Extended Data Fig. 5: Validation of the GATOR2-CASTOR1 interaction.
From: Structural basis for the dynamic regulation of mTORC1 by amino acids
(a) Validation of the CASTOR2-GATOR2 interaction. Anti-FLAG immunoprecipitates (IPs) were prepared from MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (b) The GATOR2-CASTOR1 interaction is required for overexpression of CASTOR1 to inhibit mTORC1 signaling. Anti-FLAG immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (c) The MIOS N-terminal β-propeller is not required for amino acids to activate mTORC1 signaling. Anti-FLAG immunoprecipitates (IPs) were prepared from HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (d) The GATOR2-CASTOR1 interaction is required for arginine deprivation to inhibit mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of arginine for 60 min and restimulated with arginine for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). (e) The GATOR2-CASTOR1 interaction is necessary for arginine deprivation, but not for leucine deprivation, to inhibit mTORC1 signaling. MIOS-deficient HEK293T cells transiently expressing the indicated cDNAs were starved of either leucine or arginine for 60 min and restimulated with leucine or arginine for 15 min before harvest. Anti-HA immunoprecipitates were prepared and analyzed as in (a). Data in (a)-(e) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.
