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Cited by 293Lipid Membrane Structure and BehaviorSphingolipid Metabolism and SignalingCaveolin-1 and cellular processes

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Assembly of Myelin by Association of Proteolipid Protein with Cholesterol- and Galactosylceramide-Rich Membrane Domains

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Simons, Mikael, Krämer, Eva-Maria, Thiele, Christoph, Stoffel, Wilhelm, Trotter, Jacqueline·2000·The Journal of Cell Biology
DOI 10.1083/jcb.151.1.143PMC PMC2189802
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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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Figure 2 Identification of a CIMF in primary cultures of oligodendrocytes. (A) Oligodendrocytes were extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C and floated in a density gradient (1.flotation). Six fractions were collected from the top (lane 1) to the bottom (lane 6). Proteins were separated by SDS-PAGE and, after Western blotting, were detected with the respective antibodies. The detergent-insoluble membrane fraction from the 1.flotation was reextracted, refloated in a density gradient (2.flotation), and proteins were visualized as described above. (B) Lipids were labeled overnight with [14C]acetate. Total floated membranes were incubated with 20 mM CHAPS, 1% Triton X-100, or buffer alone (total) for 30 min at 4°C and centrifuged in a density gradient. Lipids were extracted from the 0–30% interface and analyzed by TLC. Labeled lipids were detected by autoradiography and quantified by densitometric scanning. The position of the reference lipids is indicated on the left (abbreviations as in the legend to Fig. 1).
Figure 2 Identification of a CIMF in primary cultures of oligodendrocytes. (A) Oligodendrocytes were extracted with 1% Triton X-100 or 20 mM CHAPS for 30 min at 4°C and floated in a density gradient (1.flotation). Six fractions were collected from the top (lane 1) to the bottom (lane 6). Proteins were separated by SDS-PAGE and, after Western blotting, were detected with the respective antibodies. The detergent-insoluble membrane fraction from the 1.flotation was reextracted, refloated in a density gradient (2.flotation), and proteins were visualized as described above. (B) Lipids were labeled overnight with [14C]acetate. Total floated membranes were incubated with 20 mM CHAPS, 1% Triton X-100, or buffer alone (total) for 30 min at 4°C and centrifuged in a density gradient. Lipids were extracted from the 0–30% interface and analyzed by TLC. Labeled lipids were detected by autoradiography and quantified by densitometric scanning. The position of the reference lipids is indicated on the left (abbreviations as in the legend to Fig. 1).CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central
Figure 4 Association of PLP/DM20 with CIMF occurs during transport through the biosynthetic pathway. (A) Oligodendrocytes were pulse labeled for 5 min with 1 mCi/ml [35S]methionine/cysteine and chased in chase medium for the times indicated. Cells were extracted with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was immunoprecipitated from fraction one (I) or four (S) from the gradient, separated by SDS-PAGE, and visualized by autoradiography. (B) Oligodendrocytes were pulse labeled for 10 min with 1 mCi/ml [35S]methionine/cysteine and chased for 120 min at the temperature indicated.
Figure 4 Association of PLP/DM20 with CIMF occurs during transport through the biosynthetic pathway. (A) Oligodendrocytes were pulse labeled for 5 min with 1 mCi/ml [35S]methionine/cysteine and chased in chase medium for the times indicated. Cells were extracted with 20 mM CHAPS and centrifuged in a density gradient. PLP/DM20 was immunoprecipitated from fraction one (I) or four (S) from the gradient, separated by SDS-PAGE, and visualized by autoradiography. (B) Oligodendrocytes were pulse labeled for 10 min with 1 mCi/ml [35S]methionine/cysteine and chased for 120 min at the temperature indicated.CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central
Figure 7 Association of PLP/DM20 with CIMF is reduced in myelin from CGT knockout mice. (A) Myelin was prepared from CGT knockout and wild-type mice and 1 μg (protein weight) of each was extracted with 20 mM CHAPS or 1% Triton X-100 for 30 min at 4°C. Density centrifugation, SDS-PAGE, and Western blotting were performed as in the legend to Fig. 1. (B) 10 μg (protein weight) of myelin from CGT knockout mice was extracted with 20 mM CHAPS for 30 min at 4°C, or with buffer alone, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface) and resolved by TLC. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (CHAPS). The position of the reference lipids is indicated on the left. GluC, glucosylceramide; SM, sphingomyelin; additional abbreviations as in the legend to Fig. 1.
Figure 7 Association of PLP/DM20 with CIMF is reduced in myelin from CGT knockout mice. (A) Myelin was prepared from CGT knockout and wild-type mice and 1 μg (protein weight) of each was extracted with 20 mM CHAPS or 1% Triton X-100 for 30 min at 4°C. Density centrifugation, SDS-PAGE, and Western blotting were performed as in the legend to Fig. 1. (B) 10 μg (protein weight) of myelin from CGT knockout mice was extracted with 20 mM CHAPS for 30 min at 4°C, or with buffer alone, and floated in a density gradient. Lipids were extracted from the floating fraction (0–30% interface) and resolved by TLC. Myelin treated with buffer alone (total) is shown next to detergent-extracted myelin (CHAPS). The position of the reference lipids is indicated on the left. GluC, glucosylceramide; SM, sphingomyelin; additional abbreviations as in the legend to Fig. 1.CC BY-NC-SA · PubMed Central
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.
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.CC BY-NC-SA · PubMed Central

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Metadata

Authors
Simons, M., Krämer, E.-M., Thiele, C., Stoffel, W., & Trotter, J.
Affiliations
  • Heidelberg University (DE)
  • aDepartment of Neurobiology, University of Heidelberg, 69120 Heidelberg, Germany
  • University of Cologne (DE)
ORCID
Venue
The Journal of Cell Biology
Publisher
Rockefeller University Press
Published
2000-10-02
Volume
151
Issue
1
Pages
143-154
ISSN
  • 0021-9525 (ISSN-L)
  • 1540-8140
DOI
10.1083/jcb.151.1.143
PMC
PMC2189802
OpenAlex
W2098744846
Cited by
293
References
58
Citations by year
2026: 5 · 2025: 2 · 2024: 7 · 2023: 6 · 2022: 12 · 2021: 5 · 2020: 10 · 2019: 15 · 2018: 3 · 2017: 12 · 2016: 4 · 2015: 8 · 2014: 17 · 2013: 9 · 2012: 14
FWCI
5.31
Type
journal article
Language
en
Open access
Licence
cc-by-nc-sa
Locations
  • The Journal of Cell Biology (publishedVersion)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by-nc-sa)
  • PubMed Central (publishedVersion, cc-by-nc-sa)
  • PubMed (publishedVersion)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Lipid Membrane Structure and Behavior
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Sphingolipid Metabolism and Signaling
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Caveolin-1 and cellular processes
Keywords
proteolipid protein, lipid rafts, oligodendrocytes, cholesterol, galactosylceramide, glycosphingolipids, protein-lipid interactions, Golgi apparatus, sphingolipid biosynthesis
MeSH terms
Animals, Brain Chemistry, Cell Fractionation, Cholesterol, Cholic Acids, Detergents, Endoplasmic Reticulum, Galactosylceramides, Galactosyltransferases, Golgi Apparatus, Cricetinae, Myelin Sheath, Oligodendroglia, Solubility, Glycosylphosphatidylinositols, Mice, Knockout, Myelin Proteolipid Protein, Hemagglutinin Glycoproteins, Influenza Virus, Membrane Microdomains, N-Acylsphingosine Galactosyltransferase, Mice

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