Characterization of GFP‐MAL expression and incorporation in rafts

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Abstract
During myelin formation, membrane‐associated proteins have to be sorted and transported in specified membrane regions such as compact and non‐compact myelin membranes. One protein that may be involved in such a process is the Myelin and Lymphocyte protein MAL (VIP17/ MVP17). MAL was identified as a novel myelin membrane component expressed by oligodendrocytes and Schwann cells. Since MAL has been shown to be important in the apical sorting machinery of polarized cells, we have started to investigate the possible functional role of MAL in sorting myelin membrane‐associated molecules. In this study, we have generated cDNA constructs with green fluorescent protein (GFP) either at the N‐ or C‐terminus of MAL. Transfection experiments showed that GFP‐MAL expression resembles that of normal MAL, whereas the MAL‐GFP fusion construct was not properly transported within the cell. Furthermore, we could demonstrate that GFP‐MAL is enriched in detergent insoluble glycolipid‐enriched microdomains as already seen for untagged MAL. As a prerequisite for the generation of transgenic mice expressing GFP‐MAL under the control of its own regulatory elements, we have generated a cDNA construct with an 8‐kb MAL promotor fragment fused to GFP‐MAL. Transfection experiments of the Oli‐neu oligodendrocyte cell line showed that GFP‐MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summary, the results confirm that the fusion protein GFP‐MAL is incorporated into detergent‐insoluble complexes and the 8‐kb MAL promotor fragment is sufficient to be activated in oligodendrocytes. Microsc. Res. Tech. 52:645–655, 2001.