Fig 2: In vitro characterization of TECPR1 activity on all ATG8 proteins with PE and PS substrate Table showing lipid composition of liposomes used in (B) and (C).Coomassie‐stained gel depicting in vitro LC3B/GABARAP lipidation reactions facilitated by ATG16L1 using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co‐sedimentation assay. Separation of reactions into membrane bound (pellet) and supernatant allows clear distinction of ATG8‐II and ATG8‐AMP.The extent of LC3B/GABARAP‐I to ‐II conversion in (A) was determined and plotted as percentage of total LC3B.Coomassie‐stained gel depicting in vitro LC3B/GABARAP lipidation reactions facilitated by TECPR1 using the indicated combinations of proteins and liposomes (with or without SM), after being subjected to a liposome co‐sedimentation assay. Separation of reactions into membrane bound (pellet) and supernatant allows clear distinction of ATG8‐II and ATG8‐AMP.The extent of LC3B/GABARAP‐I to ‐II conversion in (A) was determined and plotted as percentage of total LC3B.

Fig 3: The role of V‐ATPase in TECPR1 recruitment to damaged lysosomes HEK293A cells transiently transfected with GFP‐SidK (aa1‐278) treated as indicated for 1 h. Cells were prepermeabilized with 0.05% Saponin on ice for 5 min to eliminate any SidK that was not involved in a membrane‐bound complex before cell lysis. The cell lysates were then subjected to GFP‐Trap immunoprecipitation, and the input, flowthrough (FT), and bound material were immunoblotted against the indicated proteins.Schematic representation of the interaction between GFP‐SidK (aa1‐278) and the soluble V1 region of the V‐ATPase before and after assembly with the membrane‐embedded VO region. The diagram highlights the protein components that are lost when the cells are subjected to prepermeabilization. The postassembly state shows the complete V‐ATPase complex, including both the V1 and VO regions, interacting with GFP‐SidK. Highlighted is also the previously shown interaction with ATG16L1.Confocal images of HeLa WT cells transiently transfected with EGFP‐SidK, with or without prepermeabilization, treated or not with LLOMe for 1 h as indicated and costained for LAMP1. Scale bars: 10 μm.

Fig 4: TECPR1 recruits the ATG5‐ATG12 complex to damaged lysosomes to catalyze LC3 lipidation onto damaged membranes Western blot analysis of LC3 lipidation status in wild‐type (WT), FIP200 KO, and ATG16L1 KO HeLa cells treated with the indicated concentrations of LLOMe for 30 min.Quantification of LC3‐II levels in HEK, HeLa, and MEF WT and ATG16L1‐deficient cell lines treated with and without 1 mM LLOMe for 30 min. Bars show mean ± SD from three biologically independent experiments represented as data points. Significance was determined from biological replicates using Student's t‐tests. *P = 0.0177, **P = 0.0082, ***P = 0.0001. Corresponding Western blots are found in EV2B.Western blot analysis of LC3 lipidation status in WT, ATG7 KO, and ATG16L1 KO HeLa cells (top) and WT, ATG5−/− and ATG16L1Δ/Δ MEFs (bottom) treated with the indicated concentration of LLOMe for 30 min.Western blot analysis of LC3 lipidation status in ATG16L1 KO HeLa cells treated with 1 mM LLOMe for 30 min followed by washout in the presence or absence of 20 μM chloroquine.Representative confocal images of HeLa WT, FIP200 KO, and ATG16L1 KO cells transfected with EGFP‐TECPR1, treated with 1 mM LLOMe for 30 min and immunostained for LC3 and LAMP1. Scale bars = 10 μm for whole images and 5 μm for the zoom. Fluorescence intensity profiles for the indicated channels across the dotted line are shown in the upper right subpanel.Western blot analysis of lyso‐IP samples collected from HeLa ATG16KO cells treated with or without 1 mM LLOMe for 30 min. Bars show mean ± SD from three biologically independent experiments represented as data points.Representative live‐cell fluorescent images of HeLa cells co‐transfected with TagBFP‐TMEM192, EGFP‐ATG5, and mCherry‐TECPR1, before and after a 15‐min treatment with 1 mM LLOMe. Scale bars = 10 μm for whole images and 2 μm for zoom. To the right is the corresponding quantification of the fold change in ATG5/TECPR1 lysosomal fluorescence intensity after a 15‐min treatment with vehicle or 1 mM LLOMe. Gray points represent individual cells from three independent experiments. Green/magenta points represent the means of individual experiments (n > 25 cells per experiment). Bars represent the mean ± SD from the three experiments. Significance was determined from biological replicates using a one‐way ANOVA with Tukey's multiple comparisons tests. ****P < 0.0001, ***P = 0.0002.Western blot analysis of LC3 lipidation status in HEK TECPR1/ATG16L1 KO cells treated with 1 mM LLOMe for 30 min. To the right is the corresponding quantification of LC3‐II protein levels. Bars show mean ± SD from three or four biologically independent experiments, which are represented as data points. Significance was determined from biological replicates using a one‐way ANOVA with Tukey's multiple comparisons tests. ***P = 0.0001, **P < 0.01, *P = 0.0467.Western blot analysis of LC3 lipidation status in HEK TECPR1/ATG16L1 double‐KO cells transfected with the indicated plasmid and treated with 1 mM LLOMe 30 min. Data are representative of three independent experiments. Source data are available online for this figure.

Fig 5: TECPR1 recruitment to damaged lysosomes precedes galectin recruitment A, BRepresentative live‐cell fluorescent images of HeLa cells co‐transfected with EGFP‐TECPR1 and mCherry‐Gal3 and treated with 1 mM LLOMe for the indicated time. Arrowheads in (B) indicate the appearance of TECPR1 (green arrow) and Gal3 (magenta arrow). Scale bars = 10 μM for (A) and 1 μM for (B).CQuantification of TECPR1 and Gal3 recruitment to damaged lysosomes. Data are presented as mean ± SD from five independent experiments (each experiment represents a single cell with at least three individual lysosomes quantified).DRepresentative confocal image of a HeLa cell transfected with EGFP‐TECPR1, treated with LLOMe for 5 min, and immunostained for CHMP2A and LAMP1. Scale bars = 10 μm for whole image and 1 μm for insets. Fluorescence intensity profiles of the indicated channels across the dotted lines are shown in the lower subpanel.ERepresentative confocal images of a HeLa cells transfected with EGFP‐TECPR1 and TagBFP‐TMEM192 (a lysosomal/late endosomal protein), treated with 1 mM LLOMe for the indicated time, and immunostained for ALIX and Gal3. Scale bars = 10 μm. Source data are available online for this figure.