Glycosphingolipid clusters and the sorting of GPI-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 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 TNG. 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.
Zurzolo
C
C
Dipartimento di Biologia e Patologia Cellulare e Molecolare, CEOS, CNR, II Policlinico, Napoli, Italy.
van't Hof
W
W
van Meer
G
G
Rodriguez-Boulan
E
E
D009327:4-Chloro-7-nitrobenzofurazan / Q000031:analogs & derivatives / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD / Q000378:metabolism; D018958:CD55 Antigens; D002460:Cell Line; D002518:Ceramides / Q000378:metabolism; D003208:Concanavalin A; D004848:Epithelium / Q000737:chemistry / Q000378:metabolism; D006028:Glycosphingolipids / Q000737:chemistry / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D011916:Rats, Inbred F344
D015703:Antigens, CD; D018958:CD55 Antigens; D002518:Ceramides; D006028:Glycosphingolipids; D017261:Glycosylphosphatidylinositols; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D003208:Concanavalin A; C037974:N-(7-(4-nitrobenzo-2-oxa-1,3-diazole))-6-aminocaproyl sphingosine; D009327:4-Chloro-7-nitrobenzofurazan