Oligomerization Is a Specific Requirement for Apical Sorting of Glycosyl‐Phosphatidylinositol‐Anchored Proteins but Not for Non‐Raft‐Associated Apical Proteins

Abstract

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 ...

Characterization of the Dimerization Interface of Membrane Type 4 (MT4)-Matrix Metalloproteinase

“…6D). Collectively, these observations indicate that the GPI-anchored MT-MMPs share a similar ability to generate disulfidelinked homodimers, a structural conformation that may regulate proteolysis and subcellular distribution (35). However, the emerging picture from multiple observations suggests that although MT6-MMP is mostly displayed as a dimer (6), surface MT4-MMP is found as a heterogeneous population of monomeric, dimeric, and oligomeric forms (Fig.…”

“…At present, the factors regulating the dynamics and extent of MT4-MMP dimerization/oligomerization remain unknown. It will be interesting to determine whether monomeric and dimeric forms of MT4-MMP display a different substrate profile and whether fluctuations in the monomer/dimer ratio will alter the subcellular distribution (basal versus apical) of MT4-MMP, as reported with other GPIanchored proteins (35)(36)(37).…”

Order of lipid phases in model and plasma membranes

“…A tenet of the model is that small, dynamic cholesterol-sphingolipid-enriched assemblies can be induced to coalesce into larger, more stable structures through clustering of domain components (2). Although experimental data support cholesterol-dependent nano-scale membrane heterogeneity (3)(4)(5)(6)(7)(8) and selective domain formation upon raft cross-linking (9)(10)(11)(12), the mechanisms that govern such associations in cell membranes remain unclear.…”

The Role of Glycans in Apical Sorting of Proteins in Polarized Epithelial Cells

“…Excess subunits often expose their more hydrophobic interaction surfaces and are thus recognized in the same way as misfolded proteins. A functional relationship between different classes of post-translational events, like formation of oligomers and glycosylation has been reported to influence apical sorting of some proteins (12)(13)(14), and will be discussed later.…”

“…However, a GPI anchor as such is not sufficient to drive the protein exclusively in the apical direction. This has been shown in many cases to require the presence of N-glycans (12,71,70) and also oligomerization of the apically sorted protein (71,13). An additional level of complexity was added, when it also was shown that the structure of the GPI-anchor itself influences whether the GPI-linked protein is transported apically or basolaterally (85,86).…”