1. Glutaminolysis Activates Rag-mTORC1 Signaling
Raúl V. Durán, Wolfgang Oppliger, Aaron M. Robitaille, Lisa Heiserich, Roswitha Skendaj, Eyal Gottlieb, Michael N. Hall 
Molecular Cell 
Volume 47, Issue 3, Pages (August 2012)
DOI: /j.molcel
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2. Molecular Cell 2012 47, 349-358DOI: (10.1016/j.molcel.2012.05.043)
Copyright © 2012 Elsevier Inc. Terms and Conditions

3. Figure 1
Leucine and Glutamine Induce Glutaminolysis and mTORC1 Activity
(A) Intracellular levels of αKG in U2OS cells incubated either in complete DMEM (control) or in DMEM without leucine and glutamine (-LQ) for 2 hr.
(B) Intracellular levels of αKG in U2OS cells incubated either in complete medium or in the absence of all amino acids (-AA) for 2 hr followed by 15 min addition of either vehicle (-LQ) or leucine and glutamine (+LQ).
(C and D) Phosphorylation status of S6K, S6, and AKT in U2OS cells starved for all amino acids for 1 hr and then restimulated with either vehicle (-LQ), leucine (L) and glutamine (Q) as indicated for 15 min.
(E) Phosphorylation status of S6K and S6 in HeLa cells upon glutamine withdrawal for different times, as indicated.
(F) Phosphorylation status of S6K and S6 in HeLa cells upon glutamine withdrawal for 24 hr followed by the addition of either leucine or glutamine for 1 hr, as indicated.
(G) mTOR localization in U2OS cells starved for all amino acids for 1 hr and restimulated with either vehicle (-LQ) or leucine and glutamine (LQ) for 15 min. Shown is a representative cell. For (A) and (B), the mean is shown; error bars represent SEM (n > 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Inhibition of Glutaminolysis Prevents the Activation of mTORC1 by Leucine and Glutamine
(A and B) Intracellular levels of αKG (A) and phosphorylation status of S6K and S6 (B) in U2OS cells starved for all amino acids for 1 hr and restimulated with leucine and glutamine (+LQ) for 15 min either in the presence or the absence of DON, as indicated.
(C) Phosphorylation status of S6K and S6 in HeLa cells upon glutamine withdrawal for 24 hr followed by glutamine addition for 1 hr either in the presence or the absence of DON.
(D) Phosphorylation status of S6K and S6 in U2OS cells treated as in (B) but using BPTES instead of DON.
(E) Phosphorylation status of S6K in U2OS cells treated as in (C) but using BPTES instead of DON.
(F) Phosphorylation status of S6K, S6, and AKT in response to leucine and glutamine (LQ) in starved U2OS cells transfected with either nontargeting siRNA (siCtrl) or with siRNA against GLS or GDH.
(G and H) Intracellular levels of ATP in U2OS cells upon either GLS/GDH knockdown or DON treatment. Glucose starvation was included as a control.
(I and J) Phosphorylation status of AMPK in U2OS cells upon either GLS/GDH knockdown or DON treatment. Glucose starvation was included as a control.
(K and L) Leucine and glutamine uptake in U2OS cells upon either GLS/GDH knockdown or DON treatment.
(M) Phosphorylation status of S6K in U2OS cell expressing the constitutively active mutant FLAG-Rheb-N153T (FLAG-RhebGTP), treated as in (F). The mean is shown; error bars represent SEM (n > 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Inhibition of Glutaminolysis Inhibits the Translocation of mTOR to the Lysosome
(A) Lysosomal localization of mTORC1 in leucine and glutamine (LQ)-stimulated U2OS cells transfected with either nontargeting siRNA (siCtrl) or with siRNA against GLS or GDH.
(B) Quantification of the lysosomal localization of mTORC1 in cells treated as in (A) in three independent experiments (for each experiment, n > 100).
(C) Lysosomal localization of RagC in U2OS cells treated as in (A). The mean is shown; error bars represent SEM (n > 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 4
Upregulation of Glutaminolysis Enhances the Response of mTORC1 to Amino Acids
(A–E) U2OS cells were transiently transfected with either empty vector (-FLAG-GDH) or with a FLAG-GDH overexpressing plasmid (+ FLAG-GDH). Forty-eight hours after transfection, the cells were starved for all amino acids for 1 hr and then restimulated with either vehicle (-LQ) or leucine and glutamine (+LQ), as indicated. FLAG-GDH expression (A), intracellular levels of αKG (B), phosphorylation status of S6K and S6 (C) and mTOR translocation to the lysosome (D and E) were measured.
(F and G) U2OS cells were incubated in the absence of all amino acids and then restimulated with either vehicle (-AA), all amino acids (+AA) or with cell-permeable αKG (+αKG) for 1 hr. Lysosomal translocation of mTOR was measured by immunofluorescence (F) and quantified (G).
(H) Phosphorylation status of S6K and S6 in U2OS cells upon 1 hr incubation in KRBB medium supplemented with either cell-permeable αKG, glutamine, or leucine as indicated. See Figures 1D and S3E for additional controls on the behavior of starved cells. The mean is shown; error bars represent SEM (n > 3, ∗p < 0.05).
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7. Figure 5 Glutaminolysis Increases GTP Loading of RagB
(A and B) U2OS cells were cotransfected with a GST-RagB-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. After GST-RagB pull-down, radiolabeled GTP and GDP were analyzed by TLC (A) and quantified using ImageJ software (B).
(C) HeLa cells were cotransfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP-expressing plasmid plus either nontargeting siRNA (siCtrl) or siRNAs targeting GLS and GDH. Forty-eight hours after transfection, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ), as indicated. Phosphorylation of S6K was measured by immunoblotting.
(D) HeLa cells were transfected with either empty vector or GST-RagB-GTP and GST-RagC-GDP. Forty-eight hours later, cells were amino acid starved for 1 hr and restimulated for 15 min with leucine and glutamine (LQ) either in the presence or absence of DON, as indicated. Phosphorylation of S6K was then measured by immunoblotting. The mean is shown; error bars represent SEM (n > 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6 Glutaminolysis Inhibition Induces Autophagy
(A) Cell size of HeLa cells incubated for 48 hr either in the presence or absence of leucine, glutamine, and DON.
(B and C) Aggregation of GFP-LC3 upon autophagosome formation in response to leucine and glutamine in cells treated with or without rapamycin. Quantification of three independent experiments (C).
(D and E) Aggregation of GFP-LC3 upon autophagosome formation in response to leucine and glutamine in cells transfected with either nontargeting siRNA (siCtrl) or with siRNAs targeting GDH and GLS. Quantification of three independent experiments (E).
(F) U2OS cells were starved for all amino acids or all amino acids except leucine and glutamine (LQ) for 5 hr in the presence or absence of DON, as indicated. Endogenous LC3-II levels were analyzed by immunoblotting.
(G) Proposed model for the regulation of mTORC1 by glutaminolysis. The mean is shown; error bars represent SEM (n = 3, ∗p < 0.05).
Molecular Cell  , DOI: ( /j.molcel )
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