Fig. 3: Membrane-interacting sites of mTOR and RAPTOR subunits.
From: Structural basis for mTORC1 activation on the lysosomal membrane
a, Cartoon representation of the mTORC1–RHEB–RAG–Ragulator–4E-BP1 complex on a membrane shown from the side view. The distance between two RAPTOR membrane-interacting sites is shown. Coloured stars indicate the membrane contact sites in the asymmetric unit. The lipidation sites of LAMTOR1 and RHEB are indicated at the end of arbitrary linkers that anchor them to membranes. b, Close-up view of RHEB density (contour level, 0.168) above the membrane (left) and overall RHEB position relative to the membrane (right). The last visible residue is shown, and the potential membrane-tethering loop is indicated with a dashed line. Close-up view of membrane-interacting sites of the RAPTOR (c) and mTOR (d) subunits. For better visualization, the unsharpened cryo-EM map from the overall refinement with C2 symmetry is used (contour level, 0.033). Residues of RAPTOR and mTOR are indicated with dots. e, Geometry of the residues involved in membrane interaction. f, In vitro mTORC1 kinase activity with different liposome sizes; quantifications were calculated with three repeats and are mean ± s.d. g, In vitro kinase activity of mTORC1 mutants; quantifications were calculated with three (M1 and M4) or four (wild type (WT), M2 and M3) repeats and are mean ± s.d. mTORC1 M1, RAPTORF1296E/M1297E; mTORC1 M2, mTOR468–476GS5 + RAPTORF1296E/M1297E; mTORC1 M3, mTORK471D/R472D/K474D + RAPTORF1296E/M1297E; mTORC1 M4, mTORK471D/R472D/K474D. h, Inducible RAPTOR knockout MEFs, treated with 0.5 μM 4-hydroxytamoxifen (4-OHT) for 48 h or left untreated, were transfected with an empty vector control or constructs expressing RAPTORWT, RAPTORF1296E or RAPTORF1296E/M1297E. After transfection (24 h), cells were starved of amino acids (a.a.) for 60 min or starved and restimulated with amino acids for 30 min and analysed by immunoblotting with the indicated antibodies. Quantifications of p-S6K/S6K and p-4E-BP1/GAPDH are shown with mean ± s.e.m. of n = 3 experiments (****P < 0.0001, two-way ANOVA, Dunnett’s multiple-comparison test). All samples derive from the same experiment, and gels and blots were processed in parallel.
