Figure 3. GTP-loading of RagA/B destabilizes Ragulator-Rag GTPase interaction.

(a)Schematic of the fluorescence in vitro binding assay for interaction of soluble, GFP-tagged Rag GTPases with non-fluorescent, bead-bound Ragulator. Excess GFP-Rags in solution allow fluorescence recovery upon photobleaching of a bead section. (b) Confocal images of glutathione beads coated with GST-Metap2 (left) or pentameric GST-Ragulator (right) and incubated with an excess of GFP-RagB + FLAG-RagC. Scale bar 100 μm. Experiment repeated 3 times. (c) In vitro FRAP experiment in which Ragulator-coated beads were incubated with GFP-RagB + FLAG-RagC^D181N (X-mutant) and loaded with the indicated guanine nucleotide combinations. Each curve is the average ± S.E.M. of [GTPγS/XTPγS N=12, GTPγS/XDP N=6, GDP/XTPγS N=6, GDP/XDP N=4] bead regions. (d) Plot of recovery fraction vs. halftime (t_1/2) from the fits in (c). Shown are best fit values with 95% confidence intervals. (e) FRAP time-lapse of single ROIs from Ragulator-coated beads incubated with excess GFP-RagB + FLAG-RagC^D181N (X-mutant) and loaded with the indicated guanine or xanthosine nucleotide combinations. Scale bar 10 μm. Experiment repeated 3 times. (f) FRAP time-lapse of single ROIs from Ragulator-coated beads incubated with excess GFP-RagB + FLAG-RagC^D181N (X-mutant) loaded with the indicated guanine nucleotide combinations and, where indicated, incubated with 1mM GTPγS or XTPγS. Scale bar 10 μm. (g) FRAP curves related to the experiment in (f). Each curve is the average ± S.E.M. of [GTPγS/XDP N=8, GDP/XTPγS N=8, GDP/XTPγS + GTPγS N=11, GTPγS/XDP + XTPγS N=12] bead regions. (h) Model displaying destabilization of Ragulator-Rag GTPase interaction upon GTP-loading of RagA/B. Interaction with Ragulator is minimally affected by the nucleotide state of Rag C/D.See Supplementary Table 1 for statistical source data.