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. Author manuscript; available in PMC: 2021 Nov 5.
Published in final edited form as: Mol Cell. 2020 Nov 5;80(3):437–451.e6. doi: 10.1016/j.molcel.2020.10.004

Figure 6. The inhibition of lysosome- or proteasome-mediated cellular degradation machinery induces lysosomal mTORC1 localization in Rag-Ragulator deficient cells independent of amino acid availability.

Figure 6.

6A. Blocking lysosome function induces lysosomal mTOR localization in p18 KO EMSC cells. The indicated cells were starved for amino acids for 15 min, then treated with a vehicle (0.1% DMSO) or the indicated lysosomal inhibitors (Concanamycin A (ConA, 5 μM), Chloroquine (CQ, 50 μM), Bafilomycin A1 (BafA, 1 μM), or NH4Cl (20 mM)) for another 45 min. Before fixing the cells, the cells were treated with a 1x amino acid mixture for 10 min as indicated (+AA). Pearson’s correlation coefficient, R values were determined for quantifying the colocalization of mTOR and LAMP2. The scale bar indicates 10 μm. **p< 0.01, ****p<0.0001 compared to p18 KO EMSC cells with vehicle treatment in the absence of amino acids. mean ± SEM, n (number of p18 KO cells analyzed) : (vehicle-AA, vehicle+AA, ConA-AA, CQ-AA, BafA-AA, NH4Cl-AA)=(16, 10, 9, 9,10,6)

6B. Blocking proteasome function also induces lysosomal mTORC1 localization. Similar experiments were performed by using MG-132 as Fig. 6A. **p< 0.01 compared to vehicle treatment in the absence of amino acids. mean ± SEM, n: (vehicle −AA, vehicle +AA, MG-AA, MG+AA)=(9, 8, 12, 14)

6C. Blocking both lysosome and proteasome function additively induces lysosomal mTOR localization. p18 KO EMSC cells were treated as Fig. 6A. **p< 0.01, ***p<0.001, ****p<0.0001 compared to vehicle treatment. mean ± SEM, n: (Veh, CQ, MG, CQ+MG)=(13,30,31,29)

6D. Blocking both lysosome and proteasome function accumulates Ub-Rheb and mTORC1 on the lysosome in Ragulator deficient cells. Lysosomes were immunopurified from p18 KO HEK293T cells in the presence or absence of CQ+MG treatment under amino acid starvation conditions.

6E. Blocking lysosome and proteasome function increases levels of Ub-Rheb and the interaction between Rheb and mTOR in p18 KO EMSC cells. p18 KO EMSC cells were starved for amino acids for 50 min then treated with or without CQ+MG for another 75 min.