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© The Rockefeller University Press, 0021-9525/2000//507 $5.00
The Journal of Cell Biology, Volume 151, Number 3, , 2000 507-518

Evidence for Segregation of Sphingomyelin and Cholesterol during Formation of Copi-Coated Vesicles

^a Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universität Heidelberg, 69120 Heidelberg, Germany
^b Institut für Anatomie, Ruprecht-Karls-Universität Heidelberg, 69120 Heidelberg, Germany
^c Deutsches Krebsforschungszentrum, 69120 Heidelberg, Germany
Biochemie-Zentrum Heidelberg (BZH), Ruprecht-Karls-Universität Heidelberg, Im Neuenheimer Feld 328, 69120 Heidelberg, Germany.49-6221-54-436649-6221-54-4160

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.

Key Words: COPI-coated vesicles • sphingomyelin • cholesterol • lipid sorting • nano-electrospray ionization tandem mass spectrometry

© 2000 The Rockefeller University Press

Abbreviations used in this paper: ARF, ADP ribosylation factor; CGN, cis-Golgi network; COP, coat protein; IC, intermediate compartment; m/z, mass/charge; nano-ESI-MS/MS, nano-electrospray ionization tandem mass spectrometry; PC, phosphatidylcholine; PREC, precursor ion scanning; SM, sphingomyelin.

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