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Niemann Pick C2 protein enables cholesterol transfer from endo-lysosomes to the plasma membrane for efflux by shedding of extracellular vesicles

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  • Alice Dupont Juhl, University of Southern Denmark, Department of Biochemistry and Molecular Biology
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  • Frederik W Lund, University of Southern Denmark, Unknown
  • Maria Louise V. Jensen, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Denmark
  • Maria Szomek, University of Southern Denmark, Department of Biochemistry and Molecular Biology
  • ,
  • Christian Würtz Heegaard
  • Peter Guttmann, Helmholtz Zentrum Berlin, Helmholtz Association, Helmholtz-Zentrum Berlin (HZB), Germany
  • Stephan Werner, Helmholtz Zentrum Berlin, Helmholtz Association, Helmholtz-Zentrum Berlin (HZB), Germany
  • James McNally, Helmholtz Zentrum Berlin, Helmholtz Association, Helmholtz-Zentrum Berlin (HZB)
  • ,
  • Gerd Schneider, Helmholtz Zentrum Berlin, Helmholtz Association, Helmholtz-Zentrum Berlin (HZB)
  • ,
  • Sergey Kapishnikov, Niels Bohr Institute, University of Copenhagen,Universitetsparken 5, 2100 Copenhagen, Denmark.
  • ,
  • Daniel Wüstner, University of Southern Denmark, Department of Biochemistry and Molecular Biology, Denmark
The Niemann-Pick C2 protein (NPC2) is a sterol transfer protein in the lumen of late endosomes and lysosomes (LE/LYSs). Absence of functional NPC2 leads to endo-lysosomal buildup of cholesterol and other lipids. How NPC2’s known capacity to transport cholesterol between model membranes is linked to its function in living cells is not known. Using quantitative live-cell imaging combined with modeling of the efflux kinetics, we show that NPC2-deficient human fibroblasts can export the cholesterol analog dehydroergosterol (DHE) from LE/LYSs. Internalized NPC2 accelerated sterol efflux extensively, accompanied by reallocation of LE/LYSs containing fluorescent NPC2 and DHE to the cell periphery. Using quantitative fluorescence loss in photobleaching of TopFluor-cholesterol (TF-Chol), we estimate a residence time for a rapidly exchanging sterol pool in LE/LYSs localized in close proximity to the plasma membrane (PM), of less than one min and observed non-vesicular sterol exchange between LE/LYSs and the PM. Excess sterol was released from the PM by shedding of cholesterol-rich vesicles. The ultrastructure of such vesicles was analyzed by combined fluorescence and cryo soft X-ray tomography (SXT), revealing that they can contain lysosomal cargo and intraluminal vesicles. Treating cells with apoprotein A1 and with nuclear receptor liver X-receptor (LXR) agonists to upregulate expression of ABC transporters enhanced cholesterol efflux from the PM, at least partly by accelerating vesicle release. We conclude that NPC2 inside LE/LYSs facilitates non-vesicular sterol exchange with the PM for subsequent sterol efflux to acceptor proteins and for shedding of sterol-rich vesicles from the cell surface.
Original languageEnglish
Article number105047
JournalChemistry and Physics of Lipids
Volume235
ISSN0009-3084
DOIs
Publication statusPublished - Mar 2021

    Research areas

  • Cholesterol efflux, Extracellular vesicles, Fluorescence, Image analysis, Niemann-Pick type C disease, Soft X-ray tomography

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