VIP21/caveolin, glycosphingolipid clusters and the sorting of glycosylphosphatidylinositol-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 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.

Characterization of a Cytosolic Heat-shock Protein-Caveolin Chaperone Complex

“…Recently, Stan et al (48) reported that an attempt to use caveolin IgG to enrich for caveolae failed to co-precipitate many of the proteins shown to be associated with caveolae by independent biochemical isolation methods (29, 49 -51), functional assays (3,4,12,52), and electron microscope methods (3,4,10,23,29,31,(53)(54)(55)(56)(57). Several studies (8,14,24,58,59) including this one demonstrate that caveolin is a mobile protein that can come out of membranes and leave behind resident proteins. Consequently, it is difficult to purify caveolae by immunoprecipitating caveolin.…”

Rafts in oligodendrocytes: Evidence and structure–function relationship

“…From a functional point of view, the specific association of a given molecular compound with a DRM has been correlated with sorting and, in polarized epithelial cells, with apical-directed membrane transport Ikonen and Simons, 1998;Ikonen, 2001;Simons and van Meer, 1988;Simons and WandingerNess, 1990;Zurzolo et al, 1994). As noted above and elsewhere (de Vries and Hoekstra, 2000), OLGs can also be considered to be polarized, given the remarkable differences in lipid composition of plasma and myelin membrane, the latter being particularly enriched in GSLs, saturated phospholipids and cholesterol, a composition typical of raft-like domains.…”

“…Particularly in membranes enriched in GSLs and cholesterol, such as in apical membranes, GSL/cholesterol-enriched microdomains or so called 'lipid rafts' may exist and/or arise. These microdomains have been postulated to be involved in signaling and to act as targeting devices in the direct transport of apical proteins from the trans-Golgi network (TGN) to the apical membrane (Brown and Rose, 1992;Hoekstra et al, 2003;Ikonen and Simons, 1998;Ikonen, 2001;Simons and van Meer, 1988;Simons and Wandinger-Ness, 1990;Zurzolo et al, 1994). Given the enrichment of GSLs and cholesterol in myelin, it is tempting to consider that transport and functioning of myelin-specific proteins towards and in the myelin sheath are accomplished in a similar fashion.…”

Detergent Insoluble Microdomains are not Involved in Transcytosis of Polymeric Ig Receptor in FRT and MDCK Cells

“…Sucrose gradient analysis of TX-100-insoluble material was performed using previously published protocols (40,45). Cells were grown to confluency in 100 mm dishes and labeled overnight with 100 mCi/ml of [ 35 S]-cysteine.…”