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

Simons M, Krämer EM, Thiele C, Stoffel W, Trotter J - J. Cell Biol. (2000)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany.

ABSTRACT
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.

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PLP copatches with influenza HA on the surface of living oligodendrocytes. Oligodendrocytes were infected for 1 h with the influenza virus or with SFV and incubated for a further 1 or 2 h, respectively. (A) Cells were extracted with 20 mM CHAPS and the distribution of influenza HA and SFV protein E2 on a density gradient was analyzed as described in the text. (B) Cells were incubated simultaneously with anti-HA and O10 anti-PLP or with anti-E2 and O10 anti-PLP followed by the respective secondary antibodies. The small bottom panels are enlargements of areas of the upper panels. Bars, 30 μm.
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Figure 5: PLP copatches with influenza HA on the surface of living oligodendrocytes. Oligodendrocytes were infected for 1 h with the influenza virus or with SFV and incubated for a further 1 or 2 h, respectively. (A) Cells were extracted with 20 mM CHAPS and the distribution of influenza HA and SFV protein E2 on a density gradient was analyzed as described in the text. (B) Cells were incubated simultaneously with anti-HA and O10 anti-PLP or with anti-E2 and O10 anti-PLP followed by the respective secondary antibodies. The small bottom panels are enlargements of areas of the upper panels. Bars, 30 μm.

Mentions: Our biochemical data using detergents suggest that PLP/DM20 is present in a membrane fraction with properties similar to lipid rafts. If this were indeed the case, PLP should colocalize on the surface of oligodendrocytes with other components of CIMF and should segregate from components that are not part of CIMF. It has been shown previously that the influenza virus HA protein is recovered to a greater extent from CHAPS-insoluble membrane than from Triton X-100–insoluble membrane (Kurzchalia et al. 1992) and thus might be a useful marker for CIMF. We confirmed that HA was also insoluble in CHAPS in primary oligodendrocytes (Fig. 5 A). HA was largely insoluble in Triton X-100 (data not shown) as well. For a non-CIMF marker, we used the SFV spike protein, E2, which we found to be soluble in CHAPS in oligodendrocytes (Fig. 5 A).


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

Simons M, Krämer EM, Thiele C, Stoffel W, Trotter J - J. Cell Biol. (2000)

PLP copatches with influenza HA on the surface of living oligodendrocytes. Oligodendrocytes were infected for 1 h with the influenza virus or with SFV and incubated for a further 1 or 2 h, respectively. (A) Cells were extracted with 20 mM CHAPS and the distribution of influenza HA and SFV protein E2 on a density gradient was analyzed as described in the text. (B) Cells were incubated simultaneously with anti-HA and O10 anti-PLP or with anti-E2 and O10 anti-PLP followed by the respective secondary antibodies. The small bottom panels are enlargements of areas of the upper panels. Bars, 30 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2189802&req=5

Figure 5: PLP copatches with influenza HA on the surface of living oligodendrocytes. Oligodendrocytes were infected for 1 h with the influenza virus or with SFV and incubated for a further 1 or 2 h, respectively. (A) Cells were extracted with 20 mM CHAPS and the distribution of influenza HA and SFV protein E2 on a density gradient was analyzed as described in the text. (B) Cells were incubated simultaneously with anti-HA and O10 anti-PLP or with anti-E2 and O10 anti-PLP followed by the respective secondary antibodies. The small bottom panels are enlargements of areas of the upper panels. Bars, 30 μm.
Mentions: Our biochemical data using detergents suggest that PLP/DM20 is present in a membrane fraction with properties similar to lipid rafts. If this were indeed the case, PLP should colocalize on the surface of oligodendrocytes with other components of CIMF and should segregate from components that are not part of CIMF. It has been shown previously that the influenza virus HA protein is recovered to a greater extent from CHAPS-insoluble membrane than from Triton X-100–insoluble membrane (Kurzchalia et al. 1992) and thus might be a useful marker for CIMF. We confirmed that HA was also insoluble in CHAPS in primary oligodendrocytes (Fig. 5 A). HA was largely insoluble in Triton X-100 (data not shown) as well. For a non-CIMF marker, we used the SFV spike protein, E2, which we found to be soluble in CHAPS in oligodendrocytes (Fig. 5 A).

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus