An essential but insufficient step for apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) in epithelial cells is their association with detergent-resistant microdomains (DRMs) or rafts. In this paper, we show that in MDCK cells both apical and basolateral GPI-APs associate with DRMs during their biosynthesis. However, only apical and not basolateral GPI-APs are able to oligomerize into high molecular weight complexes. Protein oligomerization begins in the medial Golgi, concomitantly with DRM association, and is dependent on protein–protein interactions. Impairment of oligomerization leads to protein missorting. We propose that oligomerization stabilizes GPI-APs into rafts and that this additional step is required for apical sorting of GPI-APs. Two alternative apical sorting models are presented.
To understand the mechanism involved in the apical sorting of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) we fused to the C-terminus of GFP the GPI-anchor-attachment signal of the folate receptor (FR) or of the prion protein (PrP), two native GPI-anchored proteins that are sorted apically or basolaterally, respectively, in MDCK cells. We investigated the behaviour of the resulting fusion proteins GFP-FR and GFP-PrP by analysing three parameters: their association with DRMs, their oligomerisation and their apical sorting. Strikingly, we found that different GPI-attachment signals differently modulate the ability of the resulting GFP-fusion protein to oligomerise and to be apically sorted. This is probably owing to differences in the GPI anchor and/or in the surrounding lipid microenvironment. Accordingly, we show that addition of cholesterol to the cells is necessary and sufficient to drive the oligomerisation and consequent apical sorting of GFP-PrP, which under control conditions does not oligomerise and is basolaterally sorted.
Here, we combined classical biochemistry with novel biophysical approaches to study with high spatial and temporal resolution the organization of GPI-anchored proteins (GPI-APs) at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, following sorting in the Golgi, each GPI-AP reaches the apical surface in homo-clusters. Golgi-derived homo-clusters are required for their subsequent plasma membrane organization into cholesterol-dependent hetero-clusters. By contrast, in non-polarized MDCK cells GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form hetero-clusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, different from fibroblasts, in polarized epithelial cells a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and the function of GPI-APs at the apical surface.
Protein apical sorting in polarized epithelial cells is mediated by two different mechanisms, raft dependent and raft independent. In Madin-Darby canine kidney (MDCK) cells, an essential step for apical sorting of glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs) is their coalescence into high-molecular-weight (HMW) oligomers. Here we show that this mechanism is also functional in Fischer rat thyroid cells, which possess a different sorting phenotype compared with MDCK cells. We demonstrate that, as in MDCK cells, both apical and basolateral GPI-APs associate with detergent-resistant microdomains, but that only the apical proteins are able to oligomerize into HMW complexes during their passage through the medial Golgi. We also show that oligomerization is a specific requirement for apical sorting of GPI-APs and is not used by transmembrane, non-raft-associated apical proteins.Key words: DRMs, epithelial cells, GPI-anchored proteins, oligomerization, rafts, sorting The plasma membrane of epithelial cells is divided by tight junctions into two domains, apical and basolateral, which have different protein and lipid compositions that are necessary for their proper specialized functions (1,2). The generation and maintenance of distinct apical and basolateral identities is achieved largely by the continuous sorting of newly synthesized and recycling components to one or other domain of the cell surface (3-5). Hence, sorting determinants in the cargo molecules and molecular machinery responsible for interpreting targeting signals are required.Basolateral sorting signals have been identified as short amino acid sequences, often containing tyrosine or dileucinebased motifs and confined to the cytosolic tails of membrane proteins, which are recognized by the clathrin adaptor complex (6,7). In contrast, apical signals are more variable. Lumen-localized domains, transmembrane (TM) domains and membrane-binding features have all been shown to be important for apical sorting (8-12). Furthermore, different from basolateral sorting, apical recognition is not only based on protein-protein interactions but on lipid-lipid and lipid-protein interactions also. In particular, it has been postulated that sphingolipid-and cholesterol-rich microdomains (rafts) can act as sorting platforms for inclusion of proteins into apical post-trans Golgi network (TGN) sorting vesicles (13) because of their capacity to segregate specific classes of lipids and proteins (14,15). The best example of raft-mediated apical sorting is represented by glycosyl-phosphatidylinositol (GPI)-anchored proteins (GPI-APs), which associate into detergent-resistant microdomains (DRMs) during their passage through the Golgi apparatus (13,16), where sorting is believed to occur (17,18). It was, therefore, proposed that the GPI anchor itself acts as an apical sorting determinant by mediating raft association (13,14). However, the roles of the GPI anchor and of lipid rafts as apical determinants have been recently questioned by the finding that both apically ...
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