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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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Myelin is resistant to extraction with 20 mM CHAPS. (A) Myelin was prepared from adult mice and 1 μg (protein weight) of myelin was extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C followed by flotation in an Optiprep gradient (40, 30, and 0%). Equal fractions were collected from the top (lane 1) to the bottom (lane 6). Fraction 1 (0–30% interface) represents low-density, insoluble membrane. Proteins were resolved on SDS-PAGE and, after Western blotting, were detected with anti-PLP, anti-MOG, anti-MBP, or anti-MAG antibodies. (B) For lipid analysis, 10 μg (protein weight) of myelin was extracted with Triton X-100, 20 mM CHAPS, or with respective buffer alone for 1 h at 4°C, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface), resolved by TLC, and visualized by exposure to sulfuric acid and charring. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (Triton and CHAPS). The position of the reference lipids is indicated on the left. Chol, cholesterol; GalC, hydroxylated and nonhydroxylated galactosylceramide; PE, phosphatidylethanolamine; Sulf, hydroxylated and nonhydroxylated sulfatide; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol.
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Figure 1: Myelin is resistant to extraction with 20 mM CHAPS. (A) Myelin was prepared from adult mice and 1 μg (protein weight) of myelin was extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C followed by flotation in an Optiprep gradient (40, 30, and 0%). Equal fractions were collected from the top (lane 1) to the bottom (lane 6). Fraction 1 (0–30% interface) represents low-density, insoluble membrane. Proteins were resolved on SDS-PAGE and, after Western blotting, were detected with anti-PLP, anti-MOG, anti-MBP, or anti-MAG antibodies. (B) For lipid analysis, 10 μg (protein weight) of myelin was extracted with Triton X-100, 20 mM CHAPS, or with respective buffer alone for 1 h at 4°C, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface), resolved by TLC, and visualized by exposure to sulfuric acid and charring. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (Triton and CHAPS). The position of the reference lipids is indicated on the left. Chol, cholesterol; GalC, hydroxylated and nonhydroxylated galactosylceramide; PE, phosphatidylethanolamine; Sulf, hydroxylated and nonhydroxylated sulfatide; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol.

Mentions: As shown previously (Pereyra et al. 1988; Krämer et al. 1997; van der Haar et al. 1998; Kim and Pfeiffer 1999), extraction with 1% Triton X-100 leads to solubilization of the majority of PLP/DM20, MAG, MOG, and MBP (Fig. 1 A). Only a minor fraction of these proteins was recovered from the low-density, detergent-insoluble membrane fraction (0–30% interface) (Fig. 1 A). However, when myelin was extracted with 20 mM CHAPS, the difference compared with extraction with Triton X-100 was striking. PLP/DM20, MAG, and MOG were almost exclusively recovered from the detergent-resistant interface (Fig. 1 A). In contrast, a prominent fraction of the major cytosolic protein, MBP, was found at the bottom of the gradient (Fig. 1 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)

Myelin is resistant to extraction with 20 mM CHAPS. (A) Myelin was prepared from adult mice and 1 μg (protein weight) of myelin was extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C followed by flotation in an Optiprep gradient (40, 30, and 0%). Equal fractions were collected from the top (lane 1) to the bottom (lane 6). Fraction 1 (0–30% interface) represents low-density, insoluble membrane. Proteins were resolved on SDS-PAGE and, after Western blotting, were detected with anti-PLP, anti-MOG, anti-MBP, or anti-MAG antibodies. (B) For lipid analysis, 10 μg (protein weight) of myelin was extracted with Triton X-100, 20 mM CHAPS, or with respective buffer alone for 1 h at 4°C, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface), resolved by TLC, and visualized by exposure to sulfuric acid and charring. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (Triton and CHAPS). The position of the reference lipids is indicated on the left. Chol, cholesterol; GalC, hydroxylated and nonhydroxylated galactosylceramide; PE, phosphatidylethanolamine; Sulf, hydroxylated and nonhydroxylated sulfatide; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol.
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Related In: Results  -  Collection

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Figure 1: Myelin is resistant to extraction with 20 mM CHAPS. (A) Myelin was prepared from adult mice and 1 μg (protein weight) of myelin was extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C followed by flotation in an Optiprep gradient (40, 30, and 0%). Equal fractions were collected from the top (lane 1) to the bottom (lane 6). Fraction 1 (0–30% interface) represents low-density, insoluble membrane. Proteins were resolved on SDS-PAGE and, after Western blotting, were detected with anti-PLP, anti-MOG, anti-MBP, or anti-MAG antibodies. (B) For lipid analysis, 10 μg (protein weight) of myelin was extracted with Triton X-100, 20 mM CHAPS, or with respective buffer alone for 1 h at 4°C, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface), resolved by TLC, and visualized by exposure to sulfuric acid and charring. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (Triton and CHAPS). The position of the reference lipids is indicated on the left. Chol, cholesterol; GalC, hydroxylated and nonhydroxylated galactosylceramide; PE, phosphatidylethanolamine; Sulf, hydroxylated and nonhydroxylated sulfatide; PC, phosphatidylcholine; PS, phosphatidylserine; PI, phosphatidylinositol.
Mentions: As shown previously (Pereyra et al. 1988; Krämer et al. 1997; van der Haar et al. 1998; Kim and Pfeiffer 1999), extraction with 1% Triton X-100 leads to solubilization of the majority of PLP/DM20, MAG, MOG, and MBP (Fig. 1 A). Only a minor fraction of these proteins was recovered from the low-density, detergent-insoluble membrane fraction (0–30% interface) (Fig. 1 A). However, when myelin was extracted with 20 mM CHAPS, the difference compared with extraction with Triton X-100 was striking. PLP/DM20, MAG, and MOG were almost exclusively recovered from the detergent-resistant interface (Fig. 1 A). In contrast, a prominent fraction of the major cytosolic protein, MBP, was found at the bottom of the gradient (Fig. 1 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