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© The Rockefeller University Press, 0021-9525/2000//143 $5.00
The Journal of Cell Biology, Volume 151, Number 1, , 2000 143-154

Assembly of Myelin by Association of Proteolipid Protein with Cholesterol- and Galactosylceramide-Rich Membrane Domains

^a Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany
^b Institute of Biochemistry, University of Cologne, 50931 Cologne, Germany
Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 69120 Heidelberg, Germany.49-6221-54670049-6221-548311

Myelin is a specialized membrane enriched in glycosphingolipids and cholesterol that contains a limited spectrum of proteins. We investigated the assembly of myelin components by oligodendrocytes and analyzed the role of lipid–protein interactions in this process. Proteolipid protein (PLP), the major myelin protein, was recovered from cultured oligodendrocytes from a low-density CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids. PLP associated with the CIMF after leaving the endoplasmic reticulum but before exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP with myelin lipids in CIMF was supported by the finding that it was efficiently cross-linked to photoactivable cholesterol, but not to phosphatidylcholine, which is underrepresented in both myelin and CIMF. Furthermore, depletion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocytes abolished the association of PLP with CIMF. Thus, PLP may be recruited to myelin rafts, represented by CIMF, via lipid–protein interactions. In contrast to oligodendrocytes, after transfection in BHK cells, PLP is absent from isolated CIMF, suggesting that PLP requires specific lipids for raft association. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fraction of PLP no longer associates with rafts. Formation of a cholesterol- and galactosylceramide-rich membrane domain (myelin rafts) may be critical for the sorting of PLP and assembly of myelin in oligodendrocytes.

Key Words: proteolipid protein • cholesterol • galactocerebroside • myelin • rafts

© 2000 The Rockefeller University Press

Abbreviations used in this paper: CHAPS, 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate; CGT, ceramide galactosyl transferase; CIMF, CHAPS-insoluble membrane fraction; GPI, glycosylphosphatidylinositol; HA, hemagglutinin; HS, horse serum; mβCD, methyl-β-cyclodextrin; MAG, myelin-associated glycoprotein; MBP, myelin basic protein; MOG, myelin oligodendrocyte glycoprotein; NCAM, neural cell adhesion molecule; PLP, proteolipid protein; SFV, Semliki Forest virus; VSV G, vesicular stomatitis virus G protein.

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