(A) Schematic of the dehydroergosterol (DHE) transfer assay in vitro. Plasma membrane-like liposomes (LPM) [10% phosphatidylserine (PS: DOPS), 30% cholesterol, 2.5% Dansyl-phosphatidylethanolamine (DNS-PE), 10% dehydroergosterol (DHE), 47.5% phosphatidylcholine (DOPC)] and endoplasmic reticulum-like liposomes (LER) [15% DGS-NTA (Ni), 20% phosphatidylethanolamine (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine/POPE), 65% DOPC] were incubated with either purified wild-type GRAMD1b proteins (WT) or purified mutant GRAMD1b proteins carrying GRAM domain mutations (1 mM total lipid, 0.2 μM final protein concentrations in 50 μl reaction volume unless otherwise noted). C-terminus of GRAMD1b, including the transmembrane domain, was replaced with a hexahistidine tag (His-tag) to attach purified GRAMD1b proteins to LER via His-tag DGS-NTA(Ni) interaction. A fluorometer was used to simultaneously monitor tethering of the liposomes in (B) and transfer of DHE from LPM to LER in (C). 