Fig. 2: Lipid densities sandwiched between permease and GPCR domains.

a A layer of lipid densities is observed at the interface between permease and GPCR domains. The lipid densities, contoured at 5σ, are colored light pink. b Structural modeling of two phospholipids and one cholesterol into the lipid densities. The map is contoured at 5σ. c Two perpendicular views to show the positions of lipid molecules at the interface. d A cytosolic view to show the coordination of the head groups of Lipid1 and Lipid2 at the interface. e The details of cholesterol coordination. f Biochemical verification of critical residues in cholesterol coordination. HEK-293F cells were transiently transfected with strep-tagged LYCHOS and Flag-tagged GATOR components, including DEPDC5 (Flag-DEPDC5), NPRL2 (Flag-NPRL2), and NPRL3 (Flag-NPRL3), followed by strep immunoprecipitation and immunoblotting for the indicated proteins. NPRL2 and NPRL3 were immunoblotted to represent GATOR1 complex in the assay. Multiple bands were observed for LYCHOS because of the glycosylation. The experiments were conducted independently at least three times with similar results. The lipid molecules in (d) and (e) are shown as sticks and balls and colored light pink. Two phospholipids are indicated with Lipid1 and Lipid2, and cholesterol is indicated as CHO. The potential charge interactions are indicated by dotted red lines.