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

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


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Fractionation / methods*
  • Cell Line
  • Centrifugation, Density Gradient
  • Cholesterol / metabolism*
  • Cytoplasmic Vesicles / metabolism*
  • Detergents
  • Endoplasmic Reticulum / metabolism
  • Endosomes / metabolism
  • Humans
  • Intracellular Membranes / metabolism
  • Membrane Microdomains / metabolism*
  • Minor Histocompatibility Antigens
  • Phosphatidylinositol Phosphates / biosynthesis
  • Phosphatidylinositol Phosphates / metabolism
  • Phosphotransferases (Alcohol Group Acceptor) / metabolism
  • trans-Golgi Network / metabolism*


  • Detergents
  • Minor Histocompatibility Antigens
  • Phosphatidylinositol Phosphates
  • phosphatidylinositol 4-phosphate
  • Cholesterol
  • Phosphotransferases (Alcohol Group Acceptor)
  • phosphatidylinositol phosphate 4-kinase