(A) Model of the Rheb-TSC2 GAP domain complex, built using the human Rap1-Rap1GAP complex (pdb:3BRW) as a template. The TSC2 is in cyan cartoon representation, the Rheb is in magenta cartoon representation with a transparent molecular surface shown. The bound GTP is shown in spacefilling representation and the four key mutated Rheb residues, aspartic acid (Asp) 60, glutamine (Gln) 64, asparagine (Asn) 119, and asparagine (Asn) 153, are shown in stick representation. Note that these residues are not likely to perturb the interaction between Rheb and TSC2 directly. In addition, the location of the C-terminal membrane anchor of Rheb is shown. The figure was generated with PyMOL (Schrodinger, LLC). (B) Rheb R15G mutant is GTP-bound and largely insensitive to TSC2-dependent hydrolysis. Rheb wild-type and Rheb R15G were loaded with radiolabeled GTP prior to incubation with TSC2 for 60 min. GTP and GDP were eluted from Rheb and analyzed by thin-layer chromatography. Immunoblot (IB) samples demonstrate protein loading controls for Rheb, TSC1 and TSC2. (C) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb N153T, or inactive Rheb N119I for 24 hr. Cells (in full nutrient conditions) were fixed and immunostained with antibodies against TSC2 and Flag-tagged Rheb. (D) HeLa cells were transfected with Flag-tagged wild-type Rheb, active Rheb R15G, or inactive Rheb D60K for 24 hr. Cells were starved of amino acids and serum prior to fixation. Cells were immunostained with antibodies against TSC2 and Flag-tagged Rheb. (E) HeLa cells were transfected with active and inactive constructs described in C and D. The percentage of cells with punctate TSC2 was quantified in cells expressing Rheb mutants maintained in full nutrient conditions. (F, G) Amino acids, specifically arginine regulate the interaction between Rheb and mTOR. HeLa cells were transfected with Flag-wild-type Rheb overnight, lysed and loaded onto anti-Flag beads. HeLa cells transfected with myc-mTOR were subject to starvation protocols as indicated prior to lysis. Lysates were incubated with Rheb-loaded beads at 4°C. Samples were analyzed by immunoblotting to detect interaction of mTOR with Rheb. (H) TSC complex can inhibit Rheb-mediated mTORC1 activity independent of GAP activity. Cells were transfected with Flag-wild-type Rheb, Flag-Rheb R15G or Flag-Rheb N153T with Flag-TSC2 and HA-GST-tagged S6K overnight as in Figure 4H. Cells were subjected to arginine starvation in the presence of dFCS, prior to lysis and immunoblotted for phosphorylation of S6K. (I) HeLa cells were transfected overnight with wild-type or GAP-deficient TSC2 and HA-GST-tagged S6K. Cells were lysed and immunoblotted for phosphorylation of S6K. (J) HeLa cells were transfected with GST-Rheb either in the presence of mTOR and TSC2 constructs as indicated. Lysates were subjected to pull-down with glutathione Sepharose beads and interaction between Rheb and mTOR was analyzed by immunoblot. All graphs represent an average of at least two independent experiments and error bars represent standard deviation. Scale bars: 10 μm.