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Science 334 (6056): 678-683

Copyright © 2011 by the American Association for the Advancement of Science

mTORC1 Senses Lysosomal Amino Acids Through an Inside-Out Mechanism That Requires the Vacuolar H+-ATPase

Roberto Zoncu1,2,3,4, Liron Bar-Peled1,2,3, Alejo Efeyan1,2,3, Shuyu Wang1,2,3, Yasemin Sancak1,2,3, and David M. Sabatini1,2,3,4,5,*

1 Whitehead Institute for Biomedical Research, Nine Cambridge Center, Cambridge, MA 02142, USA.
2 Department of Biology, Massachusetts Institute of Technology (MIT), Cambridge, MA 02139, USA.
3 David H. Koch Institute for Integrative Cancer Research at MIT, 77 Massachusetts Avenue, Cambridge, MA 02139, USA.
4 Broad Institute, Seven Cambridge Center, Cambridge, MA 02142, USA.
5 Howard Hughes Medical Institute, MIT, Cambridge, MA 02139, USA.


Figure 1
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Fig. 1. Requirement of the v-ATPase for mTORC1 activation by amino acids. (A) dsRNA-mediated depletion of vhaAC39 in Drosophila S2 cells. Cells were deprived for amino acids for 1.5 hours 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 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 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 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 2 μM ConA where indicated. (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 dimethyl sulfoxide (DMSO) or 2 μM SalA. Immunoblotting was used to detect the indicated proteins.

 

Figure 2
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Fig. 2. Requirement of the v-ATPase for lysosomal recruitment of mTORC1 by the Rag GTPases. (A) Immunofluorescence images of mTOR and LAMP2 in HEK-293T cells deprived of amino acids (a.a.) (top) or deprived and then stimulated (bottom) in the presence of DMSO (left) or 2.5 μM SalA (right). (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 RagBQ99L mutant (293T RagBGTP) were 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 RagBGTP 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 (RagAGTP/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, insets show selected fields that were magnified five times and their overlays. Scale bars represent 10 μm.

 

Figure 3
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Fig. 3. Interaction of the v-ATPase with the Ragulator-Rag GTPases. (A) Cartoon summarizing mass spectrometry analyses of immunoprecipitates from HEK-293T cells expressing FLAG-p18 (left), FLAG-p14 (center), and FLAG-RagB (right). 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 (IP) 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 interaction, 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. After lysis, samples were subjected to FLAG immunoprecipitation and immunoblotting for the indicated v-ATPase subunits. (F) SalA blocks regulation of the 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. Samples were lysed, FLAG-immunoprecipitated, and immunoblotted for the indicated proteins. (G) Cartoon summarizing the Ragulator–v-ATPase interactions identified in (A) to (F). Orange denotes regulation by amino acids; blue indicates lack of regulation.

 

Figure 4
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Fig. 4. In vitro analysis of mTORC1 activation by amino acids. (A) Cell-free 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 in SOM). The indicated proteins were detected by immunoblotting. The band pattern of Myc-PAT1 is probably 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-FLAGX2. Lysosomes were either left intact or permeabilized with Triton X-100 or streptolysin O before 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 2 μM SalA, activated with amino acid esters, and then incubated with Myc-Raptor. An organellar fraction from FLAG-metap2–expressing cells served as a negative control. (F) FLAG-RagB lysosomes were stimulated with amino acid esters in the presence of the proton ionophore FCCP or the nonhydrolyzable ATP analog AMP-PNP at 1 mM or 10 mM. Organelle samples were then incubated with Myc-Raptor cytosol, followed by FLAG immunoprecipitation and immunoblotting for Myc-Raptor and endogenous mTOR. (G) Model for inside-out activation of mTORC1 by lysosomal amino acids. The accumulation of amino acids inside the lysosomal lumen generates an activating signal that is transmitted to the Rag GTPases via the v-ATPase–Ragulator interaction. In turn, the Rags physically recruit mTORC1 to the lysosomal surface.

 


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