Fig 2: Cys44 and Cys135 are crucial for the transformation of the ATG10 protein structure and function. (A) The prediction of intrachain disulfide bonds in the ATG10 protein online (http://disulfind.dsi.unifi.it). Cys44 and Cys135 are indicated by a red box, respectively. (B, C) In the HCV-subgenomic replicon cells with ATG10, ATG10S, and ATG10C135S overexpression, respectively (B), or with ATG10, ATG10S, and ATG10C44S/C135S overexpression, respectively (C), co-immunoprecipitation with anti-LC3B or anti-P62 antibody shows the interactions among LC3B, P62, and LAMP2, and co-immunoprecipitation with anti-LAMP2 antibody shows the interaction between HCV subreplicon proteins (NS5B and CORE) with autophagolysosomes. Western blotting analysis shows the levels of autophagy flux proteins and HCV CORE and NS5B. (D) Subcellular co-localization of P62-LC3B and LC3B-LAMP2 among ATG10, ATG10S, ATG10C135S, and ATG10C44S/C135S overexpression in the HCV subreplicon cells by immunofluorescence. (E) The diagram of a series of N- or C-terminally truncated ATG10 mutants based on six peptides of the ATG10 protein. The dotted lines represent deletion exons. (F) Co-immunoprecipitation with anti-LC3B, anti-P62, and anti-LAMP2 antibodies shows the interaction among autophagy proteins (P62, LC3B, and LAMP2) and the HCV subreplicon proteins (NS5B and CORE). Input result shows the effect on autophagy flux and HCV subreplicon duplication in HCV-subgenomic replicon cells plus atg10, atg10s, atg101-3, atg101-4, atg101-5, atg102-6, atg103-6, or atg104-6. (G) Immunofluorescence analysis shows the subcellular colocation of P62-LC3B and LC3B-LAMP2 in HCV-subgenomic replicon cells plus atg101-3, atg102-6, atg103-6, or atg104-6. Scale bars, 15 µm.

Fig 3: KICSTOR regulates the lysosomal localization of GATOR1 but not of GATOR2 or the Rag GTPasesa) Quantitation of the imaging data in Figures 4a, b. Values are mean ± standard error.b) Loss of KICSTOR components does not affect the lysosomal localization of GATOR2. GFP-WDR24 expressing HeLa cells prepared as in Extended Data Fig. 4c were subsequently modified with the CRISPR/Cas9 system to create KPTN-deficient cells. These cells as well as wild-type and sgAAVS1-treated control cells were starved or starved and restimulated with amino acids for the noted times prior to processing for the detection of GFP and LAMP2 by immunofluorescence. Insets depict selected fields magnified 3.24× and their overlays. Scale bars represent 10 µm. Quantitation of the imaging data is shown in the bar graph below images (mean ± standard error).c) The Rag GTPases localize to lysosomes regardless of SZT2 expression. Wild-type and SZT2-deficient HEK-293T cells were treated as in (b) prior to processing for the detection of RagC and LAMP2 by immunofluorescence. Insets depict selected fields magnified 3.24× and their overlays. Scale bars represent 10 µm. Quantitation of the imaging data is shown in the bar graph on the right (mean ± standard error).

