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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 5. Mutations in Switch I of the Rag GTPases impair intersubunit communication.

Figure 5

(A) Crystal structure of Gtr2p (RagC homolog in yeast) bound to GppNHp (PDB: 3R7W). Switch I region is shown in orange. Upon GppNHp binding, Switch I forms a lid on top the nucleotide binding pocket. Leu91Hs and Lys84Hs are shown in spheres.

(B) GTP binding to RagA-RagC(L91P). Top panel: nucleotide bound to RagA and RagC(L91P). Bottom panel: quantification. Data were fit to a single-site binding equation to obtain the Kd. In comparison, wildtype binding is shown as dashed curves. RagA-RagC(L91P) binds GTP in a similar fashion as the wildtype Rags.

(C) Reaction time-course of radioactively labeled GTP remaining bound to RagA-RagC(L91P) during the off-rate measurement. Top panel: GTP bound to RagA and RagC(L91P) after using unlabeled GTP as a chase. Bottom panel: quantification. Data were fit to a single exponential decay to obtain the koff. The wildtype off-rate is shown as dashed curves for reference. GTP dissociates from RagA-RagC(L91P) faster than from the wildtype Rags.

(D) Summary of koff and Kd values for RagA-RagC(L91P), RagA-RagC(K84T), and RagA(R37P)-RagC. Wildtype data are reproduced from Figure 1Q for ease of comparison.

(E–F) Half-site on-rates for RagA-RagC(L91P) (squares) and RagA-RagC(K84T) (diamonds) (E), and RagA(R37P)-RagC (circles) (F). The subunits in bold are those with which the labeled GTP is associating in the assay to measure the half-site on-rate. The slope gives the half-site on-rate. Wildtype half-site on-rates are shown as dashed lines for comparisons. Notice that the concentration dependence for GTP association is partially restored for all the mutants. * indicates p < 0.05; ** indicates p < 0.01; ns: not significant from zero.

(G) Summary of half-site on-rates of GTP to RagA-RagC(L91P), RagA-RagC(K84T), and RagA(R37P)-RagC. On-rates for wildtype Rags are reproduced from Figure 2H for ease of comparison.

(H–I) Single (H) and multiple (I) turnover GTP hydrolysis assays for RagA-RagC(L91P) (blue circles) and RagA-RagC(K84T) (brown circles), and RagA(R37P)-RagC (red circles). Wildtype curves are shown as dashed lines.

(J) Summary of hydrolysis kinetics for RagA-RagC(L91P), RagA-RagC(K84T), and RagA(R37P)-RagC. Wildtype data are reproduced from Figure 3K for ease of comparison.

(K) Illustration of the effects of the Switch I mutations on intersubunit crosstalk within the Rag heterodimer.