VIP21 CAVEOLIN, GLYCOSPHINGOLIPID CLUSTERS AND THE SORTING OF GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEINS IN EPITHELIAL-CELLS/

We studied the role of the association between glycosylphosphatidylino sitol (GPI)-anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1- DAF, a GPI-anchored protein that is differ entially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1-DAF and glucosyl ceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-Con A(r)) gD1-DAF was mis-sorted to both surfaces, but GlcCer was still ta rgeted to the apical surface. In both MDCK and MDCK-ConA(r) cells, gD1 -DAF became associated with TX-100-insoluble GSL clusters during trans port to the cell surface. In dramatic contrast with MDCK cells, the Fi scher rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer bas olaterally. The targeting differences for GSLs in FRT and MDCK cells c annot be accounted for by a differential ability to form clusters beca use, in spite of major differences in the GSL composition, both cell l ines assembled GSLs into TX-100-insoluble complexes with identical iso pycnic densities. Surprisingly, in FRT cells, gD1-DAF did not form clu sters with GSLs and, therefore, remained completely soluble. This clus tering defect in FRT cells correlated with the lack of expression of V IP21/caveolin, a protein localized to both the plasma membrane caveola e and the trans Golgi network. This suggests that VIP21/caveolin may h ave an important role in recruiting GPI-anchored proteins into GSL com plexes necessary for their apical sorting. However, since MDCK-ConA(r) cells expressed caveolin and clustered GPI-anchored proteins normally , yet mis-sorted them, our results also indicate that clustering and c aveolin are not sufficient for apical targeting, and that additional f actors are required for the accurate apical sorting of GPI-anchored pr oteins.