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
* Author for correspondence (e-mail: msimons{at}gwdg.de)
Accepted 9 March 2005
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Summary |
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Key words: Oligodendrocytes, Myelin, Proteolipid protein, Palmitoylation, Membrane trafficking, Sorting determinants
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Introduction |
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The mechanisms by which oligodendrocytes target myelin components to myelin are not known. Studies performed in epithelial cells demonstrated that polarized protein transport to distinct apical and basolateral membrane compartments, which are separated by tight junctions, occurs after sorting in the trans-Golgi network into distinct vesicular carriers (Kreitzer et al., 2003). A polarized membrane composition and distinct trafficking routes have also been proposed for oligodendrocytes, because asymmetric membrane growth (oligodendroglial plasma membrane versus myelin membrane) requires polarized transport (de Vries et al., 1998
). This hypothesis has been supported by the identification of claudin11/OSP-based tight junctions in the interlamellar strands of myelin sheaths at the border between compact and non-compact myelin. Tight junctions could serve as a diffusion barrier and maintain the asymmetric protein distribution between the different myelin subdomains and the plasma membrane (Bronstein et al., 2000
; Gow et al., 1999
; Morita et al., 1999
). For epithelial cells, two major sorting principles have been described. Transport to the basolateral surface requires the interaction of a cytoplasmic protein transport machinery with transmembrane proteins (Mellman, 1996
; Mostov et al., 2003
; Nelson and Yeaman, 2001
). Transport to the apical membrane involves the association of proteins with glycosphingolipid- and cholesterol-enriched membrane platforms in the trans-Golgi network, followed by vesicular transport to the apical membrane (Rodriguez-Boulan and Zurzolo, 1993
; Simons and Ikonen, 1997
). Given the similarities between the lipid compositions of apical and myelin membranes, which are both highly enriched in glycosphingolipids and cholesterol, it seems likely that oligodendrocytes and epithelial cells use common mechanisms to form the specialized membranes. Indeed, there is accumulating evidence that oligodendrocytes use a raft-like transport for directing proteins to myelin (Decker et al., 2004
; Kim et al., 1995
; Kramer et al., 1997
; Simons et al., 2000
; Taylor et al., 2002
). The aim of this study was to identify sorting determinants for transport of proteins to the myelin membrane.
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Materials and Methods |
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Viral infection and transfection of primary oligodendrocytes
Green fluorescent protein (GFP)-expressing SFV (SFV-GFP) was kindly provided by P. Macchi (Max-Planck Institute for Developmental Biology, Tübingen, Germany). Primary oligodendrocytes were infected with wild-type SFV, recombinant SFV, vesicular stomatitis virus (VSV) or wild-type fowl plague virus (FPV). For viral infection, the relevant virus was diluted in maintenance medium and cells were incubated for 1 hour before replacing the virus with fresh medium. Infection was allowed to continue for a further 1-3 hours (FPV, VSV, SFV) or 5-9 hours (recombinant SFV). Primary oligodendrocytes were transfected using NupherinTM-neuron kit (Biomol, PA) according to the manufacturer's protocol. p-EGFP-F (farnesylated and double palmitoylated EGFP) and pEYFP-Mem (double palmitoylated EYFP) were from Clontech and EGFP-LDL-CD46 was from P. Keller (Max-Planck Institute for Cell Biology and Genetics, Dresden, Germany).
Primary cell cultures
Primary cultures of mouse oligodendrocytes were prepared as described previously except of a few modifications (Simons et al., 2000). After shaking, cells were plated in minimum essential medium (MEM), containing B27 supplement, 1% horse serum, L-thyroxine, tri-iodothyronine, glucose, glutamine, gentamycine, pyruvate and bicarbonate on poly-L-lysine-coated dishes or glass-coverslips. The resulting cultures were enriched in oligodendrocytes, although they still contained astrocytes. Cultures were kept without addition of growth factors for 5-8 days before virus infection, transfection or metabolic labelling.
