Fig. 7: Proximity proteomics analysis of ATG5 knockdown (KD) RPE1 cells containing p62-enwrapped lipid droplets. | Nature Communications

Fig. 7: Proximity proteomics analysis of ATG5 knockdown (KD) RPE1 cells containing p62-enwrapped lipid droplets.

From: Structural organization of p62 filaments and the cellular ultrastructure of calcium-rich p62-enwrapped lipid droplet cargo

Fig. 7

a Scheme illustrating the proximity biotinylation workflow for mass spectrometry and Western blot analysis. b Western blots analyzing the different samples that were used in the experiments; a control sample of mCherry-p62 RPE1 cells (mCh-p62) labeled with biotin, a control sample of miniTurbo-mCherry-p62 RPE1 cells (T-mCh-p62) labeled with biotin with endogenously expressed ATG5 and ATG5 knock down + miniTurbo-mCherry-p62 RPE1 cells (T-mCh-p62 ATG5 KD) labeled with biotin. Western blot was detected with streptavidin; highlighting all biotinylated proteins in the cell lysate, flow-through and wash steps of the Streptavidin pull-down column, respectively. c Western blot shows p62 expression in the different cells (mCh-p62, T-mCh-p62 and T-mCh-p62 ATG5 KD, respectively), ATG5 levels, LC3 lipidation state and a GAPDH loading control. d Volcano plot displaying relative abundance of proteins after enrichment from ATG5 KD and control samples with endogenous amount of ATG5. Autophagy related proteins, lipid droplet related proteins and calcium related proteins that were found significantly different abundance are highlighted. The proteins on either side of the volcano plot were only present in the control sample (in blue) or only present in the ATG5 KD sample (in red), y-axis indicates protein LFQ intensity. e Working model of the early events of selective autophagy captured in this study: after calcium efflux from the ER, together with calcium, p62 filaments form a coat around the lipid droplet cargo to be degraded. Due to the ATG5 KD condition preventing LC3b conjugation to phosphatidyl ethanolamine (PE) residing in the membrane, we can visualize this early structure of selective autophagy that otherwise is rapidly degraded after complete membrane enwrapment and fusion with the lysosome. All mass spectrometry experiments were performed on biological quadruplicates. The workflow figure was created in BioRender (Huesgen, lab (2025) https://BioRender.com/zh44l17).

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