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J Lipid Res. 2011 Mar;52(3):582-9. doi: 10.1194/jlr.D012807. Epub 2010 Dec 29.

Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.

Author information

1
Centre for Molecular Cell Biology, Department of Inflammation, Division of Medicine, University College London, Royal Free Campus, Rowland Hill Street, London, United Kingdom NW3 2PF. m.waugh@medsch.ucl.ac.uk

Abstract

Cholesterol is an abundant lipid of the trans-Golgi network (TGN) and of certain endosomal membranes where cholesterol-rich microdomains are important in the organization and compartmentalization of vesicular trafficking. Here we describe the development of a rapid method to isolate a cholesterol-rich endomembrane fraction. We show that widely used subcellular fractionation techniques incompletely separate cholesterol-rich membranes, such as the TGN, from organelles, such as late endosomes and lysosomes. To address this issue, we devised a new subcellular fractionation scheme involving two rounds of velocity centrifugation, membrane sonication, and discontinuous sucrose density gradient centrifugation. This strategy resulted in the isolation of a cholesterol and GM1 glycosphingolipid-enriched membrane fraction that was completely cleared of plasma membrane, endoplasmic reticulum, and mitochondria. This buoyant fraction was enriched for the TGN and recycling endosome proteins Rab11 and syntaxin-6, and it was well resolved from cis-Golgi and early and late endosomal membranes. We demonstrate that this technique can give useful insights into the compartmentation of phosphoinositide synthesis, and it facilitates the isolation of cholesterol-rich membranes from a population of TGN-trafficking vesicles.

PMID:
21191144
PMCID:
PMC3035695
DOI:
10.1194/jlr.D012807
[Indexed for MEDLINE]
Free PMC Article

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