Fig. 6

CLUH-proximal mitochondrial proteins accumulate overtime in a translation-dependent manner. A Schematic representation of the TurboID experimental design using HCT116 cells stably expressing the TurboID protein fused to CLUH or GFP proteins. The proximity labeling is performed for 30 min or 16 h in the presence of 50 μM biotin in the culture medium. Biotinylated proteins, from both the specific (TurboID-CLUH) and control (TurboID-GFP-BioID2) samples, are isolated using streptavidin-coupled magnetic beads and identified by LC-MS/MS. B Parallel coordinates plot comparing the fold change (FC) enrichment of TurboID identified proteins at 30 min and 16 h (n=3 and threshold: FDR<0.1 and FC >2, Fig. S5 C and D). The FC variation corresponds to the ratio of FC between 30 min and 16h. The y-axis corresponds to the log2 transformed values. Group 1 (purple) is defined as containing proteins with a FC ratio < 0.5 and group 2 (yellow) as protein with FC variation >=0.5. The full datasets and analysis are available in Table S7. C Venn diagram showing the intersection between group 1 (purple), group 2 (yellow), and human mitochondrial proteins (gray) listed in Mitocarta 3.0 database. D Schematic representation of the TurboID experimental design using HCT116 cells stably expressing the TurboID protein fused to CLUH in translation inhibition conditions. Cells are pre-treated with 100μg/mL of puromycin for 20 min prior to biotin pulse labeling. The proximity labeling is performed for 30 min in the presence of 50 μM biotin in the culture medium. E Streptavidin enriched proteins are analyzed by western blot. Biotinylated proteins are pulled down (PD) from total input extracts (INP) using streptavidin-coupled magnetic beads. The loaded samples correspond to 0.5% of the input and 20% of the pulled-down samples. The CPMPs (LRPPRC, IMMT) and other indicated proteins are revealed using specific antibodies. Two different exposures for SPAG5 are shown. Biotinylated proteins are revealed using HRP-coupled streptavidin