Extended Data Fig. 3: Validation of the GATOR2-Sestrin2 interaction.
From: Structural basis for the dynamic regulation of mTORC1 by amino acids
(a) Validation of the GATOR2-Sestrin2 interface. Anti-FLAG immunoprecipitates (IPs) were prepared from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. (b) The GATOR2-Sestrin2 interaction is required for overexpression of Sestrin2 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, d) Validation of the (c) Sestrin1-GATOR2 and (d) Sestrin3-GATOR2 interactions. Anti-FLAG immunoprecipitates (IPs) were prepared from WDR24-deficient HEK293T cells transiently expressing the indicated cDNAs and were analyzed as in (a). (e) Identification of WDR24 mutants that interfere with the GATOR2-Sestrin2 interaction but not activation of mTORC1 by nutrients. WDR24-deficient HEK293T cells stably expressing the indicated cDNAs were starved of all amino acids for 60 min and restimulated with all amino acids for 15 min before harvest. Cell lysates were analyzed as in (a). (f) The GATOR2-Sestrin2 interaction is required for mTORC1 to sense leucine, but not arginine, deprivation. WDR24-deficient HEK293T cells stably 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. Cell lysates were analyzed as in (a). Data in (a)-(f) are representative of two independent experiments. For gel source data, see Supplementary Fig. 1.
