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Segregation of sphingolipids and sterols during formation of secretory vesicles at the trans-Golgi network

¹ Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany
² Center for Brain Science, Harvard University, Cambridge, MA 02138

Correspondence to Kai Simons: simons{at}mpi-cbg.de

The trans-Golgi network (TGN) is the major sorting station in the secretory pathway of all eukaryotic cells. How the TGN sorts proteins and lipids to generate the enrichment of sphingolipids and sterols at the plasma membrane is poorly understood. To address this fundamental question in membrane trafficking, we devised an immunoisolation procedure for specific recovery of post-Golgi secretory vesicles transporting a transmembrane raft protein from the TGN to the cell surface in the yeast Saccharomyces cerevisiae. Using a novel quantitative shotgun lipidomics approach, we could demonstrate that TGN sorting selectively enriched ergosterol and sphingolipid species in the immunoisolated secretory vesicles. This finding, for the first time, indicates that the TGN exhibits the capacity to sort membrane lipids. Furthermore, the observation that the immunoisolated vesicles exhibited a higher membrane order than the late Golgi membrane, as measured by C-Laurdan spectrophotometry, strongly suggests that lipid rafts play a role in the TGN-sorting machinery.

Abbreviations used in this paper: COPI, coat protein I; GP, general polarization; GPI, glycosyl-PI; HDSV, heavy density secretory vesicle; InvRFP, invertase-RFP; IPC, inositolphosphoceramide; LDSV, light density secretory vesicle; M(IP)₂C, mannosyl-di-IPC; MIPC, mannosyl-IPC; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PM, plasma membrane; PS, phosphatidylserine; TEV, tobacco etch virus; TGN/E, TGN/endosome system.

© 2009 Klemm et al.
This article is distributed under the terms of an Attribution–Noncommercial–Share Alike–No Mirror Sites license for the first six months after the publication date (see http://www.jcb.org/misc/terms.shtml). After six months it is available under a Creative Commons License (Attribution–Noncommercial–Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/).

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This article has been cited by other articles:

• Kaiser, H.-J., Lingwood, D., Levental, I., Sampaio, J. L., Kalvodova, L., Rajendran, L., Simons, K. (2009). Order of lipid phases in model and plasma membranes. Proc. Natl. Acad. Sci. USA 106: 16645-16650 [Abstract] [Full Text]  
• Short, B. (2009). Lipid rafts inflate at the trans-Golgi. JCB 185: 567-567 [Full Text]  

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