A trimeric Rab7 GEF controls NPC1-dependent lysosomal cholesterol export

Nature Communications, vol. 11, pp. 5559

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.

Authors 8

1. Dick J. H. van den Boomen corresponding

   University of Cambridge

   Affiliation as printed

   Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK. djhv2@cam.ac.uk

   Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK

2. Agata Sienkiewicz

   University of Cambridge

   Affiliation as printed

   Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK

3. Ilana Berlin Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden University, Leiden, The Netherlands

4. Marlieke L.M. Jongsma Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden University, Leiden, The Netherlands

5. Daphne M. van Elsland Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden University, Leiden, The Netherlands

6. J. Paul Luzio

   University of Cambridge

   Affiliation as printed

   Cambridge Institute for Medical Research, University of Cambridge, Cambridge, UK

7. Jacques Neefjes Aachen

   Leiden University · Leiden University Medical Center

   Affiliation as printed

   Leiden University Medical Centre, Leiden University, Leiden, The Netherlands

8. Paul J. Lehner corresponding

   University of Cambridge

   Affiliation as printed

   Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK. pjl30@cam.ac.uk

   Cambridge Institute of Therapeutic Immunology & Infectious Disease, University of Cambridge, Cambridge, UK