Requirement of the v-ATPase for mTORC1 activation by amino acids. (A) Double-stranded RNA (dsRNA)-mediated depletion of vhaAC39 in Drosophila S2 cells. Cells were deprived for amino acids for 1.5 h and then stimulated with complete medium for 30 min. Proteins from cell lysates were analyzed for phosphorylation of dS6K at threonine 398 (T398). Depletion of vhaAC39 by two distinct dsRNAs is compared to that of dRagC. (B) dsRNA-mediated depletion of both vha100-1 and vha100-2 in S2 cells suppresses amino acid-induced T398 phosphorylation of dS6K. (C) Cell size measurement after depletion of vhaAC39 in S2 cells with two dsRNAs (red and blue) compared to a control dsRNA (black). (D) S6K1 phosphorylation at T389 in HEK-293T cells treated with short-hairpin RNA (shRNA) targeting GFP, RagC and RagD, and V0c. Cells were deprived of amino acids for 50 min and, where indicated, stimulated for 10 min. Immunoblotting was used to detect the indicated proteins. (E) S6K1 phosphorylation in HEK-293T cells deprived of amino acids for 50 min in the presence of the indicated concentrations of Concanamycin A (ConA) and then stimulated for 10 min with amino acids. (F) S6K1 phosphorylation in HEK-293T cells deprived of amino acids for 50 min in the presence of the indicated concentrations of Salicylihalamide A (SalA) and restimulated for 10 min with amino acids. (G) ConA blocks mTORC1 activation by alcohol esters of amino acids. HEK-293T cells were deprived of amino acids for 50 min and then stimulated for 10 min with amino acids or alcohol esters of amino acids in the presence of the indicated concentration of ConA. (H) Activation of mTORC1 by intracellular amino acids. HEK-293T cells were deprived of amino acids for 50 min and stimulated with amino acids or cycloheximide in DMSO or 2 µM SalA. Immunoblotting was used to detect the indicated proteins.

Requirement of the v-ATPase for lysosomal recruitment of mTORC1 by the Rag GTPases. (A) Immunofluorescence images of mTOR and LAMP1 in HEK-293T cells deprived of amino acids (top) or deprived and then stimulated (bottom) in the presence of DMSO (left) or 2.5 µM SalA (right). The mTOR and LAMP2 channels are shown separately. Inset shows a higher magnification of a selected field. In the merge, yellow indicates co-localization. (B) HEK-293T cells expressing a lentivirally-encoded shRNA targeting GFP (left) or V0c (right) were deprived of amino acids (top) or deprived and then stimulated (bottom). (C) Staining for RagC and LAMP2 in HEK-293T cells deprived of amino acids (top) or deprived and then stimulated (bottom) in the presence of DMSO (left) or 2.5 µM SalA (right). (D) HEK-293T cells stably expressing the constitutively active RagB^Q99L mutant (293T RagB^GTP) deprived of amino acids (top) or deprived and stimulated (bottom) in the presence of DMSO (left) or 2.5 µM ConA (right). (E) S6K1 phosphorylation in HEK-293T cells and HEK-293T RagB^GTP cells deprived of amino acids for 50 min in the presence of DMSO or 2 µM SalA and stimulated for 10 min with amino acids. (F) S6K1 phosphorylation in wild-type MEFs (RagA^+/+) or in MEFs homozygous for the constitutive active RagA Q66L mutant (RagA^GTP/GTP); cells were deprived of amino acids for 50 min in the presence of DMSO or 2.5 µM SalA and stimulated for 10 min with amino acids.
In all images, scale bars represent 10 µm.

