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. Author manuscript; available in PMC: 2018 Nov 2.
Published in final edited form as: Mol Cell. 2017 Oct 19;68(3):552–565.e8. doi: 10.1016/j.molcel.2017.09.026

Figure 4. Regulators of the Rag GTPase heterodimer control its nucleotide loading state.

Figure 4

(A) SDS-PAGE followed by Coomassie blue staining of recombinant GATOR1 and FLCN-FNIP2 complexes indicating their subunits.

(B–C) Single turnover reactions of GATOR1- (B) and FLCN-FNIP- (C) stimulated GTP hydrolysis of wildtype Rags. GATOR1 and FLCN-FNIP dose-dependently increase GTP hydrolysis by wildtype Rags.

(D) GATOR1-stimulated GTP hydrolysis of RagA(Q66L)-RagC mutant in a single turnover condition. The leucine substitution in RagA abolishes the stimulation by GATOR1.

(E) Concentration dependence of GATOR1-stimulated GTP hydrolysis under single turnover conditions for wildtype Rags (black), RagA(Q66L)-RagC (blue), RagA-RagC(Q120L) (orange), and RagA(Q66L)-RagC(Q120L) (red).

(F) FLCN-FNIP2-stimulated GTP hydrolysis of RagA-RagC(Q120L) mutant in a single turnover condition. The leucine substitution in RagC abolishes the stimulation by FLCN-FNIP2.

(G) Concentration dependence of FLCN-FNIP2-stimulated GTP hydrolysis under single turnover conditions for wildtype Rags (black), RagA(Q66L)-RagC (blue), RagA-RagC(Q120L) (orange), and RagA(Q66L)-RagC(Q120L) (red).

(H) Summary of hydrolysis kinetics for GATOR1- or FLCN-FNIP2-stimulated reactions.