1. Volume 49, Issue 1, Pages 172-185 (January 2013)
Metabolic Stress Controls mTORC1 Lysosomal Localization and Dimerization by Regulating the TTT-RUVBL1/2 Complex 
Sang Gyun Kim, Gregory R. Hoffman, George Poulogiannis, Gwen R. Buel, Young Jin Jang, Ki Won Lee, Bo-Yeon Kim, Raymond L. Erikson, Lewis C. Cantley, Andrew Y. Choo, John Blenis 
Molecular Cell 
Volume 49, Issue 1, Pages (January 2013)
DOI: /j.molcel
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2. Molecular Cell 2013 49, 172-185DOI: (10.1016/j.molcel.2012.10.003)
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3. Figure 1
Glucose and Glutamine Are Essential, but Redundant, Regulators of mTORC1 Activation through a TSC- and AMPK-Independent Mechanism
(A) Tsc2−/− MEFs were deprived of glucose, glutamine, or glucose and glutamine for 12 hr. mTORC1 and AMPKα activities were then monitored by immunoblotting.
(B) Tsc2−/− and Tsc1−/− MEFs were deprived of the indicated nutrients for 12 hr.
(C) The kinetics of the inhibitory effect of glucose/glutamine deprivation on mTORC1.
(D) Tsc2−/− MEFs were treated with AMPK inhibitor for 1 hr prior to cell lysis at the indicated concentrations following 12 hr of glucose/glutamine deprivation.
(E) Tsc2−/− MEFs, which have the endogenous Raptor knocked down and have either the WT or S722A/S792A Raptor expressed, were deprived of the indicated nutrients and analyzed as in (A).
(F) Ampkα1,2+/+ and Ampkα1,2−/− MEFs deprived of the indicated nutrients for 10 hr were analyzed as in (A).
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 2
Deprivation of Glucose and Glutamine Inhibits mTORC1 Independently of the Rag Proteins
(A) Tsc2−/− MEFs were nondeprived (Con), deprived of aa for 2 hr, or deprived of glucose and glutamine for 12 hr. Cells were coimmunostained for mTOR (green) and the lysosomal protein LAMP1 (red) and then processed for images. See supporting data in Figure S2.
(B) Ampkα−/− MEFs stably expressing empty, RagB WT, or Q99L were starved of the indicated nutrients or treated with rapamycin (20 nM). Phosphorylation of S6K1 was measured 10 hr postdeprivation.
(C) Tsc2−/− MEFs stably expressing RagB WT or Q99L were deprived of the indicated nutrients and processed as in (A).
Molecular Cell  , DOI: ( /j.molcel )
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5. Figure 3
Mitochondrial Inhibitors Suppress mTORC1 Activity Independently of AMPK and Rags
(A) Ampk+/+ and Ampk−/− MEFs were treated with phenformin (1 or 10 mM), rotenone (1 or 10 μg/ml), or FCCP (4 or 20 μM) for 1 hr. mTORC1 and AMPKα activity were measured.
(B) Ampk+/+ and Ampk−/− MEFs were treated as in (A) and ATP levels were measured. The data are represented as mean ± SD.
(C and D) Ampk+/+ and Ampk−/− MEFs were treated with phenformin at the indicated concentrations for 1 hr (C) or treated with 5 mM phenformin for the indicated times (D).
(E) ATP levels were measured in Ampk+/+ and Ampk−/− MEFs after treatments as in (D). Each value represents the normalized mean ± SD for n = 3.
(F) Ampk−/− MEFs expressing either RagB WT or Q99L were deprived of aa (2 hr) or glucose/glutamine (6 hr), or treated with phenformin (5 mM), rotenone (10 μg/ml), or FCCP (20 μM) for 1 hr.
(G) Ampk−/− MEFs expressing either RagB WT or Q99L were treated as in (F) and coimmunostained for mTOR (green) and LAMP1 (red).
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6. Figure 4
Energetic Stress Regulates the Assembly of the TTT-RUVBL1/2 Complex and its Interaction with mTOR
(A) List of the subunits of the TTT-RUVBL1/2 complex scored in the siRNA screen. Fold increase was calculated based on changes in phospho-rpS6 signals when the gene was knocked down.
(B–D) Tsc2+/+ and Tsc2−/− MEFs were transfected with the indicated siRNAs, and mTORC1 activity was measured 60 hr posttransfection.
(E) Tsc2−/− MEFs were starved with glucose/glutamine (12 hr) or aa (2 hr), or treated with 30 μM FCCP (1 hr). Cells were subjected to immunoprecipitation with anti-mTOR antibody followed by immunoblotting with the indicated antibody.
(F) Tsc2−/− MEFs expressing either human RUVBL1 WT or D302N were transfected with nontargeting control or RUVBL1 siRNA for 48 hr. Immunoprecipitation was analyzed as in (E).
(G) Tsc2−/− MEFs expressing HA-Tel2 along with either human RUVBL1 WT or D302N were transfected with siRNAs as in (F). Anti-HA immunoprecipitates from the cell lysates were analyzed by immunoblotting with the indicated antibodies.
