Fig 1: Quantitative analysis of the lysosomal proteome in response to damage.(A) Scheme depicting major steps in lysophagy and the approaches employed to elucidate components of the pathway. (B) Scheme for tandem mass tagging (TMT)-based proteomics of lysosomes from HeLa cells in response to lysosome rupture by glycyl-l-phenylalanine 2-naphthylamide (GPN). Cells expressing TMEM192-HA were left untreated or treated with GPN for the indicated period of time (in duplicate) and cell lysates subjected to a Lyso-IP protocol prior to TMT-based proteomics. (C) Volcano plot for GPN (22.5 min)-treated cells versus untreated Lyso-IP samples (Log2 FC versus -Log10 p-value) based on the TMT experiment in (B). Specific categories of proteins are indicated by colored circles. (D) GO enrichment (component) for proteins that accumulate on lysosomes in response to GPN treatment. (E) Time course reflecting the dynamics of recruitment or loss of selected proteins from lysosomes in response to GPN treatment. Error bars represent SD from two biological replicates. (F) Dynamics of recruitment or loss of proteins linked with autophagy (top), ESCRT (middle), and MTOR (lower) pathways in association with lysosomes upon GPN treatment. All the lines for each category represent individual proteins (see Supplementary file 2), and proteins with the most highly dynamic changes are indicated as dashed lines. (G) HeLa cells were either left untreated or treated with GPN for 45 min prior to isolation of lysosomes by Lyso-IP. Samples were then subjected to immunoblotting with the indicated antibodies. Figure 1—source data 1.Uncropped blots for Figure 1G. Figure 1—source data 2.TMT ratios from GPN time course for Figure 1E.