Glycosphingolipid clusters and the sorting of GPI-anchored proteins in epithelial cells

ABSTRACT

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 sorted differentially 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 contrast to MDCK cells, the Fischer rat thyroid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. Both MDCK and FRT cells had the ability to assemble GSLs into TX-100-insoluble complexes, but, surprisingly, in FRT cells, gD1-DAF did not associate with GSLs and, therefore, remained completely soluble in TX100. This clustering defect in FRT cells correlated with the absence of VIP21/caveolin, a protein localized to both the plasma membrane caveolae and the TGN. 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