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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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The CIMF represents a subdomain of the membrane in oligodendrocytes. (A) Oligodendrocytes were labeled overnight with 100 μCi/ml [35S]methionine/cysteine; a total membrane fraction was then prepared and treated with 20 mM CHAPS or buffer alone (total). After centrifugation in a density gradient, six equal fractions were collected from the top (lane 1) to the bottom (lane 6) and the radioactive counts were determined in each fraction. The percent of radioactivity in each fraction is shown. (B) Oligodendrocytes were extracted with 1% Triton X-100 or with 20 mM CHAPS and centrifuged in a density gradient as described in the text. The GPI-anchored proteins NCAM120 and F3 are enriched in the Triton X-100, but not in the CHAPS–insoluble membrane fraction.
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Figure 3: The CIMF represents a subdomain of the membrane in oligodendrocytes. (A) Oligodendrocytes were labeled overnight with 100 μCi/ml [35S]methionine/cysteine; a total membrane fraction was then prepared and treated with 20 mM CHAPS or buffer alone (total). After centrifugation in a density gradient, six equal fractions were collected from the top (lane 1) to the bottom (lane 6) and the radioactive counts were determined in each fraction. The percent of radioactivity in each fraction is shown. (B) Oligodendrocytes were extracted with 1% Triton X-100 or with 20 mM CHAPS and centrifuged in a density gradient as described in the text. The GPI-anchored proteins NCAM120 and F3 are enriched in the Triton X-100, but not in the CHAPS–insoluble membrane fraction.

Mentions: Having shown that the major components of myelin were resistant to extraction with CHAPS, it was important to rule out that the conditions for detergent extraction were so mild that large fragments of the plasma membrane remained completely intact. Oligodendrocytes were subjected to metabolic radiolabeling with 35S-Translabel, and the total floated membrane fraction was either extracted with 20 mM CHAPS or left untreated; fractions were then separated on a density gradient. The majority of the radioactivity was recovered at the 0–30% interface from membranes that had not been incubated with CHAPS (Fig. 3 A). In contrast, the radioactivity was mainly found in the bottom fractions (especially fraction 4) from membranes that had been incubated with CHAPS, demonstrating that CIMF at the 0–30% interface represents a subdomain of the oligodendrocyte membrane (Fig. 3 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)

The CIMF represents a subdomain of the membrane in oligodendrocytes. (A) Oligodendrocytes were labeled overnight with 100 μCi/ml [35S]methionine/cysteine; a total membrane fraction was then prepared and treated with 20 mM CHAPS or buffer alone (total). After centrifugation in a density gradient, six equal fractions were collected from the top (lane 1) to the bottom (lane 6) and the radioactive counts were determined in each fraction. The percent of radioactivity in each fraction is shown. (B) Oligodendrocytes were extracted with 1% Triton X-100 or with 20 mM CHAPS and centrifuged in a density gradient as described in the text. The GPI-anchored proteins NCAM120 and F3 are enriched in the Triton X-100, but not in the CHAPS–insoluble membrane fraction.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: The CIMF represents a subdomain of the membrane in oligodendrocytes. (A) Oligodendrocytes were labeled overnight with 100 μCi/ml [35S]methionine/cysteine; a total membrane fraction was then prepared and treated with 20 mM CHAPS or buffer alone (total). After centrifugation in a density gradient, six equal fractions were collected from the top (lane 1) to the bottom (lane 6) and the radioactive counts were determined in each fraction. The percent of radioactivity in each fraction is shown. (B) Oligodendrocytes were extracted with 1% Triton X-100 or with 20 mM CHAPS and centrifuged in a density gradient as described in the text. The GPI-anchored proteins NCAM120 and F3 are enriched in the Triton X-100, but not in the CHAPS–insoluble membrane fraction.
Mentions: Having shown that the major components of myelin were resistant to extraction with CHAPS, it was important to rule out that the conditions for detergent extraction were so mild that large fragments of the plasma membrane remained completely intact. Oligodendrocytes were subjected to metabolic radiolabeling with 35S-Translabel, and the total floated membrane fraction was either extracted with 20 mM CHAPS or left untreated; fractions were then separated on a density gradient. The majority of the radioactivity was recovered at the 0–30% interface from membranes that had not been incubated with CHAPS (Fig. 3 A). In contrast, the radioactivity was mainly found in the bottom fractions (especially fraction 4) from membranes that had been incubated with CHAPS, demonstrating that CIMF at the 0–30% interface represents a subdomain of the oligodendrocyte membrane (Fig. 3 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