期刊
JOURNAL OF LIPID RESEARCH
卷 52, 期 3, 页码 582-589出版社
ELSEVIER
DOI: 10.1194/jlr.D012807
关键词
caveolae; cholesterol/cell tissue; fluorescence microscopy; Golgi apparatus
资金
- Cancer Research UK [C25540/A8562]
- Biotechnology and Biological Sciences Research Council (BBSRC) [BB/G021163/1]
- BBSRC [BB/G021163/1] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/G021163/1] Funding Source: researchfish
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.jlr 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.-Waugh, M. G., K. M. E. Chu, E. L. Clayton, S. Minogue, and J. J. Hsuan. Detergent-free isolation and characterization of cholesterol-rich membrane domains from trans-Golgi network vesicles. J. Lipid Res. 2011. 52: 582-589.
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