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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 does not associate with CIMF in BHK cells. Cells were transiently transfected for expression of PLP or PLP/DM20, or infected with influenza virus for expression of HA. Cells were extracted with 20 mM CHAPS, subjected to density gradient centrifugation, and proteins were analyzed from each fraction.
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Figure 8: PLP does not associate with CIMF in BHK cells. Cells were transiently transfected for expression of PLP or PLP/DM20, or infected with influenza virus for expression of HA. Cells were extracted with 20 mM CHAPS, subjected to density gradient centrifugation, and proteins were analyzed from each fraction.

Mentions: The above results suggest that galactosylceramide and/or sulfatide are required for the association of PLP with CIMF. We tested whether PLP is associated with CIMF in cells that generate rafts, but do not synthesize galactosylceramide and sulfatide as major sphingolipids (Scheiffele et al. 1999). For this purpose, we transiently transfected BHK cells with PLP and prepared CIMF as described above. In contrast to oligodendrocytes, PLP was not associated with CIMF in BHK cells (Fig. 8 A). Exogeneously expressed HA was recovered from CIMF in BHK cells (Fig. 8 C), thus showing that this cell type does indeed generate rafts. In case the transport of PLP is dependent on the presence of the DM20 isoform, we cotransfected cells with both PLP and DM20. However, PLP still did not associate with the isolated CIMF (Fig. 8 B). We ensured that exogenously expressed PLP had reached the plasma membrane surface by labeling living BHK cells at 4°C with O10 mAb, which recognizes an extracellular epitope of PLP (Jung et al. 1996). The positive staining with O10 of transfected BHK cells confirmed that PLP had reached the cell surface and suggests that the protein was correctly folded (data not shown). Hence, we conclude that association of PLP with CIMF is not an intrinsic property of PLP, but depends on the specialized lipid environment generated by oligodendrocytes.


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 does not associate with CIMF in BHK cells. Cells were transiently transfected for expression of PLP or PLP/DM20, or infected with influenza virus for expression of HA. Cells were extracted with 20 mM CHAPS, subjected to density gradient centrifugation, and proteins were analyzed from each fraction.
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Related In: Results  -  Collection

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

Figure 8: PLP does not associate with CIMF in BHK cells. Cells were transiently transfected for expression of PLP or PLP/DM20, or infected with influenza virus for expression of HA. Cells were extracted with 20 mM CHAPS, subjected to density gradient centrifugation, and proteins were analyzed from each fraction.
Mentions: The above results suggest that galactosylceramide and/or sulfatide are required for the association of PLP with CIMF. We tested whether PLP is associated with CIMF in cells that generate rafts, but do not synthesize galactosylceramide and sulfatide as major sphingolipids (Scheiffele et al. 1999). For this purpose, we transiently transfected BHK cells with PLP and prepared CIMF as described above. In contrast to oligodendrocytes, PLP was not associated with CIMF in BHK cells (Fig. 8 A). Exogeneously expressed HA was recovered from CIMF in BHK cells (Fig. 8 C), thus showing that this cell type does indeed generate rafts. In case the transport of PLP is dependent on the presence of the DM20 isoform, we cotransfected cells with both PLP and DM20. However, PLP still did not associate with the isolated CIMF (Fig. 8 B). We ensured that exogenously expressed PLP had reached the plasma membrane surface by labeling living BHK cells at 4°C with O10 mAb, which recognizes an extracellular epitope of PLP (Jung et al. 1996). The positive staining with O10 of transfected BHK cells confirmed that PLP had reached the cell surface and suggests that the protein was correctly folded (data not shown). Hence, we conclude that association of PLP with CIMF is not an intrinsic property of PLP, but depends on the specialized lipid environment generated by oligodendrocytes.

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