Fig 2: Cyst(e)ine promotes GPX4 protein synthesis partly through Rag-mTORC1-4EBP signaling.a UMRC6 cells were cultured in media with indicated concentrations of cystine for 24 h followed by Western blotting to analyze protein levels. b UMRC6 cells were treated with indicated concentrations of Rapamycin (Rapa) or Torin1 for 24 h. GPX4 and mTOR signaling protein levels were assessed by Western blotting. c UMRC6 cells were treated with indicated concentrations of AZD8055 for 24 h. GPX4 and mTOR signaling protein levels were assessed by Western blotting. d UMRC6 cells were treated with 1 µM Torin1 or AZD8055 for 24 h followed by polyribosome fractionation and RT-PCR to analyze GPX4 and ACTB (Actin) mRNA distribution profiles during protein translation. n = 3 biologically independent repeats. Error bars are mean ± SD. P value was determined by two-tailed unpaired Student’s t test. e Western blotting analysis of indicated protein levels in UMRC6 control (sgC) and RagA/RagB double-knockout (A/B-DKO) cell lines. LE long exposure. f sgC and A/B-DKO cells were cultured in cystine-free media for 24 h followed by 200 µM cystine stimulation for 8 h and Western blotting analysis. g, h Wild-type (shown as “/”) and 4EBP1-4A–overexpressing (shown as “4 A”) UMRC6 or 786-O cells were treated with or without 100 ng/ml Doxycycline (Dox.) for 30 h followed by Western blotting analysis. i Western blotting analysis of protein expression in control (sgCon) and 4EBP1/2-double-knockout (DKO-1 and DKO-2) UMRC6 cells. j Western blotting analysis of protein expression in indicated cell lines treated with or without 1 µM Torin1 for 24 h. k Western blotting analysis of protein expression in indicated cell lines cultured in complete media or cystine-free media for 24 h. l Representative images from immunohistochemical staining of kidney tumor tissue microarray for GPX4 and P-4EBP1. m Summary and statistical analysis of immunohistochemical staining results for GPX4 and P-4EBP1 in tumor tissue microarray.

Fig 3: Sestrin2 controls mTORC1 through GATOR complexes.(A) Sestrin2 inhibits phosphorylation of mTORC1 targets through GATOR. HEK293 cells stably transduced with indicated shRNA were infected with GFP or Sestrin2-overexpressing lentiviruses. After 48 h, cells were analyzed by IB with indicated antibodies against endogenous proteins. (B and C) Silencing Npr2, the Drosophila homologue of NPRL2, relieves Drosophila Sestrin (dSesn)-induced growth arrest. Anterior (B, upper panels) and dorsal (B, lower panels) views of wing blades with ap-GAL4–driven expression of indicated transgenic elements were imaged under a dissection microscope and a transmitted light microscope, respectively. Dorsal wing area were quantified and presented as a bar graph (C, n = 7, means ± s.e.m.). P value was calculated using Student's t-test. (D and E) Autophagy defects in dSesn-null animals were corrected by heterozygotic mutations of GATOR2 components. Thoraces from 5 day-old flies of indicated strains were analyzed through immunoblotting of dAtg8 and Actin proteins (D). Relative levels of unprocessed (dAtg8-I) and processed (dAtg8-II) dAtg8 proteins were quantified by densitometry and presented as a bar graph (E, n = 3, means ± s.e.m.). P values were calculated using Student's t-test. (F and G) Proposed model of the Sestrin2-GATOR-RagB pathway that mediates stress-induced mTORC1 silencing. In unstressed conditions, GATOR1 is constitutively inhibited by GATOR2, and RagB recruits mTORC1 to lysosomal surface and activates it as well as its downstream targets (F). Upon stress-induced expression of Sestrin2, GATOR1 is released from GATOR2-mediated inhibition and inactivates RagB (G). In the absence of active RagB, mTORC1 is released from lysosomal surface and mTORC1 signaling is subsequently inactivated. Cropped gel images are used in this figure and the gels were run under the same experimental conditions.

Fig 4: Structural comparison between RagA and RagB suggests main differences localize to the unstructured N-terminal tail.(a) Structure-based sequence alignment of RagA and RagB prepared with ESPript. Similar and identical residues are marked by yellow and red boxes, respectively. Secondary structure assignment is based on PDBID: 6S6D. (b) Residue differences in the structures of RagA and RagB are not predicted to cause overall structural changes. Superposition of the structure of RagA (from PDBID: 6S6D; shown in blue) with RagB (modelled; shown in red) shows high structural similarity between the two structures. Side chains of variable positions shown as dark grey (RagA) or light grey (RagB) sticks. (c) Minimal surface residue differences between RagA and RagB. Surface representation of the model of RagB (red) / RagD (yellow) heterodimer in the active conformation (modelled based on PDBID: 6S6D). Variable positions between RagA and RagB are coloured blue. (d) Ribbon representation of the model of the RagB (red) / RagD (yellow) heterodimer in the active conformation (modelled based on PDBID: 6S6D). Variable positions between RagA and RagB are coloured blue and side chains are shown as sticks. (e) Same as in (d), but for the inactive RagB/D dimer (modelled based on PDBID: 6ULG).

Fig 5: The RagA and RagB paralogues differentially control mTORC1 activity upon AA starvation.a, Immunoblots with lysates from HEK293FT WT, or qKO cells stably expressing RagA/D or RagB/D, treated with medium containing (+) or lacking (–) AA, in basal (+), starvation (–) or add-back (–/+) conditions, probed with the indicated antibodies. Arrowheads indicate bands corresponding to different protein forms, when multiple bands are present. P, phosphorylated form; S, SUMOylated form. b–e, Quantification of TFEB (b and c), TFE3 (d) and S6K (e) phosphorylation from the blots shown in a. n = 4 independent experiments. f, Co-localization analysis of mTOR with LAMP2 (lysosomal marker) in HEK293FT WT or qKO cells stably expressing RagA/D or RagB/D, using confocal microscopy. Magnified insets shown to the right. Scale bars, 10 μm. g, Quantification of mTOR/LAMP2 co-localization from n = 40 individual cells per condition from a representative experiment out of two 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