(F) A model of the molecular basis of accessible PM cholesterol recognition by the GRAM domain of GRAMD1b. At steady state, limited amount of accessible cholesterol (i.e. chemically active cholesterol) in the inner leaflet of the PM is not sufficient for the recruitment of the GRAM domain to the PM. Upon additional cholesterol loading to the PM or liberation of cholesterol from sphingomyelin (SM)-sequestered pool of inaccessible PM cholesterol (i.e. chemically inactive cholesterol), accessible PM cholesterol transiently increases in the inner leaflet of the PM beyond a certain threshold (as unsequestered cholesterol can spontaneously flip flop between the outer and inner leaflets), resulting in increased co-distribution of accessible cholesterol and anionic lipids, including phosphatidylserine (PS), in this leaflet, which serves as a platform for PM recruitment of the GRAM domain. The GRAM domain then binds to both accessible cholesterol and anionic lipids, using distinct but synergistic binding sites for cholesterol and anionic lipids, to tether the PM to the ER, and facilitates the StART-like domain-dependent extraction and transport of accessible PM cholesterol to the ER. Accessible cholesterol transport to the ER results in suppression of SREBP-2 cleavage, preventing overaccumulation of cholesterol in cells. Our study identified amino acid residues that are important for cholesterol sensing (G187, R189) and anionic lipid sensing (K161, R191) of the GRAM domain. G187 is critical for determining the sensitivity of the GRAM domain to accessible PM cholesterol, while K161 and R191 contribute to a basic patch of the GRAM domain that is required for sensing anionic lipids, including PS. An intellectual disability-associated R189W mutation, which specifically results in cholesterol-sensing defect, impairs the GRAM domain's ability to sense transient expansion of the accessible pool of PM cholesterol and abolishes GRAMD1b-mediated PM to ER cholesterol transport. 