Fig. 5: Lysosomal cystine regulates mTORC1 activity through Ragulator-Rag GTPase lysosomal scaffold.

(a–d) mPTCs from Ctns^KO mice were transduced with a Null or hemagglutinin-tagged CTNS^WT (CTNS^WT-HA) or HA-tagged-mutant CTNS (CTNS^G339R-HA) bearing adenoviral particles for 2 days. b Cystine levels in Ctns mPTCs; n = 6 biologically independent samples. (c, h) Cells were cultured under fed or starved conditions or restimulated by adding back amino acids and glucose (AA/G stim). c Immunoblotting of the indicated proteins. d Fed Ctns^KO mPTCs transiently expressing tagged CTNS^WT or CTNS^G339R were lysed, and the samples were subjected to HA immunoprecipitation and immunoblotting for the indicated proteins. (e–i) Starved Ctns^WT mPTCs were exposed to CDME (0.1 mM) or with unmodified l-cystine (0.1 mM) for the indicated times. f Cystine levels in the Ctns mPTCs; n = 7 (time point 0) and n = 5 (time points 0.5 h and 6 h) biologically independent samples. g Cystine levels in whole cell lysates and purified lysosomes; n = 2 biologically independent samples. h Immunoblotting of the indicated proteins; n = 3 biologically independent samples. Quantification of the indicated proteins is shown in Supplementary Fig. 9g. i The cells were lysed, and the samples were subjected to HA immunoprecipitation and immunoblotting for the indicated proteins. All data in c, d, i were obtained from 2 independent biological replicates. Plots represent mean ± SEM. Statistics calculated by one-way ANOVA followed by Tukey’s or Sidak’s multiple comparisons test in b, f. Source data are provided as a Source Data file. Images in a, e were drawn using pictures from Servier Medical Art, which is licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).