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First published online May 28, 2005
doi: 10.1242/10.1242/jcs.02365

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Palmitoylation is a sorting determinant for transport to the myelin membrane

¹ Department of Neurology, Hertie Institute for Clinical Brain Research, University of Tübingen, Hoppe-Seyler-Strausse 3, 72076 Tübingen, Germany
² Center for Biochemistry and Molecular Cell Biology, University of Göttingen, Humboldtallee 23, 37075 Göttingen, Germany
³ Institute of Pathology, Liebermeisterstr. 8, University of Tübingen, 72076 Tübingen, Germany
⁴ Max-Planck-Institute for Experimental Medicine, Hermann-Rein-Strausse 3, 37075 Göttingen, Germany
⁵ Department of Neurodegeneration and Restorative Research, Center of Neurological Medicine and CMPB, Waldweg 33, 37073 Göttingen, Germany

View larger version (65K): [in a new window]   Fig. 1. Enriched oligodendrocyte cultures form MLMs even in the absence of neurons. (A) Primary cultures of oligodendrocytes with membrane sheets at an early stage of differentiation were stained for MBP and PLP. (B) Immunofluorescence analysis of the same culture at a later stage of differentiation. Notice the many thin processes, which contain pearl-like membrane patches (arrows). These patches are recognized by antibodies directed at both major myelin proteins MBP and PLP. Membrane patches also contain the myelin lipids cholesterol (C-E), galactosylceramide (C) and sulfatide (E), as shown by reactivity with filipin (C-E) and antibodies against the O1 (C) and O4 (E) epitopes. (F) Electron micrograph of differentiated oligodendrocyte shows multiple thin processes containing MLMs. (G,H) Multilamellar membranes at different stages of compaction. MLMs were seen in almost all of the cells. The interior of these structures were most often electron lucent and did not contain axons. Bar, 20 µm or as indicated.

View larger version (97K): [in a new window]   Fig. 2. The viral surface glycoproteins HA and VSVG, but not E2, are transported to MLMs. After infection of primary oligodendrocytes with FPV, VSV or SFV, cells were fixed and processed for immunofluorescence microscopy. Cells were triple stained for PLP (O10), MBP and the viral glycoproteins. Both HA (A) and VSVG (B) were found in MLMs (arrows), from which E2 (C) was largely excluded. Bar, 10 µm.

View larger version (55K): [in a new window]   Fig. 3. Palmitoylation enhances transport of PLP and DM20 to MLMs. Primary oligodendrocytes were infected with recombinant SFV to express the palmitoylation-deficient form of DM20, DM20(C5,6,9,108S)-myc (A), and DM20-myc (B). Cells were stained for PLP (O10) and MBP to visualize MLM (arrows). Staining with anti-myc antibody revealed preferential targeting of DM20-myc to MLMs compared with DM20(C5,6,9,108S)-myc. Primary oligodendrocytes were infected with SFV-DM20-myc or SFV-DM20(C5,6,9,108S)-myc and light-membrane fractions were isolated according to the myelin-isolation protocol to enrich for MLMs. The amount of virus-derived DM20-myc and DM20(C5,6,9,108S)-myc in the myelin preparations was determined by immunoblotting with an anti-myc antibody (C). (D) DM20-myc and DM20(C5,6,9,108S)-myc with palmitoylation sites are shown in red. Bar, 20 µm.

View larger version (63K): [in a new window]   Fig. 4. The 13 amino acid N-terminus of PLP is sorted into MLMs in a palmitoylation-dependent way. Primary oligodendrocyte cultures were infected with recombinant SFV producing 1-13PLP-EYFP or its palmitoylation-deficient mutant 1-13PLP(C5,6,9S)-EYFP, and processed for immunofluorescence 5 hours after infection. Cells were stained for PLP (O10) to visualize MLMs (arrows). 1-13PLP(C5,6,9S)-EYFP showed a diffuse staining of the oligodendroglial soma and some processes (A), whereas 1-13PLP-EYFP was distributed over the entire oligodendrocyte membrane and was detectable in MLMs (B). 1-13PLP-EYFP was also found on intracellular membranes in the Golgi region. Confocal microscopy analysis showed colocalization of 1-13PLP-EYFP with GM130, a marker for the Golgi apparatus (C). Treatment with BFA (10 µg ml–1) for the last 4 hours during post-infection time resulted in a redistribution of 1-13PLP-EYFP from the distal membrane extensions into the soma and proximal part of some processes (D). Primary oligodendrocytes were infected with SFV-1-13PLP-EYFP, SFV-1-13PLP(C5,6,9S)-EYFP or SFV-EYFP, and light-membrane fractions were isolated according to the myelin-isolation protocol to enrich MLMs. The amounts of virus-derived 1-13PLP-EYFP, 1-13PLP(C5,6,9S)-EYFP and EYFP in the myelin preparations were determined by immunoblotting with an anti-GFP antibody (E). The sequence of the N-terminal 13 amino acids is shown for 1-13PLP-EYFP and 1-13PLP(C5,6,9S)-EYFP (F). Bars, 20 µm.

View larger version (63K): [in a new window]   Fig. 5. Transport of acylated GFP fusion proteins to MLMs. Primary oligodendrocytes were transiently transfected with plasmids coding for various fluorescent fusion proteins containing different acylation sites. We used a EGFP fusion protein with the C-terminal 20 amino acids of H-Ras (EGFP-tH), which contain a combined farnesylation/palmitoylation signal, and an EYFP fusion protein with the N-terminal 20 amino acids of neuromodulin (tN-EYFP), which contain a consensus sequence for dual palmitoylation. To visualize MLMs, cells were stained for PLP (O10) and MBP. Both EGFP-tH (A) and tN-EYFP (B) were found in MLMs. By contrast, an LDL-receptor construct in which the cytoplasmic domain of the LDL receptor had been replaced by the cytoplasmic domain of CD46 to remove its endocytic sorting determinant and the extracellular LDL receptor domain had been replaced by EGFP was not localized to MLMs (C,D). (E) The constructs used. Bar, 10 µm.

View larger version (52K): [in a new window]   Fig. 6. Palmitoylation-deficient mutants of PLP/DM20 are less efficiently targeted to myelin in myelinating co-cultures of oligodendrocytes and neurons. Myelinating co-cultures of oligodendrocytes and neurons were infected with recombinant SFV to produce 1-13PLP(C5,6,9S)-EYFP (A), 1-13PLP-EYFP (B), DM20(C5,6,9,108S)-myc (C) and DM20-myc (D). The myelin sheaths and axons were identified by labelling with anti-MBP antibody and with an antibody specific for neuronal tubulin or neurofilament, respectively. Bars, 20 µm.