VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol‐anchored proteins in epithelial cells

We studied the role of the association between glycosylphosphatidylinositol (GPI)‐anchored proteins and glycosphingolipid (GSL) clusters in apical targeting using gD1‐DAF, a GPI‐anchored protein that is differentially sorted by three epithelial cell lines. Differently from MDCK cells, where both gD1‐DAF and glucosylceramide (GlcCer) are sorted to the apical membrane, in MDCK Concanavalin A‐resistant cells (MDCK‐ConAr) gD1‐DAF was mis‐sorted to both surfaces, but GlcCer was still targeted to the apical surface. In both MDCK and MDCK‐ConAr cells, gD1‐DAF became associated with TX‐100‐insoluble GSL clusters during transport to the cell surface. In dramatic contrast with MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1‐DAF and GlcCer basolaterally. The targeting differences for GSLs in FRT and MDCK cells cannot be accounted for by a differential ability to form clusters because, in spite of major differences in the GSL composition, both cell lines assembled GSLs into TX‐100‐insoluble complexes with identical isopycnic densities. Surprisingly, in FRT cells, gD1‐DAF did not form clusters with GSLs and, therefore, remained completely soluble. This clustering defect in FRT cells correlated with the lack of expression of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the trans Golgi network. This suggests that VIP21/caveolin may have an important role in recruiting GPI‐anchored proteins into GSL complexes necessary for their apical sorting. However, since MDCK‐ConAr cells expressed caveolin and clustered GPI‐anchored proteins normally, yet mis‐sorted them, our results also indicate that clustering and caveolin are not sufficient for apical targeting, and that additional factors are required for the accurate apical sorting of GPI‐anchored proteins.