Figure 7.
Mammalian ATG8sparticipate in recruitment of NUFIP2 to damaged lysosomes. (A) Summary of the detected autophagy factors and their quantitative changes based on DIA LC/MS/MS analysis of LysoIP in HEK293T cells (see Table S1, Tab 5). FC, fold change. (B) DIA LC/MS/MS analysis of lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLaWT or mATG8 knockout (hexaKO) treated with or without 4 mM LLOMe for 30 min. Scatter (volcano; log2 fold change and −Log10 P values) plot of stress granule core proteins; n = 3 (see Table S1, Tab 9). Dashed line, significance cut-off (P < 0.05). (C) Analysis of indicated proteins associated with purified lysosomes. Lysosomes were purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLa (WT), LC3TKO, GBRPTKO, and hexaKO cells treated with 4 mM LLOMe for 30 min. (D) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged mATG8. GABARAP (GABA); GABARAPL1 (GABAL1); GABARAPL2 (GABAL2). (E) Quantification of D. Data (% binding) represents the percentage of the corresponding protein relative to its input. (F) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged mATG8. GABARAP (GABA); GABARAPL1 (GABAL1); GABARAPL2 (GABAL2). (G) Quantification of F. Data (% binding) represents the percentage of the corresponding protein relative to its input. (H) Quantification of GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged GABARAP deletions. Blots in Fig. S4 C. Data (% binding) represents the percentage of the corresponding protein relative to its input. (I) Quantification of GST pull-down assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged GABARAP deletions. Blots in Fig. S4 F. Data (% binding) represents the percentage of the corresponding protein relative to its input. AR, autoradiography. Data, means ± SEM; *, P < 0.05; **, P < 0.01, ANOVA. See also Fig. S4. Source data are available for this figure: SourceData F7.

Mammalian ATG8s participate in recruitment of NUFIP2 to damaged lysosomes. (A) Summary of the detected autophagy factors and their quantitative changes based on DIA LC/MS/MS analysis of LysoIP in HEK293T cells (see Table S1, Tab 5). FC, fold change. (B) DIA LC/MS/MS analysis of lysosomes purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLaWT or mATG8 knockout (hexaKO) treated with or without 4 mM LLOMe for 30 min. Scatter (volcano; log2 fold change and −Log10 P values) plot of stress granule core proteins; n = 3 (see Table S1, Tab 9). Dashed line, significance cut-off (P < 0.05). (C) Analysis of indicated proteins associated with purified lysosomes. Lysosomes were purified by anti-HA immunoprecipitation (LysoIP; TMEM192-3xHA) from parental HeLa (WT), LC3TKO, GBRPTKO, and hexaKO cells treated with 4 mM LLOMe for 30 min. (D) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged mATG8. GABARAP (GABA); GABARAPL1 (GABAL1); GABARAPL2 (GABAL2). (E) Quantification of D. Data (% binding) represents the percentage of the corresponding protein relative to its input. (F) GST pulldown assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged mATG8. GABARAP (GABA); GABARAPL1 (GABAL1); GABARAPL2 (GABAL2). (G) Quantification of F. Data (% binding) represents the percentage of the corresponding protein relative to its input. (H) Quantification of GST pulldown assay of in vitro translated and radiolabeled Myc-tagged NUFIP2 with GST or GST-tagged GABARAP deletions. Blots in Fig. S4 C. Data (% binding) represents the percentage of the corresponding protein relative to its input. (I) Quantification of GST pull-down assay of in vitro translated and radiolabeled Myc-tagged G3BP1 with GST or GST-tagged GABARAP deletions. Blots in Fig. S4 F. Data (% binding) represents the percentage of the corresponding protein relative to its input. AR, autoradiography. Data, means ± SEM; *, P < 0.05; **, P < 0.01, ANOVA. See also Fig. S4. Source data are available for this figure: SourceData F7.

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