Interaction of the v-ATPase with the Ragulator-Rag GTPases. (A) Cartoon summarizing mass spectrometry analyses of immunoprecipitates from FLAG-p18 (left), FLAG-p14 (center) and FLAG-RagB (right) expressing HEK-293T cells. v-ATPase subunits are color-coded according to their peptide representation (scale at the far right). (B) Binding of Ragulator to the V0 domain. HEK-293T cells stably expressing FLAG-tagged p18 and p14 were lysed and subjected to FLAG-immunoprecipitation followed by immunoblotting for V0c and V0d1. FLAG-LAMP1 and FLAG-Metap2 served as negative controls. (C) Binding of Ragulator to the V1 domain. HEK-293T cells stably expressing FLAG-tagged p18, p14, LAMP1 and Metap2 were lysed and subjected to FLAG-immunoprecipitation followed by immunoblotting for V1A, V1B2 and V1D. (D) (Top) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated with recombinant V0d1 fused to glutathione S-transferase (HA-GST-V0d1), immobilized on glutathione agarose beads. Samples were subjected to immunoblotting for FLAG to detect bound Ragulator components. HA-GST-Rap2A served as a negative control. (Bottom) In vitro binding assays in which purified FLAG-p18 and FLAG-p14 were incubated with recombinant V1D fused to glutathione S-transferase (HA-GST-V1D). HA-GST-metap2 served as a negative control.
(E) The Ragulator-V1 interaction, but not the Ragulator-V0, is regulated by amino acids. HEK-293T cells stably expressing FLAG-tagged p18, p14 and Metap2 were deprived of amino acids for 90 min, or deprived and then stimulated with amino acids for 15 min. Following lysis, samples were subjected to FLAG-immunoprecipitation and immunoblotting for the indicated v-ATPase subunits. (F) SalA blocks regulation of Ragulator-V1 interaction by amino acids. HEK-293T cells stably expressing FLAG-p14 were deprived of amino acids for 90 min, or deprived and then stimulated with amino acids for 15 min, in the presence of DMSO or 2 µM SalA. (G) Cartoon summarizing the Ragulator-v-ATPase interactions identified in (A)–(F). Orange denotes regulation by amino acids, blue indicates lack of regulation.

In vitro analysis of mTORC1 activation by amino acids. (A) In vitro binding of myc-raptor to FLAG-RagB- but not to FLAG-Rap2A-containing vesicles. Organelle preparations were left unstimulated, or were stimulated with amino acids or amino acid esters and incubated with myc-raptor-containing cytosol. After FLAG immunoprecipitation, bound myc-raptor was detected by immunoblotting (B) Intact FLAG-RagB lysosomes, FLAG-RagB lysosomes permeabilized with Streptolysin-O, and FLAG-RagB lysosomes permeabilized with Triton X-100, were left unstimulated, stimulated with amino acids, or stimulated with amino acid esters. Myc-raptor was detected by immunoblotting (C) S6K1 phosphorylation at T389 in HEK-293T cells transiently expressing FLAG-S6K1, FLAG-S6K1 + myc-PAT1, FLAG S6K1 + HAGST-tagged active Rag mutants, or FLAG-S6K1 + myc-PAT1 + HAGST-active Rags. Cells were deprived of amino acids for 50 min, or starved and then stimulated for 10 min (see methods). The indicated proteins were detected by immunoblotting. The band pattern of myc-PAT1 is likely due to glycosylation. (right) Immunofluorescence images of lysosomes from HEK-293T cells transiently expressing myc-PAT1 and stained for myc tag (top, red in the merge) and for LAMP2 (center, green in the merge). (D) Accumulation of ¹⁴C-labeled amino acids into lysosomes immunopurified from HEK-293T cells expressing LAMP1-mRFP-FLAG^X2. Lysosomes were either left intact or permeabilized with Triton X-100 or Streptolysin O prior to measurement. Overexpression of PAT1 largely abolished amino acid accumulation inside lysosomes. Each value represents the mean ± SD of three independent samples. (E) FLAG-RagB lysosomes were treated with DMSO or SalA, activated with amino acid esters and then incubated with myc-raptor. An organellar fraction from FLAG-metap2 expressing cells served as negative control. (F) FLAG-RagB lysosomes were stimulated with amino acid esters in the presence of the proton ionophore FCCP or the non-hydrolyzable ATP analog AMP-PNP at 1 mM or 10 mM. Organelle samples were then incubated with mycraptor cytosol, followed by FLAG-IP and immunoblotting for myc-raptor and endogenous mTOR. (G) Model for inside-out activation of mTORC1 by lysosomal amino acids. Accumulation of amino acids inside the lysosomal lumen generates an activating signal that is transmitted to the Rag GTPases via the v-ATPase-Ragulator. In turn, the Rags physically recruit mTORC1 to the lysosomal surface.