1. A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export.
- Author
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van den Boomen DJH, Sienkiewicz A, Berlin I, Jongsma MLM, van Elsland DM, Luzio JP, Neefjes JJC, and Lehner PJ
- Subjects
- Biological Transport, CRISPR-Cas Systems genetics, Cholesterol, LDL metabolism, Endosomes metabolism, Endosomes ultrastructure, Fluorescent Dyes metabolism, Genome, Human, Guanine Nucleotide Exchange Factors metabolism, HEK293 Cells, HeLa Cells, Homeostasis, Humans, Hydroxymethylglutaryl-CoA Synthase metabolism, Lysosomes ultrastructure, Models, Biological, Multiprotein Complexes metabolism, Niemann-Pick C1 Protein, Protein Binding, rab7 GTP-Binding Proteins, Cholesterol metabolism, Intracellular Signaling Peptides and Proteins metabolism, Lysosomes metabolism, Protein Multimerization, rab GTP-Binding Proteins metabolism
- Abstract
Cholesterol import in mammalian cells is mediated by the LDL receptor pathway. Here, we perform a genome-wide CRISPR screen using an endogenous cholesterol reporter and identify >100 genes involved in LDL-cholesterol import. We characterise C18orf8 as a core subunit 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 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.
- Published
- 2020
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