Extended Data Fig. 8: GATOR2 complex does not exhibit E3 ubiquitin ligase activity.

(a), (b) In vitro ubiquitination assay does not demonstrate GATOR2-dependent ubiquitin chain formation or modification of GATOR1. GATOR1 (500 nM) was incubated with a panel of E2 enzymes in the absence (a) or presence of GATOR2 (250 nM; b). Reactions were incubated at 37 °C for 1 h and analyzed by immunoblotting for the indicated proteins. (c) Coomassie-stained SDS-PAGE analysis of GATOR1 and GATOR2 complexes assayed in (a) and (b). (d) Ubiquitin chains formed in the presence of GATOR2 and UBE2D3 are not affected by GATOR2 inhibitors Sestrin2 or CASTOR1. UBE4B was used as a positive control for E3-dependent ubiquitin chain forming activity. Reactions were analyzed as in (a). (e) GATOR2 does not promote ubiquitin discharge from E2-Ub conjugates. Ubiquitin charged-UBE2D3 (UBE2D3-Ub) was incubated in the presence of 10 mM lysine and UBE4B, HOIP catalytic domain, or GATOR2 (100 nM each) at 22 °C for the indicated amounts of time. Samples were harvested in SDS-PAGE buffer without reducing agent, except where otherwise indicated, to preserve the E2-Ub thioester linkage. (f) Pharmacological inhibition of the ubiquitination cascade does not affect activation of mTORC1 by amino acids. HEK293T cells were treated with 0.5 µM TAK-243 for a total of 4 h. Cells were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest. Samples were harvested in SDS-PAGE sample buffer without reducing agent to preserve the E2-Ub thioester linkage and analyzed by immunoblotting. (g) NPRL2 lysine residues are not required for activation and regulation of mTORC1 by amino acids. NPRL2-deficient cells stably expressing the indicated constructs were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest and were analyzed by immunoblotting for the indicated proteins. HA, hemagglutinin. (h) NPRL3 lysine residues are not required for activation and regulation of mTORC1 by amino acids. NPRL3-deficient cells stably expressing the indicated constructs were starved of amino acids for 60 min and restimulated with amino acids for 15 min prior to harvest and were analyzed as in (g). Data in (a), (b), (d)-(h) are representative of two independent experiments. For gel source data, see Supplementary Figure 1.