Myelinating co-cultures of neurons and oligodendrocytes were prepared according to a modified protocol of Lubetzki et al. (Lubetzki et al., 1993). Cultures were grown on glass coverslips in oligodendrocyte growth medium as described above and virus infection was done after 24 days in culture.
Immunofluorescence and electron microscopy
Immunofluorescence and electron microscopy were performed using a standard protocol. Primary antibodies were against: haemagglutinin (HA) (Harder et al., 1998); VSV glycoprotein (VSVG) (Harder et al., 1998
); MBP [monoclonal IgG (Sternberger) or polyclonal antibody (DAKO)]; PLP [polyclonal antibody P6 (Linington and Waehneldt, 1990
); O10, monoclonal mouse IgM]; galactosylceramide (monoclonal IgM, O1); galactosylsulfatide (monoclonal IgM, O4); tubulin-ßIII (Promega); neurofilament (MN2E4, monoclonal IgM, Alexis); and myc tag (monoclonal IgG, Cell Signaling). For filipin labelling, cells were incubated for 20 minutes at room temperature with filipin (Sigma) diluted in PBS (0.5 mg ml1) and washed twice for 5 minutes in PBS. For quantification of transport to MLMs, more then 50 cells were counted from two to five independent experiments. For quantification of transport to myelin in oligodendroglial and neuronal co-cultures, more than 30 myelinating cells were evaluated from three or four independent experiments. Data are reported as dichotomized categories (colocalization vs no colocalization). Statistical differences were determined with Fischer's exact test.
Immunoblotting
Proteins were separated on 15% SDS-PAGE and detected by using the ECL western-blotting system (BioRad). Anti-GFP antibody was obtained from Santa Cruz Biotechnology.
MLM preparation
MLMs were prepared from enriched oligodendrocyte cultures 8 hours after infection with SFV according to Norton and Poduslo (Norton and Poduslo, 1973). A postnuclear supernatant was subjected to 12 minutes of centrifugation (JA20, 4°C) at 12,250 g. The membrane-containing pellet was resuspended in 0.85 M sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF) and subjected to discontinuous sucrose-gradient centrifugation (11.5 ml 0.85 M sucrose overlaid with 0.5 ml 0.25 M sucrose) using a SW41 Ti rotor at 50,000 g at 4°C for 30 minutes. The light membrane was collected from the interface and washed twice with buffer containing 50 mM Tris, pH 7.5, 5 mM EDTA, 2 mM PMSF by centrifugation with SW41 Ti rotor at 50,000 g at 4°C for 30 minutes. The purified membrane fraction was resuspended in lysis buffer (1% Triton X-100, 0.1% SDS in PBS).
Viral production rates were determined by immunoblotting of 1% total cell lysates with antibodies against the myc or GFP tags. The amount of endogenous PLP in each myelin preparation was measured by immunoblotting with anti-PLP (P6) antibody. In case of virally produced PLP, the amount of endogenous DM20 served as a measure for the preparation yield. The ratio of virus-derived PLP or DM20 mutants compared with the corresponding virus-derived wild-type protein in the myelin fraction was calculated by adjusting the for different production levels and preparation yields. For quantification, blots were scanned and evaluated with the ScionImage. Values are shown as means ± SD. Statistical differences were determined with unpaired, two-tailed Student's t tests.
