Figure 3.

GATOR regulates mTORC1 localization to the lysosomal surface and functions upstream of the Rag GTPases. (A) RNAi-mediated depletion of the GATOR2 components Mios and Seh1L prevents amino acid-induced mTOR lysosomal translocation. HEK-293T cells expressing the indicated shRNAs were starved or starved and restimulated with amino acids for the specified times prior to co-immunostaining for mTOR (red) and Lamp2 (green). (B) Reduced expression of DEPDC5 in HEK-293T cells results in constitutive mTOR localization to the lysosomal surface. HEK-293T cells treated with the indicated lentiviral shRNAs were processed as described in (A). (C) Images of HEK-293T cells stably expressing FLAG-DEPDC5 starved for or starved and restimulated with amino acids. Cells were processed as described in (A). In all images, insets show selected fields that were magnified five times and their overlays. Scale bar equals 10 μM. (D) GATOR1 functions upstream of the nucleotide binding state of the Rags. HEK-293T cells transfected with the indicated cDNAs in expression vectors were starved of amino acids for 50 min or starved and restimulated with amino acids for 10 min. The indicated proteins were detected by immunoblotting.