Fig. 6: mTORC1 maintains activity towards its non-lysosomal substrates in cellular models of diminished lysosomal mTOR localization.
From: Spatial and functional separation of mTORC1 signalling in response to different amino acid sources
a, Immunoblots with lysates from HEK293FT WT and RagA/B KO cells, treated with media containing or lacking AAs, in basal (+AA), starvation (−AA) or add-back (–/+AA) conditions, probed with the indicated antibodies. For basal (+AA) conditions, culture media were replaced by +AA treatment media 90 min before lysis. For AA starvation (−AA), culture media were replaced by starvation media 1 h before lysis. For AA add-back samples (–/+AA), cells were first starved as described above and then starvation media were replaced by +AA treatment media for 30 min. The composition of all media is described in Methods. b, In vitro kinase assays with mTORC1 immunopurified from WT or RagA/B KO HEK293FT cells and recombinant 4E-BP1 protein used as substrate, with 4E-BP1 phosphorylation detected by immunoblotting. No ATP samples (−ATP) used as negative controls. c, Lyso-IP experiments in WT and RagA/B KO HEK293FT cells stably expressing HA-tagged TMEM192 (or FLAG-TMEM192 as negative control). Intact lysosomes immunopurified by anti-HA IPs under native conditions, and the presence of the indicated proteins in the lysosomal and non-lysosomal fractions, as well as in whole-cell lysates, analysed by immunoblotting. Note the absence of S6K from lysosomal fractions and the presence of phospho-TFEB in the lysosomal fractions only of control cells. n = 2 independent experiments. d,e, Phosphorylation of multiple mTORC1 substrates is largely unaffected by BafA1 treatment (100 nM, 6 h) (d) or loss of Rag GTPases (e). In e, Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. f,g, GRASP55 phosphorylation by mTORC1 is retained in RagA/B KO (f) or BafA1-treated cells (100 nM, 6 h) (g), similarly to that of S6K. In g, starvation was performed as in a. Torin1 (250 nM, 1 h) was used as a control for mTOR inhibition. h, RagC is an additional lysosomal mTORC1 substrate that requires properly functioning lysosomes for its phosphorylation, similarly to TFEB/TFE3. AA starvation or blockage of lysosomal function with BafA1 (100 nM, 6 h) decrease RagC phosphorylation (shown as elevated RagC signal with #5466). Treatments performed as in a. i,j, Lysosomal localization of RagC is unaffected by BafA1 treatment (100 nM, 6 h) (i). Quantification of RagC/LAMP2 colocalization in (j). Scale bars, 25 μm and for insets, 5 μm. n = 50 individual cells from five independent fields per condition. Arrowheads indicate bands corresponding to different protein forms when multiple bands are present. P, phosphorylated form. Data in graphs shown as mean ± s.e.m. n.s., non-significant. Source numerical data and unprocessed blots are available in Source data. See also Extended Data Figs. 3–8.