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Results |
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Palmitoylation of PLP/DM20 is important for transport to myelin
We designed palmitoylation-deficient PLP and DM20 mutants, which allowed us to study the particular effects of distinct palmitoylation sites on the targeting of PLP. Palmitoylation of PLP occurs N-terminally at C5, C6 and C9, and in the cytoplasmic loop at C108, C138 and C140. DM20, the minor isoform of PLP, lacks 35 amino acids in the cytosolic loop and therefore lacks cysteines 138 and 140. We exchanged the N-terminal palmitoylation sites of PLP (C5, C6 and C9) for serines and added a myc tag to its C-terminus to obtain a mutant form of PLP that is still palmitoylated in the cytosolic loop [PLP(C5,6,9S)-myc]. Furthermore, we constructed a mutant form of DM20 that lacks C5, C6, C9 and C108, and is therefore devoid of all palmitoylation sites [DM20(C5,6,9,108S)-myc]. All constructs were cloned into an SFV vector. To ensure that the proteins were folded properly and transported to the plasma membrane, we performed surface stainings of living BHK cells with a conformation-dependent antibody against PLP/DM20 (O10) (Jung et al., 1996). All of the above proteins were stained on the cell surface with O10, demonstrating their transport through the biosynthetic pathway (data not shown). Next, we tested their targeting to MLMs. Immunofluorescence microscopy revealed that targeting of PLP(C5,6,9S)-myc and DM20(C5,6,9,108S)-myc to MLMs was not completely abolished. However, compared with their respective wild-type constructs, both proteins appeared to be more restricted to the soma and fewer MBP/PLP patches were positive for DM20(C5,6,9,108S)-myc compared with DM20-myc (localization to MLMs in 67% of cells for DM20-myc and in 38.5% of cells for DM20(C5,6,9,108S)-myc; P<0.005) (Fig. 3A,B). The same applies to PLP(C5,6,9S)-myc compared with PLP-myc (localization to MLMs in 90% of the cells for PLP-myc and in 69% of the cells for PLP(C5,6,9S)-myc; data not shown; P<0.05).
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To obtain further support for the different targeting to MLMs, a biochemical approach was used. Primary cultures of oligodendrocytes were infected with recombinant SFV and a light membrane fraction was isolated according to the myelin isolation protocol of Norton and Poduslo (Norton and Poduslo, 1973) to enrich MLMs. Consistent with the immunocytochemical results, we repeatedly found less DM20(C5,6,9,108S)-myc and PLP(C5,6,9S)-myc in the MLM-enriched fraction than we did of the respective wild-type forms (Fig. 3C). Quantification revealed a reduced incorporation of DM20(C5,6,9,108S)-myc (reduced to 48±14%, n=4, P<0.01) and PLP(C5,6,9S)-myc (reduced to 32±31%, n=3, P<0.05; data not shown) into the light membrane fraction.
Both morphological and biochemical experiments indicate that transport to MLMs is facilitated by palmitoylation of PLP/DM20. However, targeting of the palmitoylation-deficient PLP/DM20 to MLM still occurs, albeit less efficiently than wild-type PLP/DM20. This points to the presence of additional transport signals in PLP/DM20 that can substitute for the absent palmitoylation sites. Another explanation could be that the mutant form of PLP is routed to MLMs by associating with wild-type endogenous PLP/DM20. To test this possibility, enriched primary oligodendrocyte cultures were prepared from PLP knockout animals. As in the wild-type cultures, patches of MBP and galactosylceramide (O1) were observed along processes, which is consistent with the finding that myelin assembly occurs despite the absence of PLP (Klugmann et al., 1997). Targeting of PLP(C5,6,9S)-myc to MLM in PLP knockout cultures was not completely abolished (data not shown), indicating that the transport of palmitoylation-deficient PLP is not facilitated by co-transport with endogenous wild-type PLP.
