Lipid rafts are essential for peroxisome biogenesis in HepG2 cells

Authors

  • Jannes Woudenberg,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
    Current affiliation:
    1. Department of Gastroenterology and Hepatology, Radbound University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands
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  • Krzysztof P. Rembacz,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Mark Hoekstra,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Antonella Pellicoro,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
    Current affiliation:
    1. Edinburgh University Center for Inflammation Research, The Queen's Medical Research Institute, Edinburgh, UK
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  • Fiona A. J. van den Heuvel,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Janette Heegsma,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Sven C. D. van IJzendoorn,

    1. Section of Membrane Cell Biology, Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Andreas Holzinger,

    1. Department of Neonatology, Dr. von Hauner's Children's Hospital, Ludwig-Maximilians University, Munich, Germany
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  • Tsuneo Imanaka,

    1. Department of Biological Chemistry, Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, Toyama, Japan
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  • Han Moshage,

    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
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  • Klaas Nico Faber

    Corresponding author
    1. Department of Gastroenterology and Hepatology and Department of Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
    • Department of Gastroenterology and Hepatology, University Medical Center Groningen, University of Groningen, P.O. Box 30.001, 9700 RB Groningen, The Netherlands
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    • fax: 0031-50-3619306


  • Potential conflict of interest: Nothing to report.

Abstract

Peroxisomes are particularly abundant in the liver and are involved in bile salt synthesis and fatty acid metabolism. Peroxisomal membrane proteins (PMPs) are required for peroxisome biogenesis [e.g., the interacting peroxisomal biogenesis factors Pex13p and Pex14p] and its metabolic function [e.g., the adenosine triphosphate–binding cassette transporters adrenoleukodystrophy protein (ALDP) and PMP70]. Impaired function of PMPs is the underlying cause of Zellweger syndrome and X-linked adrenoleukodystrophy. Here we studied for the first time the putative association of PMPs with cholesterol-enriched lipid rafts and their function in peroxisome biogenesis. Lipid rafts were isolated from Triton X-100–lysed or Lubrol WX–lysed HepG2 cells and analyzed for the presence of various PMPs by western blotting. Lovastatin and methyl-β-cyclodextrin were used to deplete cholesterol and disrupt lipid rafts in HepG2 cells, and this was followed by immunofluorescence microscopy to determine the subcellular location of catalase and PMPs. Cycloheximide was used to inhibit protein synthesis. Green fluorescent protein–tagged fragments of PMP70 and ALDP were analyzed for their lipid raft association. PMP70 and Pex14p were associated with Triton X-100–resistant rafts, ALDP was associated with Lubrol WX–resistant rafts, and Pex13p was not lipid raft–associated in HepG2 cells. The minimal peroxisomal targeting signals in ALDP and PMP70 were not sufficient for lipid raft association. Cholesterol depletion led to dissociation of PMPs from lipid rafts and impaired sorting of newly synthesized catalase and ALDP but not Pex14p and PMP70. Repletion of cholesterol to these cells efficiently reestablished the peroxisomal sorting of catalase but not ALDP. Conclusion: Human PMPs are differentially associated with lipid rafts independently of the protein homology and/or their functional interaction. Cholesterol is required for peroxisomal lipid raft assembly and peroxisome biogenesis. HEPATOLOGY 2010

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