TY - JOUR T1 - A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export JF - bioRxiv DO - 10.1101/835686 SP - 835686 AU - Dick J.H. van den Boomen AU - Agata Sienkiewicz AU - Ilana Berlin AU - Marlieke L.M. Jongsma AU - Daphne M. van Elsland AU - J. Paul Luzio AU - Jacques J.C. Neefjes AU - Paul J. Lehner Y1 - 2019/01/01 UR - http://biorxiv.org/content/early/2019/11/08/835686.abstract N2 - Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, using an endogenous cholesterol reporter in a genome-wide CRISPR screen we identify >70 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core component of the mammalian Mon1-Ccz1 guanidine exchange factor (GEF) for Rab7, required for complex stability and function. C18orf8-deficient cells lack Rab7 activation and show severe defects in late endosome morphology and endosomal LDL trafficking, resulting in cellular cholesterol deficiency. Unexpectedly, free cholesterol accumulates within swollen lysosomes, suggesting a critical additional defect in lysosomal cholesterol export. We find that active Rab7 interacts with the NPC1 cholesterol transporter and licenses lysosomal cholesterol export. This process is abolished in C18orf8-, Ccz1- and Mon1A/B-deficient cells and restored by a constitutively active Rab7. The trimeric Mon1-Ccz1-C18orf8 (MCC) GEF therefore plays a central role in cellular cholesterol homeostasis coordinating Rab7 activation, endosomal LDL trafficking and NPC1-dependent lysosomal cholesterol export. ER -