Palmitoylation of the N-terminal 13 amino acids of PLP is required for sorting into myelin
A previous study has shown that the N-terminal 13 amino acids of PLP fused to LacZ are sufficient for targeting the cytoplasmic fusion protein PLP-LacZ to the myelin membrane (Wight et al., 1993). Its sequence harbours three cysteine residues, which are palmitoylated in full-length PLP in vivo. Because palmitoylation-deficient PLP/DM20 was targeted less efficiently to MLMs, we studied whether transport of the N-terminal PLP peptide was also dependent on palmitoylation. For this purpose, we constructed recombinant SFV either with the wild-type N-terminal 13 amino acids of PLP fused to EYFP (1-13PLP-EYFP) or with a mutant form in which the cysteines had been replaced by serines [1-13PLP(C5,6,9S)-EYFP]. To find out whether the N-terminal 13 amino acids of PLP were still palmitoylated when fused to EYFP, cells were infected with recombinant SFV and metabolically labelled with [3H]-labelled palmitic acid. The autoradiography revealed palmitoylation of 1-13PLP-EYFP but not of 1-13PLP(C5,6,9S)-EYFP (data not shown). To evaluate the subcellular localization of 1-13PLP-EYFP and 1-13PLP(C5,6,9S)-EYFP, primary cultures of oligodendrocytes were infected with recombinant SFV and processed for immunofluorescence 5 hours after infection. Whereas 1-13PLP(C5,6,9S)-EYFP showed a diffuse staining of the oligodendroglial soma and some processes (Fig. 4A), 1-13PLP-EYFP was distributed over the entire oligodendrocyte membrane and was readily detectable in MLMs (Fig. 4B). In addition, it was found on intracellular membranes in the Golgi region. Confocal microscopy analysis proved colocalization of 1-13PLP-EYFP with GM130, a marker for the Golgi apparatus (Fig. 4C). To test whether 1-13PLP-EYFP transport to oligodendroglial membrane extensions occurred by a Golgi-dependent pathway, cells were treated with the Golgi-disrupting agent, brefeldin A (BFA) (Fig. 4D). Treatment with BFA resulted in a redistribution of 1-13PLP-EYFP from the distal membrane extensions into the soma and some processes, suggesting that 1-13PLP-EYFP traffics by the exocytic pathway to the plasma membrane (targeting of 1-13PLP-EYFP to MLM was reduced by 62.5% in BFA-treated cells; P<0.0001).
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Palmitoylation is a general sorting determinant for transport to myelin
To test whether palmitoylation can act as a general targeting motif for transport to MLMs, primary oligodendrocytes were transiently transfected with plasmids coding for various fluorescent fusion proteins containing different acylation sites. For transfection, a peptide that combines a non-classical nuclear localization sequence (M9) with a nucleotide-binding domain was used to improve delivery of plasmids into differentiated oligodendrocytes (Subramanian et al., 1999). The distribution of enhanced green fluorescent protein (EGFP) fused to the C-terminal 20 amino acids of H-Ras (EGFP-tH) was investigated first. This 20-residue sequence contains a combined farnesylation/palmitoylation signal, and is sufficient to target the cognate full-length protein to lipid rafts (Prior et al., 2001
; Zacharias et al., 2002
). We found that EGFP-tH was efficiently transported to MLM (Fig. 5A). In a similar way, the N-terminal 20 amino acids of neuromodulin (which contain a consensus sequence for dual palmitoylation) fused to EYFP (tN-EYFP) were highly enriched in MLMs (Fig. 5B). By contrast, EYFP lacking any additional targeting information was absent from MLMs (data not shown). As an additional control and to rule out unspecific targeting, we expressed a low density lipoprotein (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. Moreover, the extracellular LDL receptor domain had been replaced by EGFP. This fusion protein was not targeted to MLMs, demonstrating selective transport to MLM (Fig. 5C).
Palmitoylation-deficient mutants of PLP/DM20 are less efficiently targeted to myelin in myelinating co-cultures of oligodendrocytes and neurons
Having identified a role for palmitoylation in the targeting to MLMs, we next characterized the function of this targeting motif in the context of myelinating co-cultures. Co-cultures were infected with recombinant SFV and processed for immunofluorescence 8 hours after infection. The myelin sheaths and axons were identified by labelling with anti-MBP antibody and with an antibody specific for neuronal tubulin or neurofilament, respectively. We found that only 1-13PLP-EYFP but not 1-13PLP(C5,6,9S)-EYFP was efficiently delivered to myelin [localization in myelin in 65% of the cells for 1-13PLP-EYFP and 35% of the cells for 13PLP(C5,6,9S)-EYFP; P<0.05] (Fig. 6A,B). Next, we tested the targeting of the palmitoylation-deficient mutant of DM20 to myelin. Immunofluorescence microscopy revealed that DM20(C5,6,9,108S)-myc appeared to be more restricted to the soma and was less efficiently targeted to myelin than wild-type DM20 [localization in myelin in 79% cells for DM20-myc; in 50% cells for DM20(C5,6,9,108S)-myc; P<0.05] (Fig. 6C,D). These results strengthen our conclusion that palmitoylation is a sorting determinant to myelin.
