GLYCOSPHINGOLIPID CLUSTERS AND THE SORTING OF GPI-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 sorted differentially by three epithelial cell lines. Differently from MDCK c ells, where both gD1-DAF and glucosylceramide (GlcCer) are sorted to t he apical membrane, in MDCK Concanavalin A-resistant cells (MDCK-ConA( r)) gD1-DAF was mis-sorted to both surfaces but GlcCer was still targe ted to the apical surface. In both MDCK and MDCK-ConA(r) cells, gD1-DA F became associated with TX-100 insoluble GSL clusters during transpor t to the cell surface. In contrast to MDCK cells, the Fischer rat thyr oid (FRT) cell line targeted both gD1-DAF and GlcCer basolaterally. Bo th MDCK and FRT cells had the ability to assemble GSLs into TX-100-ins oluble complexes, but, surprisingly, in FRT cells, gD1-DAF did not ass ociate with GSLs and,therefore, remained completely soluble in TX100. This clustering defect in FRT cells correlated with the absence of VIP 21/caveolin, a protein localized to both the plasma membrane caveolae and the TGN. This suggests that VIP21/caveolin may have an important r ole in recruiting GPI-anchored proteins into GSL complexes,necessary f or their apical sorting. However, since MDCK-ConA(r) cells expressed c aveolin and clustered GPI-anchored proteins normally, yet mis-sorted t hem, our results also indicate that clustering and caveolin are not su fficient for apical targeting and that additional factors are required for the accurate apical sorting of GPI-anchored proteins.