Fig 4: ROCK1 knockdown eliminates the accumulation of autophagosomes and autolysosomes induced by GAK disruption. (A) ROCK1 expression in wild-type, GAK-KO, GAK-KO/shNT and GAK-KO/shROCK1 cells was confirmed by immunoblotting. (B) Immunofluorescence analysis of LAMP2 (magenta) and LC3B (green) in wild-type, GAK-KO, GAK-KO/shNT and GAK-KO/shROCK1 cells under basal-fed or starvation (RPMI-Gln for 8 h) conditions. The dashed boxed regions are shown at a high magnification (×3) in the inset. Scale bar, 20 µm. (C) Number of enlarged LC3B-positive puncta in wild-type, GAK-KO, GAK-KO/shNT and GAK-KO/shROCK1 cells was quantified using Imaris. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. The box extends from the lower to the upper quartile, the middle line indicates the median, the X indicates the mean and the whiskers represent the minimum to maximum values, except for outliers, which are indicated by dots (wild-type/fed, n=16; GAK-KO/fed, n=11; GAK-KO/shNT/fed, n=9; GAK-KO/shROCK1#1/fed, n=18; GAK-KO/shROCK1#2/fed, n=22; wild-type/starved, n=27; GAK-KO/starved, n=13; GAK-KO/shNT/starved, n=13; GAK-KO/shROCK1#1/starved, n=28; GAK-KO/shROCK1#2/starved, n=22). **P<0.01 vs. wild-type. ##P<0.01 vs. GAK-KO. (D) Lysosomal size in wild-type, GAK-KO, GAK-KO/shNT and GAK-KO/shROCK1 cells was quantified using ZEN 2012 software. Data were analyzed using one-way ANOVA followed by Tukey's post hoc test. The box extends from the lower to the upper quartile, the middle line indicates the median, the X indicates the mean and the whiskers represent the minimum to maximum values, except for outliers, which are indicated by dots (n=100). *P<0.05 and **P<0.01 vs. wild-type; #P<0.05 and ##P<0.01 vs. GAK-KO. ROCK, Rho-associated protein kinase; GAK, cyclin G-associated kinase; KO, knockout; sh, short hairpin RNA; NT, non-targeting; LAMP, lysosomal associated membrane protein; Gln, glutamine; ns, not significant.

Fig 5: GAK disruption causes the formation of enlarged LAMP2-positive vesicles. (A) Representative transmission electron microscopy images of wild-type and GAK-KO cells under basal-fed conditions. Dashed boxed regions are shown at a high magnification (×3) in the inset. Scale bar, 5 µm. (B) Immunofluorescence analysis of LAMP2 (magenta) in wild-type and GAK-KO cells under basal-fed conditions. Dashed boxed regions are shown at a high magnification (×3) in the inset. Scale bar, 10 µm. (C) Lysosomal size in wild-type and GAK-KO cells under basal-fed conditions was quantified using ZEN 2012 software. Data were analyzed using two-way ANOVA followed by Bonferroni's post hoc test. Data are presented as the mean ± SD (wild-type, n=204; GAK-KO, n=295). *P<0.05 and **P<0.01 vs. wild-type. (D) Immunofluorescence analysis of LAMP2 (magenta) in wild-type, GAK-KO, sham-rescued and GAK-rescued cells under basal-fed or starvation (RPMI-Gln for 8 h) conditions. Dashed boxed regions are shown at a high magnification (×3) in the inset. Scale bar, 10 µm. (E) GAK expression in wild-type, GAK-KO, sham-rescued and GAK-rescued cells was determined by immunoblotting. (F) Lysosomal size in wild-type, GAK-KO, sham-rescued and GAK-rescued cells was quantified using ZEN 2012 software. Data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. The box extends from the lower to the upper quartile, the middle line indicates the median, the X indicates the mean and the whiskers represent the minimum to maximum values, except for outliers, which are indicated by dots (wild-type/fed, n=212; GAK-KO/fed, n=240; sham-rescued/fed, n=140; GAK-rescued/fed, n=277; wild-type/starved, n=195; GAK-KO/starved, n=112; sham-rescued/starved, n=172; GAK-rescued/starved, n=180). **P<0.01 vs. wild-type. (G) Number of lysosomes in wild-type, GAK-KO, sham-rescued and GAK-rescued cells was quantified using Imaris. Data were analyzed using one-way ANOVA followed by Dunnett's post hoc test. The box extends from the lower to the upper quartile, the middle line indicates the median, the X indicates the mean and the whiskers represent the minimum to maximum values, except for outliers, which are indicated by dots (wild-type/fed, n=25; GAK-KO/fed, n=10; sham-rescued/fed, n=9; GAK-rescued/fed, n=12; wild-type/starved, n=21; GAK-KO/starved, n=15; sham-rescued/starved, n=10; GAK-rescued/starved, n=16). *P<0.05 and **P<0.01 vs. wild-type. GAK, cyclin G-associated kinase; KO, knockout; LAMP, lysosomal associated membrane protein; ns, not significant; N, nucleus; Gln, glutamine.