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Discussion |
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Our data are supported by a previous in vivo study that revealed targeting of ß-galactosidase to the myelin membrane of transgenic animals expressing a PLP-LacZ fusion gene that contained the portion of the PLP sequence coding for the first 13 amino acids of PLP (Wight et al., 1993). By contrast, transgenic mice producing GFP under the control of the same PLP promoter but lacking the sequence coding for the N-terminal 13 amino acids of PLP produced GFP in the cell body of oligodendrocytes (Fuss et al., 2000
). Taken together with our findings that define the palmitoylated cysteines as the crucial sorting determinants of these 13 amino acids, these data clearly indicate that palmitoylation acts as a sorting signal to myelin that probably also operates in vivo. It is interesting to compare our findings using genetic tools with a previous report in which palmitoylation of PLP in brain slices was partially inhibited by various drugs (DeJesus and Bizzozero, 2002
). Here, a reduction of PLP palmitoylation by less then 50% was probably too low to have a significant effect on PLP targeting. It is possible that PLP contains additional sorting determinants and palmitoylation acts together with these signals to enhance the fidelity of sorting to myelin. We have previously shown that PLP tightly associates with the major myelin lipids galactosylceramide and cholesterol, and that this step might be crucial for sorting of PLP (Simons et al., 2000
). Association of transmembrane proteins with lipids also involves direct protein-lipid interactions, as has been shown for HA, in which hydrophobic amino acid residues in contact with the exoplasmatic leaflet of the membrane play a crucial role in mediating protein interaction with an ordered lipid environment (Scheiffele et al., 1997
). It is therefore conceivable that association of PLP with the myelin membrane is also governed by direct interaction of the transmembrane domains of PLP with myelin lipids. Palmitoylation might be important to stabilize these protein-lipid interactions. The preferential association of palmitate, a 16-carbon saturated acyl chain, with ordered lipid environments might be the molecular basis for the affinity to the myelin membrane.
It is important to realize, however, that palmitoylation is not sufficient to transport all proteins to myelin. For example, the SFV glycoprotein E2 is palmitoylated close to its transmembrane domain (Scharer et al., 1993) but is not transported to MLMs. Palmitoylation might therefore be overruled by additional sorting signals in proteins that are not destined for myelin. This is reminiscent to sorting in epithelial cells, in which, for example, N- and O-glycosylation (a sorting determinant for transport to the apical membrane) is recessive to basolateral sorting signals (Scheiffele et al., 1995
). However, we found that palmitoylation of the peripheral membrane protein 1-13PLP-WT-EYFP, which is diffusely localized through the cytoplasm, is sufficient for its targeting to the myelin membrane; this clearly shows that palmitoylation can act as the sole sorting determinant to myelin in some cases. This is supported by the sorting of palmitoylated fusion proteins of EYFP to MLMs. Given the complexity of the myelin membrane with subdomains containing different proteins (e.g. abaxonal, adaxonal and periaxonal myelin), a range of sorting determinants are likely to operate in oligodendrocytes. General post-translational modifications, such as protein palmitoylation, might be important for enhancing the affinity of proteins for the myelin membrane. However, additional molecular interactions are required for the generation of myelin in its full complexity, including its different subdomains. For example, ligation of NF-155 to the axonal contactin/Caspr complex is crucial for the formation of paranodal myelin and leads to the recruitment of NF-155 into oligodendroglial lipid rafts (Schafer et al., 2004
; Tait et al., 2000
). Myelin biogenesis must be considered to be a multistep process involving hierarchies of sorting events. The challenge for the future will be to define additional molecular interactions and to identify the underlying sorting machinery required for the formation of the myelin membrane.
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Acknowledgments |
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