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: Cancer-associated RagC mutations show increased lysosomal localization and p18 binding.(a-b) Colocalization analysis between the Rags and LAMP2 (lysosomal marker) in HEK293FT qKO cells stably expressing HA-tagged RagA with WT RagC, RagD, or the T90N and W115R RagC mutants, using confocal microscopy. Magnified insets shown to the right. Scale bars = 10 μm (a). Quantification of HA/LAMP2 colocalization from n = 50 individual cells per condition from a representative experiment out of 2 independent replicates is shown in (b). Data shown as mean ± SEM. *** p<0.005, **** p<0.001. (c) The cancer-associated RagC mutants bind more strongly to p18/LAMTOR1, compared to wild-type RagC. Co-immunoprecipitation experiments in HEK293FT qKO cells, transiently expressing FLAG-tagged p18 or Luciferase (Luc) as negative control, and HA-tagged RagA with WT RagC, RagD, or the T90N and W115R RagC mutants. Binding of the Rags to p18 was analyzed by immunoblotting as indicated. n = 2 independent experiments. Source numerical data and unprocessed blots are available in Source Data. Source data

Fig 4: Cancer-associated RagC mutations enhance TFE3/TFEB phosphorylation and mTOR lysosomal recruitment.a, Schematic representation of HA-tagged WT RagC and the cancer-related T90N, W115R RagC point mutants. b, Immunoblots with lysates from HEK293FT qKO cells stably expressing HA-tagged RagA with WT RagC or RagD, or the T90N, W115R RagC mutants, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P, phosphorylated form; S, SUMOylated form. c–f, Quantification of TFEB (c and d), TFE3 (e) and S6K (f) phosphorylation from b. n = 3 independent experiments. g, Co-localization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT qKO cells stably expressing HA-tagged RagA with the RagC proteins shown in a or RagD, using confocal microscopy. Magnified insets shown to the right. Scale bars, 10 μm. h, Quantification of mTOR/LAMP2 co-localization 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 and unprocessed blots are available in source data.Source data

Fig 5: Interaction properties of RagC- and RagD-containing dimers.(a) RagC and RagD bind similarly to RagA. Co-immunoprecipitation experiments in HEK293FT qKO cells, transiently expressing HA-tagged RagA with FLAG-tagged RagC, RagD, or Luciferase (Luc) as negative control. Binding of RagA to RagC or RagD was analyzed by immunoblotting as indicated. n = 2 independent experiments. (b-f) Co-immunoprecipitation experiments in HEK293FT qKO cells transiently expressing FLAG-tagged RagA/C, RagA/D, or Luciferase (Luc) as a negative control. Binding of the Rags to the indicated proteins was analyzed by immunoblotting. P: phosphorylated protein form; S: SUMOylated protein form(s) (b). Quantification of Rag binding to mTOR, Raptor, TFEB, and TFE3, shown in (c), (d), (e), (f), respectively. n = 3 independent experiments. Data in graphs shown as mean ± SEM. * p<0.05. Source numerical data and unprocessed blots are available in Source Data. Source data