Figure 3(A) Chemical structures of the sterols tested in (B). (B) Sterol specificity of the GRAM domain of GRAMD1b (GRAM1b) as assessed via liposome sedimentation assays. Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as indicated. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining (mean ± SEM, n = 6 independent experiments for liposomes containing only DOPC, n = 3 independent experiments for the rest). DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine). (C) Magnified ribbon diagrams of the modelled GRAM1b showing amino acid side chains that contribute to anionic lipid sensitivity (K161, R191) (blue) and an amino acid side chain of R189 (red), whose substitution to tryptophan (W) is associated with intellectual disability in humans. The bottom panel shows the diagram that was rotated 45° around the indicated axis as shown. (D-E) Liposome sedimentation assays of wild-type GRAM1b proteins (WT) and mutant versions of GRAM1b proteins carrying indicated mutations (R189W, R189W/R191A). Liposomes containing the indicated mole% lipids were incubated with purified GRAM1b proteins as shown. Bound proteins [pellet, (P)] were separated from the unbound proteins [supernatant, (S)], run on SDS-PAGE and visualized by colloidal blue staining [mean ± SEM, n = 3 independent experiments for all conditions in the co-incidence detection assay; n = 3 independent experiments for R189W and R189W/R191A, n = 6 independent experiments for WT in phosphatidylserine (PS) sensitivity assay; n = 3 independent experiments for R189W and R189W/R191A, n = 5 independent experiments for WT in cholesterol sensitivity assay]. DOPC, phosphatidylcholine (1,2-dioleoyl-sn-glycero-3-phosphocholine); DOPS, phosphatidylserine (1,2-dioleoyl-sn-glycero-3-phospho-L-serine); Chol., cholesterol. (F) Confocal images of live GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-tagged GRAM domain of GRAMD1b (EGFP-GRAM1b) constructs as indicated. Cells were treated with SMase (100 mU/ml for 1 hour at 37°C) before imaging. Insets show at higher magnification the regions indicated by white dashed boxes. Anionic lipid sensing defective mutants (K161A, R191A, and K161A/R191A), a cholesterol sensing defective mutant (R189W), and a cholesterol/anionic lipid sensing defective mutant (R189W/R191A) are shown. Note the absence of (R189W, R189W/R191A, and K161A/R191A) or significantly reduced (K161A, R191A) PM recruitment of the mutant versions of EGFP-GRAM1b compared to the strong PM recruitment of wild-type EGFP-GRAM1b (WT). Scale bars, 10 µm. (G) Quantification of the ratio of PM EGFP-GRAM1b signals to the cytosolic EGFP-GRAM1b signals, as assessed by confocal microscopy and line scan analysis from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b with or without SMase treatment (100 mU/ml for 1 hour at 37°C) as shown in (F) and Appendix Fig S1B (mean ± SEM, n = 40 cells for WT, and n = 30 cells for other conditions; data are pooled from four independent experiments for WT and three independent experiments for the rest; two-tailed unpaired Student's t-test, **P < 0.0001. n.s. denotes not significant). (H) Left: Time course of normalized EGFP signal, as assessed by TIRF microscopy, from GRAMD1 TKO HeLa cells expressing either wild-type (WT) or mutant versions of EGFP-GRAM1b constructs as indicated. SMase treatment (100 mU/ml) is indicated. Right: Values of ΔF/F0 corresponding to the end of the experiment as indicated by the arrow [mean ± SEM, n = 126 cells (WT), n = 38 cells (K161A), n = 33 cells (R191A), n = 43 cells (K161A/R191A), n = 46 cells (R189W), n = 44 cells (R189W/R191A); data are pooled from six independent experiments for WT and two independent experiments for the rest; Dunnett's multiple comparisons test, **P = 0.0002 (WT vs. K161A) and **P < 0.0001 for the rest].