Fig 2: 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 3: Nuciferine inhibits mTORC1 activity and decreases TFEB phosphorylation. (A) Representative co-immunoprecipitation (Co-IP) analysis to assay interactions between HA-TFEB and RagA, RagC, mTOR and 14-3-3 in HepG2 cells. HepG2 cells were transfected with HA-TFEB and treated with 100 µmol/L nuciferine for 12 h or starved of amino acids (AA) for 1 h. (B) Representative immunoblotting of p-TFEB-Ser211, GFP-TFEB, p70 S6 kinase (S6K), p-S6K-Thr389, ribosomal protein S6 (S6), p-S6-Ser240/244, EIF4E-binding protein 1 (4E-BP1) and p-4E-BP1-Ser65 in HEK293T cells and quantification of p-TFEB-Ser211/TFEB, p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to control (no treatment) group. HEK293T cells were transfected with GFP-TFEB, and then treated with different concentrations of nuciferine (0, 10, 25, 50, 100 or 200 µmol/L) for 12 h. (C) Representative immunoblotting of HA-RagC, TFEB, unc-51-like kinase 1 (ULK1), p-ULK1-Ser757, S6K, p-S6K-Thr389, S6, p-S6-Ser240/244, 4E-BP1 and p-4E-BP1-Ser65 in HepG2 cells and quantification of p-ULK1-Ser757/ULK1, p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to control (no treatment) group. HepG2 cells were transfected with or without active RagC (S75L), and then treated with 100 µmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (D) HEK293T cells were analyzed by GFP-TFEB fluorescence and quantified to calculate the percentage of cells showing TFEB unclear localization. Scale bar, 20 µm. HEK293T cells were transfected with GFP-TFEB, and then treated with 100 µmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (E) HepG2 cells were analyzed by immunofluorescence and quantified to calculate the percentage of cells showing TFEB unclear localization. Scale bar, 20 µm. Cells were transfected with RagC or active RagC (S75L), and then treated with 100 µmol/L nuciferine for 12 h or 250 nmol/L Torin1 for 1 h. (F) Representative images of mTOR immunofluorescence staining and LysoTracker in HepG2 cells and quantification of colocalization coefficient. HepG2 cells were starved of AA for 1 h in the presence or absence of 100 µmol/L nuciferine. Scale bar, 10 µm. (G) Representative immunoblotting of S6K, p-S6K-Thr389, S6, p-S6-Ser240/244, 4E-BP1 and p-4E-BP1-Ser65 in the livers from different groups and quantification of p-S6K-Thr389/S6K, p-S6-Ser240/244/S6, p-4E-BP1-Ser65/4E-BP1 and expressed as fold change relative to ND group. Mice were treated as described in Fig. 1. n = 6 mice per group were used to analyze the results. All experiments were repeated at least 3 times. Data were expressed as the mean ± SEM; *P < 0.05, **P < 0.01.

Fig 4: Nuciferine binds to HBXIP. (A) Representative Co-IP analysis to assay interactions between Flag-P14 and RagA, RagC, P18, MP1, C7orf59, hepatitis B X-interacting protein (HBXIP) and mTOR in HepG2 cells. HepG2 cells were transfected with Flag-P14 and treated with 100 µmol/L nuciferine for 12 h or starved of AA for 1 h. (B) Cellular thermal shift assay of P14, P18, MP1, C7orf59 and HBXIP in HEK293T cells. Cells were treated with or without100 µmol/L nuciferine for 12 h. (C) The complex structure of HBXIP with nuciferine and the binding pocket with nuciferine. (D) Proposed model for the beneficial effects of nuciferine on NAFLD. Nuciferine interacts with HBXIP, blocks the function of Ragulator complex and reduces lysosomal localization of Rag GTPases and mTORC1, which activates TFEB-mediated ALP and further improves hepatic steatosis and insulin resistance in mice with NAFLD (created with BioRender.com.). All experiments were repeated at least 3 times. Data were expressed as the mean ± SEM; *P < 0.05, **P < 0.01.

Fig 5: Rap1 depletion increases the interaction between mTORC1 and lysosomes.a Immunofluorescence images from a single confocal plane of Rap1A+B-depleted HEK293A cells. Samples were coimmunostained for RagC (green) and LAMP2 (red). b Lysates from control or Rap1A+B siRNA-depleted HEK293A cells were separated into lysosome-enriched heavy membranes and cytosolic/light membranes fractions, and analyzed by immunoblotting for the endogenous amounts of the indicated proteins. Graph shows quantification of lysosome fraction. c, d Samples were processed as in a and coimmunostained for mTOR (green) and LAMP2 (red). The relative change in mTOR-associated lysosomes was quantified using 3D-reconstruction analysis (d). The number of cells analyzed to quantify lysosome abundance is shown in Supplementary Fig. 14. e S6K1 phosphorylation in Rap1-depleted cells expressing dominant negative RagB-T54N cDNA to prevent mTOR localization to lysosomes. f Model: Rap1 suppresses lysosome abundance and peripheral expansion when amino acids are limited (left). When nutrient levels are sufficient, mTORC1 is recruited to and activated on lysosomes (left and right). Rap1 depletion leads to an aberrant increase in lysosomes and the surface available for mTORC1 activation. The enhanced association between mTORC1 and its lysosome-bound activators results in mTORC1 hyperactivity (right). Also, see Supplementary Fig. 13g. The microscopic fields imaged were randomly selected. All experiments were repeated at least three times and graphs represent relative immunoblot band intensity from n individual experiments. Statistical data are presented as mean values ± s.e.m; Student’s t-test; two-sided, unpaired. Scalebars:10 µm. Uncropped images of blots are shown in Supplementary Fig. 17. pS6K T389 and total S6K1 were processed on separate blots due to technical reasons. See Data Source File for statistics source data.