STARD3 mediates endoplasmic reticulum-to-endosome cholesterol transport at membrane contact sites

StAR-related lipid transfer domain-3 (STARD3) is a sterol-binding protein that creates endoplasmic reticulum (ER)-endosome contact sites. How this protein, at the crossroad between sterol uptake and synthesis pathways, impacts the intracellular distribution of this lipid was ill-defined. Here, by using in situ Cholesterol labeling and quantification, we demonstrated that STARD3 induces Cholesterol accumulation in endosomes at the expense of the plasma membrane. STARD3-mediated Cholesterol routing depends both on its lipid transfer activity and its ability to create ER-endosome contacts. Corroborating this, in vitro reconstitution assays indicated that STARD3 and its ER-anchored partner, Vesicle-associated membrane protein-associated protein (VAP), assemble into a machine that allows a highly efficient transport of Cholesterol within membrane contacts. Thus, STARD3 is a Cholesterol transporter scaffolding ER-endosome contacts and modulating cellular Cholesterol repartition by delivering Cholesterol to endosomes.