title: Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles.
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.
authors.affiliation: 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
authors.forename: Mark G
authors.initials: MG
authors.lastname: Waugh
authors.forename: K M Emily
authors.initials: KM
authors.lastname: Chu
authors.forename: Emma L
authors.initials: EL
authors.lastname: Clayton
authors.forename: Shane
authors.initials: S
authors.lastname: Minogue
authors.forename: J Justin
authors.initials: JJ
authors.lastname: Hsuan
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D002784:Cholesterol / Q000378:metabolism*; D022162:Cytoplasmic Vesicles / Q000378:metabolism*; D003902:Detergents; D004721:Endoplasmic Reticulum / Q000378:metabolism; D011992:Endosomes / Q000378:metabolism; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D021962:Membrane Microdomains / Q000378:metabolism*; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates / Q000096:biosynthesis / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism; D021601:trans-Golgi Network / Q000378:metabolism*
chemical_list: D003902:Detergents; D015778:Minor Histocompatibility Antigens; D018129:Phosphatidylinositol Phosphates; C037178:phosphatidylinositol 4-phosphate; D002784:Cholesterol; D017853:Phosphotransferases (Alcohol Group Acceptor); C402182:phosphatidylinositol phosphate 4-kinase