Fig 2: RagC and RagD differentially regulate TFE3 localization, target gene expression and lysosomal biogenesis.a, TFE3 localization analysis in HEK293FT WT or qKO cells, stably expressing HA-tagged RagA/C, RagA/D or Luc as a negative control, using confocal microscopy. Nuclei stained with DAPI. Magnified insets shown to the right. Scale bars, 10 μm. n = 3 independent experiments. b, Scoring of TFE3 localization from a. Individual cells were scored for nuclear, intermediate or cytoplasmic TFE3 localization, as indicated in the example images. Scale bars, 10 μm. n = 3 independent experiments. c, Expression analysis of the TFE3 target genes GPNMB and UAP1L1 in HEK293FT WT, qKO cells or qKOs stably expressing HA-tagged RagA/C or RagA/D. nGPNMB = 7 independent experiments; nUAP1L1 = 6 independent experiments. d, LysoTracker staining in HEK293FT WT, qKO cells or qKOs stably expressing HA-tagged RagA/C or RagA/D. e, Quantification of LysoTracker signal intensity from n = 50 individual cells per condition from a representative experiment out of three independent replicates. Data in graphs shown as mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.001. Source numerical data are available in source data.Source data

Fig 3: RRAGC mutation renders SUDHL-4 sensitive to cardamonin. Normal, RagCWT and RagCT90N-mutant HEK-293T or SUDHL-4 cells were treated with cardamonin (12 µM). (A) Immunoprecipitates pulled down by an anti-FLAG antibody were collected from cells expressing the indicated cDNAs. Cell lysates and immunoprecipitates were analysed by western blotting (n = 3). (B) Cell viability of normal, RagCWT and RagCT90N-mutant SUDHL-4 cells was determined by CCK-8 assay. Data are presented as mean ± SD (n = 3). **p < 0.01 compared with the control group; ##p < 0.01 compared with the cardamonin-treated RagCWT group. (C) RagCWT and RagCT90N-mutant SUDHL-4 cells were treated with cardamonin (12 µM) or everolimus (0.5 µM). Cell lysates were analysed by western blotting to measure the level of the indicated proteins (n = 3)

Fig 4: Generation of Ragulator-deficient clone with total mTORC1 activity.A, schematic representation of the findings of this study, depicting the phosphorylation of the TFEB substrate by mTORC1 bound to the Ragulator-Rag scaffold. B, the results of Western blotting studies on ΔRagulator and parental RAW264.7 cells. Lamtor1, Lamtor2, Lamtor3, Lamtor4, and Lamtor5 are components of the Ragulator complex; β-actin served as the internal control; mTOR and Raptor are components of mTORC1; RagC is necessary for the docking of TFEB to Ragulator. C, representative result of Western blotting studies for determining the activity of mTORC1. The control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. The phosphorylation of S6K in these cells was abolished following treatment with the mTOR kinase inhibitor, Torin1, at the indicated concentrations for 1 h. D, the densities of the p-S6K and S6K bands in panel C were quantified. The ratio of densities of the p-S6K band to S6K band is shown. Four independent experiments were performed. Bars indicate the median. Statistical significance was calculated using Mann–Whitney U test. E, the results of Upstream Regulator Analysis, as determined using the web-based software, Ingenuity Pathway Analysis. The pathways with z scores less than −2 are significantly inactivated in the ΔRagulator cells, compared to the RAW264.7 cells. The z score for the TFEB pathway was >2, which indicates that TFEB is significantly activated in the ΔRagulator cells than in the control RAW264.7 cells. F, the sizes of the ΔRagulator cells were larger than those of the parental RAW264.7 cells. The cell sizes of 1 × 104 cells were estimated using flow cytometry. Statistical significance was calculated using Welch’s t test. G, Western blotting for determining the phosphorylation of TSC2. Control RAW264.7 cells and ΔRagulator cells were cultured in nutrient-sufficient and serum-supplemented DMEM. Three biological triplicates were examined. H, the densities of the p-TSC2 and the TSC2 bands in panel G were quantified. The ratio of densities of the p-TSC2 band to TSC2 band is shown. Statistical significance was calculated using the Mann–Whitney U test. mTORC1, mammalian target of rapamycin complex 1; TFEB transcription factor EB.

Fig 5: Effects of different treatments on the AP-5/SPG11/SPG15 complex versus mTORC1. (A) IF labeling of SPG15-GFP (amplified with anti-GFP) in cells that had been treated with siRNA to knock down RagA, the RagC/D GAP FLCN, or the RagA/B GATOR1 GAP subunit NPRL2. To better visualize the labeling and any effects of siRNA depletion, all cells were starved (1 h). These knockdowns have a slight effect on SPG15-GFP recruitment, but they are not as dramatic as a RagC or Ragulator knockdown. Scale bar: 20 µm. (B) Western blots of whole-cell lysates from SPG15-GFP cells treated with siRNA as indicated, with mTOR as a loading control. The blot shows mTOR labeling in a control versus FLCN knockdown, but it is representative of the loading for both knockdowns. The blots are representative of at least two independent experiments. (C) IF labeling of SPG15-GFP (amplified with anti-GFP) and mTOR in cells treated in various ways, showing an inverse relationship between the two. Starvation (1 h) increases SPG15 recruitment while preventing mTOR recruitment, while the drugs rapamycin and Torin1 lock mTOR on the membrane and prevent recruitment of SPG15. Scale bar: 20 µm. (D) IF labeling of SPG11-GFP (amplified with anti-GFP), showing that, like SPG15-GFP, its membrane association is inhibited by rapamycin and Torin1. Scale bar: 20 µm. kd, knockdown.