Extended Data Fig. 12: Functional analysis of the core GATOR2 β-propellers.

(a) The β-propellers of MIOS, WDR59, and WDR24 are required for interactions between GATOR2 and CASTOR1, GATOR1 and KICSTOR, and Sestrin2, respectively. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient, WDR59-deficient or WDR24-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed by immunoblotting for the indicated proteins. CASTOR1 D304A and Sestrin2 E451Q mutants do not bind arginine or leucine, respectively, and bind GATOR2 irrespective of amino acid availability. HA, hemagglutinin. (b) Schematic comparing WDR24 Nβ and SEH1L β-propeller organization in wild-type and β-single chain (βsc) constructs. (c) Schematic comparing WDR59 Nβ and SEC13 β-propeller organization in wild-type and β-single chain (βsc) constructs. The fused construct contains the intervening RWD domain. (d) The GATOR2 single chain β-propeller pairs of MIOS-SEH1L, WDR59-SEC13, and WDR24-SEH1L are sufficient to interact with CASTOR1, GATOR1 and KICSTOR, and Sestrin2, respectively. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (e) GATOR2 mediates the interaction between Sestrin2 and CASTOR1, indicating that they can co-occupy the same GATOR2 particle. Anti-FLAG immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (f) Sestrin2 and CASTOR1, but not SAR1B, interact with GATOR2 and suppress mTORC1 signaling. Anti-FLAG and anti-HA immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (g) GATOR2 interacts with Sestrin2 and CASTOR1, but not SAR1B. Anti-FLAG immunoprecipitates were prepared from HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (h) GATOR2 does not immunoprecipitate the Rag GTPases independent of GATOR1. Anti-FLAG immunoprecipitates were prepared from wild-type of DEPDC5-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (i) MIOS Nβ is not absolutely required for activation of mTORC1 or the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from MIOS-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). (j) WDR59 Nβ is necessary for activation of mTORC1 and the GATOR2-GATOR1 and GATOR2-KICSTOR interactions. Anti-FLAG and anti-HA immunoprecipitates were prepared from WDR59-deficient HEK293T cells that transiently expressed the indicated cDNAs and were analyzed as in (a). Data in (a), (d)-(j) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.