKate2 (bottom row). Scale bar, 10 µm. c, Confocal images showing that BTZ treatment at 5 µM or 10 µM concentration stabilizes mitoUFD at the OMM. Scale bar, 10 µm. d, Western blot analysis showing that BTZ treatment stabilizes mitoUFD but not mitoGFP. e, Western blot showing chase assay of mitoUFD (degraded) versus mitoGFP (stable). f, Quantification of GFP levels in worms by fluorescence imaging over a 9 h CHX-chase assay. Mean ± s.e.m., n = 5 independent biological replicates. Two-way ANOVA; the P value indicates the effect of substrates (mitoUFD versus mitoGFP). g, A volcano plot showing enriched binding partners of mitoUFD compared with mitoGFP, which includes ubiquitin associated/related proteins and proteasome subunits. h, Mitochondrial enrichment followed by pulldown of polyubiquitylated proteins by TUBE agarose in L4440 control and cdc-48 RNAi worms; western blot (WB) against GFP to show polyubiquitylated mitoUFD substrate. Input: isolated and lysed mitochondria before TUBE IP. The same number of worms was used for both conditions.
