A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Nat Commun. 2020 Nov 3;11(1):5559. doi: 10.1038/s41467-020-19032-0.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biological Transport
  • CRISPR-Cas Systems / genetics
  • Cholesterol / metabolism*
  • 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
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Lysosomes / metabolism*
  • Lysosomes / ultrastructure
  • Models, Biological
  • Multiprotein Complexes / metabolism
  • Protein Binding
  • Protein Multimerization*
  • rab GTP-Binding Proteins / metabolism*

Substances

  • Cholesterol, LDL
  • Fluorescent Dyes
  • Guanine Nucleotide Exchange Factors
  • Intracellular Signaling Peptides and Proteins
  • Multiprotein Complexes
  • NPC1 protein, human
  • rab7 protein
  • Cholesterol
  • HMGCS1 protein, human
  • Hydroxymethylglutaryl-CoA Synthase
  • rab GTP-Binding Proteins