Characterization of GFP-MAL expression and incorporation in rafts

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 n ovel myelin membrane component expressed by oligodendrocytes and Schwann ce lls. Since MAL has been shown to be important in the apical sorting machine ry of polarized cells, we have started to investigate the possible function al role of MAL in sorting myelin membrane-associated molecules. In this stu dy, 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 fusio n construct was not properly transported within the cell. Furthermore, we c ould 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 th e Oli-neu oligodendrocyte cell line showed that GFP-MAL was expressed, but only in cells, which were stimulated for differentiation with cAMP. In summ ary, the results confirm that the fusion protein GFP-MAL is incorporated in to detergent-insoluble complexes and the 8-kb MAL promotor fragment is suff icient to be activated in oligodendrocytes. (C) 2001 Wiley-Liss, Inc.