(H) Cell lysates were prepared from Tsc2−/− MEFs expressing HA-Tel2 deprived of (12 hr) or restimulated with (1 hr) 20 mM glucose and 4 mM glutamine. Anti-HA immunoprecipitates were analyzed as in (G). See also Figure S4.
(I) Tsc2−/− MEFs expressing either human RUVBL1 WT or D302N were transfected with siRNAs as in (F). mTORC1 activity was addressed 48 hr posttransfection.
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7. Figure 5
Energetic Stress and Loss of RUVBL1/2 Prevent mTORC1 Recruitment to the Lysosomes and mTORC1-Rag Interaction
(A) Tsc2−/− MEFs were transfected with siRNA against RUVBL1 or RUVBL2 for 42 hr or deprived of glucose and glutamine for 12 hr. Cells were then coimmunostained for mTOR and LAMP1 and processed for images.
(B) Quantitation of mTOR localized to the lysosomes was measured based on percentages of the mTOR area overlapped with the LAMP1 area using the Measure Colocalization function under the MetaMorph program. At least three images processed as in (A) were analyzed per sample. The data are represented as mean ± SD and p value is indicated.
(C) Tsc2−/− MEFs expressing either Flag-RagB WT or Q99L were deprived of aa (2 hr) or glucose/glutamine (12 hr) and then stimulated with aa (30 min) or glucose/glutamine (1 hr). After 1 hr treatment with 1 mg/ml DSP prior to lysis, anti-Flag immunoprecipitates were analyzed by immunoblotting with the indicated antibodies.
(D) Tsc2−/− MEFs expressing Flag-RagB WT were transfected with siRNAs against Tti1 or RUVBL1, then starved (12 hr) and stimulated with glucose and glutamine (1 hr). Anti-Flag immunoprecipitates were prepared 40 hr post-siRNA transfection and analyzed as in (C).
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 6
Energetic Stress and Loss of the TTT-RUVBL1/2 Complex Inhibit mTORC1 through Mislocalization and Prevention of mTORC1 Dimerization
(A) Tsc2−/− MEFs expressing Myc-Raptor WT or Myc-Raptor-CAAX were deprived of glucose/glutamine (12 or 24 hr) or of aa/serum (2 hr), and mTORC1 activity was measured.
(B) Glucose/glutamine-deprived Tsc2−/− MEFs (12 or 24 hr) were stimulated with glucose (20 mM)/glutamine (4 mM) for 1 hr, and mTOR levels and activity were measured.
(C) Myc-Raptor- or Myc-Raptor-CAAX-expressing cells were transfected with siRNAs against Tti1 or RUVBL1, and mTORC1 activity was measured 36 hr posttransfection.
(D and E) Tsc2+/+ MEFs following 12 hr glucose/glutamine deprivation (D) or 36 hr siRNA transfection (E) were lysed with the lysis buffer containing the reduced salts. mTOR-Raptor interaction was measured using anti-Raptor immunoprecipitates followed by immunoblotting with the indicated antibodies. See also Figure S6.
(F) Tsc2−/− MEFs expressing HA-Raptor and Myc-Raptor were deprived of (12 hr) and stimulated with (1 hr) glucose/glutamine. Anti-HA immunoprecipitates were prepared from cells treated with DSP for 1 hr prior to lysis and analyzed by immunoblotting with the indicated antibodies.
(G) Tsc2−/− MEFs expressing HA-Raptor and Myc-Raptor were transfected with the indicated siRNAs and treated and processed as in (F). Anti-HA immunoprecipitates were prepared 42 hr post-siRNA transfection.
Molecular Cell  , DOI: ( /j.molcel )
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9. Figure 7
Bioinformatic Analysis of the TTT-RUVBL1/2 Complex Genes in Cancer
(A and B) Box-plots indicate significantly higher expression of the complex’s genes in breast (n = 593) and colorectal (n = 237) carcinomas compared to normal (p < 0.05) based on TCGA database.
(C) Heatmap diagram of unsupervised hierarchical clustering of mRNA levels of the complex’s genes indicates two clearly defined groups of breast tumors with high and low expression of the complex’s genes.
(D) Spearman’s rank correlation coefficient matrix indicates high positive correlation (p < 0.001) in the expression of the complex’s genes in breast carcinomas, in particular between TEL2-TTI1 and RUVBL1-RUVBL2 genes.
(E–G) Spearman’s rank correlation coefficient matrices indicate high positive correlation (p < 0.001) between the expression of the complex’s genes and putative target genes of mTORC1 signaling encoding glycolytic (E), pentose phosphate pathway (F), and lipid/sterol biosynthesis enzymes (G). See also Figure S7.
(H) Model for glucose/glutamine (energy)-induced mTORC1 activation. Through glycolysis and glutaminolysis, glucose and glutamine provide carbons to the TCA cycle and consequently control ATP production. Upon availability of enough energy, the TTT-RUVBL1/2 complex gets stabilized and thus plays major roles in formation of the mTORC1 complex into the mature dimeric form, which may be necessary for Rag-dependent lysosomal localization of mTORC1.
Molecular Cell  , DOI: ( /j.molcel )
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