Fig 2: RagBshort and RagBlong are resistant to GATOR1.a,b, RagABKO cells expressing each RagA/B isoform were transiently transfected with increasing amounts of GATOR1 plasmids (5 ng, 25 ng or 100 ng of each GATOR1 subunit) or metap2 (100 ng) as negative control: representative example (a) and quantification of four independent experiments, with RagA-expressing cells transfected with metap2 set to 1 (b). Bar height indicates average, and error bars represent standard deviation; n = 4 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. c,d, Control and RagAKO cells were transiently transfected with high (200 ng) levels of each GATOR1 subunit or metap2 as negative control and treated with amino-acid-rich medium or starved of amino acids for 30 min before lysis: representative example (c) and quantification of three independent experiments, with unstarved metap2-transfected control cells set to 1 (d). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. Two-way ANOVA and Sidak’s post-hoc test. e, Schematic representation of the two binding interfaces of GATOR1 to RagA/B. f–h, Rag interaction with the inhibitory interface (depicted in f) is assessed by co-immunoprecipitating the whole GATOR1 complex: representative example (g) and quantification of three independent experiments, with the inactive mutant of RagA set to 1 (h). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. One-way ANOVA and Tukey’s post-hoc test. i–k, Rag interaction with the GAP interface (depicted in i) is assessed through co-IP with the Nprl2/3 dimer in DEPDC5KO cells: representative example (j) and quantification of three independent experiments, with the inactive mutant of RagA set to 1 (k). Bar height indicates average, and error bars represent standard deviation; n = 3 biological replicates. One-way ANOVA and Tukey’s post-hoc test. -aa, amino-acid-free DMEM + 10% dFBS. +aa, -aa medium supplemented with 1× amino acids. Exact P values are shown in the graphs. NS, not significant. Source numerical data and unprocessed blots are available in source data.Source data

Fig 3: RagBlong acts as a ‘sponge’ for the GAP interface of GATOR1.(a, b) Coomassie staining of RagBlong (a) or RagA (b) purified from RagABKO HEK293T cells as dimers with FLAG-tagged RagCS75N (1 µg dimer per lane). The experiment was repeated once. (c, d) GTPase activity of RagA or RagBlong proteins dimerized to RagCS75N (1 µM) and mixed in solution with the indicated concentrations of GATOR1. RagBlong was purified from mammalian cells and RagA was purified from either (c) bacteria or (d) mammalian cells. Data from three (c) or two (d) independent experiments were plotted and a curve was fit with non-linear regression analysis following a hyperbola model. Circle=average (c) or individual replicates (d), error bars=standard deviation. (e, f) in vitro interaction of purified Rag dimers with GATOR1 (containing DEPDC5Y775A) in the presence of GDP. (e) Representative example. (f) Quantification of 3 independent experiments normalized for the background in the empty beads control. Bar Height=average, error bars=standard deviation, n = 3 replicates. One-way ANOVA and Tukey’s post-hoc test. (g) Quantification of the total RagA/B protein levels in Fig. 5a-i, with the RagA condition set to 1. Line=average, error bars=standard deviation, n = 9 biological replicates. One-way ANOVA and Tukey’s post-hoc test. (h-i) S6K1 phosphorylation in RagABKO cells transiently transfected with RagA, RagA and RagBshort, or all three RagA/B isoforms during starvation (amino-acid free DMEM + 10% dFBS) for the indicated time points. (h) Representative example. (i) Quantification of six independent experiments, with RagA-expressing cells at time point 0 set to 1. Circle=average, error bars=standard deviation, n = 6 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. (j-m) Amino-acid titration in Neuro-2a cells knockout for RagA (j-k) or RagB (l-m) treated with the indicated amino-acid concentrations for one hour. (j,l) Representative examples. (k.m) Quantifications of 3 independent experiments, with unstarved control cells set to 1. Circle=average, error bars=standard deviation, n = 3 biological replicates. Two-way ANOVA and Tukey’s post-hoc test. Exact p values are shown in the graphs. n.s., not significant. Source numerical data and unprocessed blots are available in source data. Source data

Fig 4: 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 5: Characterization of the Rag genomic alterations in qKO HEK293FT cells.(a) CRISPR/Cas9-mediated knockout of RRAGA. The associated genomic changes in the two RRAGA alleles and the resulting changes in the RagA protein are shown. (b) CRISPR/Cas9-mediated knockout of RRAGB. The associated genomic changes in RRAGB are shown. (c) CRISPR/Cas9-mediated knockout of RRAGC. The associated genomic changes in RRAGC and the resulting changes in the RagC protein are shown. (d) CRISPR/Cas9-mediated knockout of RRAGD. The associated genomic changes in RRAGD are shown. Source data