Interaction of Rag heterodimers with recombinant and endogenous mTORC1 in a manner that depends on the nucleotide binding state of RagB. In (A) through (D) HEK-293T cells were transfected with the indicated cDNAs in expression vectors, cell lysates prepared, and lysates and HA- or FLAG-immunoprecipitates were analyzed by immunoblotting for the amounts of the specified recombinant or endogenous proteins. (E) In vitro binding of purified FLAG-raptor with wild type RagB-D or RagB^GTP-D^GDP.

Effects of overexpressed RagB^GTP-containing heterodimers on the mTORC1 pathway and its response to leucine, amino acids, or insulin. Effects of expressing the indicated proteins on the phosphorylation state of co-expressed S6K1 in response to deprivation and stimulation with (A) leucine, (B) total amino acids, or (C) insulin. Cell lysates were prepared from HEK-293T cells deprived for 50 minutes of serum, and (A) leucine or (B) amino acids, and then, where indicated, stimulated with leucine or amino acids for 10 minutes. HEK-293E cells (C) were deprived of serum for 50 minutes and where indicated stimulated with 150 nM insulin for 10 minutes. Lysates and FLAG-immunoprecipitates were analyzed for the levels of the specified proteins and the phosphorylation state of S6K1. (D) Effects of amino acid deprivation on insulin-mediated activation of mTORC1. HEK-293E cells were starved for serum and amino acids or just serum for 50 minutes, and where specified, stimulated with 10 or 150 nM insulin. Cell lysates were analyzed for the level and phosphorylation state of S6K1.

Insensitivity of the mTORC1 pathway to amino acid deprivation in cells stably expressing RagB^GTP. (A) Cell size distributions (graphs) and S6K1 phosphorylation (immunoblot) of cells stably expressing RagB, Rheb1, Rag^GTP, or Rap2A. Mean cell diameters ± S.D. (μm) are: Rap2A, 16.05 ± 0.07; Rheb1, 16.79 ± 0.06; RagB, 16.40 ± 0.08; and RagB^GTP, 16.68 ± 0.06 (n = 4 and p < 0.0008 for all comparisons to Rap2A-expressing cells). HEK-293T cells transduced with lentiviruses encoding the specified proteins were deprived for 50 minutes for serum and (B) leucine or (C) total amino acids, and, where indicated, re-stimulated with leucine or amino acids for 10 minutes. Cell lysates were analyzed for the levels of the specified proteins and the phosphorylation state of S6K1. (D) Amino acid-stimulated interaction of the Rag proteins with mTORC1. HEK-293T cells stably expressing FLAG-tagged RagB, RagD, or RagB^GTP were starved for amino acids and serum for 50 minutes and, where indicated, re-stimulated with amino acids for 10 minutes. Cells were then processed with a chemical cross-linking assay and cell lysates and FLAG-immunoprecipitates analyzed for the levels of the indicated proteins. (E) Effects of amino acid stimulation on GTP loading of RagB. Values are mean ± s.d. for n = 3 (p < 0.02 for increase in GTP loading caused by amino acid stimulation). (F) Abundance of RagA, RagB, RagC, and RagD in HeLa cells expressing the indicated shRNAs. (G) S6K1 phosphorylation in HeLa cells expressing shRNAs targeting RagC and RagD. Cells were deprived of serum and leucine for 50 minutes, and, where indicated, re-stimulated with leucine for 10 minutes. (H) Effects of dsRNA-mediated knockdowns of Drosophila orthologues of RagB or RagC on amino acid-induced phosphorylation of dS6K.

Rag-dependent regulation by amino acids of the intracellular localization of mTOR. (A) HEK-293T cells were starved for serum and amino acids for 50 minutes or starved and then re-stimulated with amino acids for the indicated times in the presence or absence of rapamycin. Cells were then processed in an immunofluorescence assay to detect mTOR (green), co-stained with DAPI for DNA content (blue), and imaged. 80–90% of the cells exhibited the mTOR localization pattern shown. (B) and (C) mTOR localization in HEK-293T cells expressing the indicated shRNAs and deprived and re-stimulated with amino acids as in (A). Immunoblot of raptor expression levels. (D) mTOR localization in HEK-293T cells stably expressing RagB, Rheb1, RagB^GTP, or Rheb1^GTP and deprived and re-stimulated with amino acids as in (A).

Amino acids promote the localization of mTOR to a Rab7-positive compartment that also contains Rheb. (A) mTOR and Rab7 localization in cells deprived or stimulated with amino acids. HEK-293T cells transiently transfected with a cDNA for dsRed-Rab7 were starved for serum and amino acids for 50 minutes and, where indicated, stimulated with amino acids for 10 minutes. Cells were then processed to detect mTOR (green), Rab7 (red), and DNA content (blue), and imaged. Two examples are shown of mTOR localization in the presence of amino acids. (B) HEK-293T cells stably expressing RagB^GTP and transiently transfected with a cDNA for dsRed-Rab7 were treated and processed as in (A). (C) Rheb1 and Rab7 localization in cells deprived or stimulated with amino acids. HEK-293T cells transiently transfected with 1–2 ng of cDNAs for GFP-Rheb1 and dsRed-Rab7 were treated as in (A), processed to detect Rheb1 (green), Rab7 (red), and DNA content (blue), and imaged. (D) Model for role of Rag GTPases in signaling amino acid availability to mTORC1.