Assembly of myelin by association of proteolipid protein with cholesterol-and galactosylceramide-rich membrane domains

Myelin is a specialized membrane enriched in glycosphingolipids and cholest erol that contains a limited spectrum of proteins. We investigated the asse mbly of myelin components by oligodendrocytes and analyzed the role of lipi d-protein interactions in this process. Proteolipid protein (PLP), the majo r myelin protein, was recovered from cultured oligodendrocytes from a low-d ensity CHAPS-insoluble membrane fraction (CIMF) enriched in myelin lipids, PLP associated with the CIMF after leaving the endoplasmic reticulum but be fore exiting the Golgi apparatus, suggesting that myelin lipid and protein components assemble in the Golgi complex. The specific association of PLP w ith myelin lipids in CIMF was supported by the finding that it was efficien tly cross-linked to photoactivable cholesterol, but not to phosphatidylchol ine, which is underrepresented in both myelin and CIMF. Furthermore, deplet ion of cholesterol or inhibition of sphingolipid synthesis in oligodendrocy tes abolished the association of PLP with CIMF Thus, PLP may be recruited t o myelin rafts, represented by CIMF via lipid-protein interactions. In cont rast to oligodendrocytes, after transfection in BHK cells, PLP is absent fr om isolated CIMF, suggesting that PLP requires specific lipids for raft ass ociation. In mice deficient in the enzyme ceramide galactosyl transferase, which cannot synthesize the main myelin glycosphingolipids, a large fractio n of PLP no longer associates with rafts. Formation of a cholesterol- and g alactosylceramide-rich membrane domain (myelin rafts) may be critical for t he sorting of PLP and assembly of myelin in oligodendrocytes.