Supplementary Fig. 6: GPR137B as an activator of lysosomal RagA/C.
From: The lysosomal GPCR-like protein GPR137B regulates Rag and mTORC1 localization and activity
a, GPR137B co-expression with Rap2A does not enable Rap2A expression-dependent recruitment of mTORC1 under amino acid starvation (2 h). Heatmaps show that co-expression of GPR137B-mCherry with Rap2A does not induce Rap2A expression-dependent recruitment of mTORC1 translocation (left), but co-expression of GPR137B with RagA/C causes mTORC1 translocation at low levels of RagA/C expression (right). HeLa cells were amino acid starved for 2 h in dialysed serum, and cells co-expressing GPR137B-mCherry and CFP-Rap2A (n = 958 cells) or GPR137B-mCherry and RagA/CFP-RagC (n = 1,466 cells) were binned into 8 equal bins of CFP intensities and mCherry intensities. The average of the mTOR translocation score for each bin is plotted as a blue-yellow heatmap scaled from 50 to 450. Grey indicates bins for which there was an insufficient number of cells. Heatmaps are plotted from translocation scores pooled from two independent experiments. b, Repeat of experiment in Fig. 5e demonstrating synergistic activation of mTORC1 by GPR137B and wild-type RagA/C. See the Methods section. c, Calibration of wild-type Turquoise-RagC (paired with HA-tagged wild-type RagA) and Q120L CFP-RagC (paired with HA-tagged DN RagA) fluorescence intensity using an endogenous RagA antibody (see Methods). d, Histograms of all single-cell measurements for the expression levels of Lamp1 and GPR137B; the levels of mTOR translocation show dynamic ranges for these three components in Fig. 5e. The red bracket in each histogram shows that most of the values for each parameter is included in the heatmap and therefore represents the population accurately. e,f, Lysosomal Venus-RagC intensity recovers similarly when co-expressed with GPR137B or with Lamp1 in NPRL3−/− HEK293E cells in the absence of amino acids after photobleaching (e). Lamp1, n = 9 cells and GPR137B, n = 9 cells. (f) A bar graph representation of data in e is also shown (f). n = 3 independent experiments with data representing mean ± s.d. (e,f); a two-tailed Student’s t-test was used in e.
