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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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Cholesterol interacts with PLP/DM20 and both cholesterol and sphingolipids are required for integrity of CIMF. (A) Oligodendrocytes were incubated for 30 min with 5 mM mβCD, for 5 d with 50 μM fumonisin B1 (+FB1), or left untreated, before extraction with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was visualized by Western blotting. (B) Oligodendrocytes were grown in the presence of 3H-photocholesterol or 10-azisteric acid and [3H]-choline (PC), as indicated. Cells were UV irradiated for cholesterol and PC cross-linking and were prepared as described in the legend to Fig. 4 A. Cell lysates were either directly applied to SDS-PAGE (5–20%) or first immunoprecipitated (IP) with anti-PLP antibody. Labeled proteins were visualized by fluorography. Note that photoaffinity labeling reveals binding of PLP/DM20 to photocholesterol, but not to PC.
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Figure 6: Cholesterol interacts with PLP/DM20 and both cholesterol and sphingolipids are required for integrity of CIMF. (A) Oligodendrocytes were incubated for 30 min with 5 mM mβCD, for 5 d with 50 μM fumonisin B1 (+FB1), or left untreated, before extraction with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was visualized by Western blotting. (B) Oligodendrocytes were grown in the presence of 3H-photocholesterol or 10-azisteric acid and [3H]-choline (PC), as indicated. Cells were UV irradiated for cholesterol and PC cross-linking and were prepared as described in the legend to Fig. 4 A. Cell lysates were either directly applied to SDS-PAGE (5–20%) or first immunoprecipitated (IP) with anti-PLP antibody. Labeled proteins were visualized by fluorography. Note that photoaffinity labeling reveals binding of PLP/DM20 to photocholesterol, but not to PC.

Mentions: To study the involvement of cholesterol in the formation of CIMF, we analyzed whether PLP/DM20 was still associated with CIMF in cholesterol-depleted membranes. To deplete oligodendrocytes of cholesterol, we used mβCD, which is known to extract cholesterol with a higher specificity than other lipids (Klein et al. 1995). Oligodendrocytes were cultured in the presence of 5 mM mβCD for 30 min, extracted with CHAPS, and run on a density gradient gel, where the distribution of PLP/DM20 was compared with untreated cells. After cholesterol depletion, PLP/DM20 was no longer associated with CIMF to the same extent as in control cells (Fig. 6 A). These results demonstrate that cholesterol is an essential component of CIMF.


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)

Cholesterol interacts with PLP/DM20 and both cholesterol and sphingolipids are required for integrity of CIMF. (A) Oligodendrocytes were incubated for 30 min with 5 mM mβCD, for 5 d with 50 μM fumonisin B1 (+FB1), or left untreated, before extraction with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was visualized by Western blotting. (B) Oligodendrocytes were grown in the presence of 3H-photocholesterol or 10-azisteric acid and [3H]-choline (PC), as indicated. Cells were UV irradiated for cholesterol and PC cross-linking and were prepared as described in the legend to Fig. 4 A. Cell lysates were either directly applied to SDS-PAGE (5–20%) or first immunoprecipitated (IP) with anti-PLP antibody. Labeled proteins were visualized by fluorography. Note that photoaffinity labeling reveals binding of PLP/DM20 to photocholesterol, but not to PC.
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Related In: Results  -  Collection

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Figure 6: Cholesterol interacts with PLP/DM20 and both cholesterol and sphingolipids are required for integrity of CIMF. (A) Oligodendrocytes were incubated for 30 min with 5 mM mβCD, for 5 d with 50 μM fumonisin B1 (+FB1), or left untreated, before extraction with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was visualized by Western blotting. (B) Oligodendrocytes were grown in the presence of 3H-photocholesterol or 10-azisteric acid and [3H]-choline (PC), as indicated. Cells were UV irradiated for cholesterol and PC cross-linking and were prepared as described in the legend to Fig. 4 A. Cell lysates were either directly applied to SDS-PAGE (5–20%) or first immunoprecipitated (IP) with anti-PLP antibody. Labeled proteins were visualized by fluorography. Note that photoaffinity labeling reveals binding of PLP/DM20 to photocholesterol, but not to PC.
Mentions: To study the involvement of cholesterol in the formation of CIMF, we analyzed whether PLP/DM20 was still associated with CIMF in cholesterol-depleted membranes. To deplete oligodendrocytes of cholesterol, we used mβCD, which is known to extract cholesterol with a higher specificity than other lipids (Klein et al. 1995). Oligodendrocytes were cultured in the presence of 5 mM mβCD for 30 min, extracted with CHAPS, and run on a density gradient gel, where the distribution of PLP/DM20 was compared with untreated cells. After cholesterol depletion, PLP/DM20 was no longer associated with CIMF to the same extent as in control cells (Fig. 6 A). These results demonstrate that cholesterol is an essential component of CIMF.

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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Related in: MedlinePlus