The biosynthetic-secretory pathway, supplemented by recycling routes, specifies epithelial membrane polarity

In prevailing epithelial polarity models, membrane-based polarity cues (e.g., the partitioning-defective PARs) position apicobasal cellular membrane domains. Intracellular vesicular trafficking expands these domains by sorting apicobasal cargo towards them. How the polarity cues are polarized and how sorting confers long-range vesicle directionality is still unclear. Here, a systems-based approach using two-tiered C. elegans genomics-genetics screens identifies trafficking molecules that are not implicated in apical sorting yet polarize apical membrane and PAR complex components. Live tracking of polarized membrane biogenesis suggests that the biosynthetic-secretory pathway, linked to recycling routes, is asymmetrically oriented towards the apical domain during its biosynthesis, upstream of PARs and independent of polarized target domains. This mode of membrane polarization could offer solutions to questions of current models of polarity and polarized trafficking.

Calcium levels in the Golgi complex regulate clustering and apical sorting of GPI-APs in polarized epithelial cells

Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are lipid-associated luminal secretory cargoes selectively sorted to the apical surface of the epithelia where they reside and play diverse vital functions. Cholesterol-dependent clustering of GPI-APs in the Golgi is the key step driving their apical sorting and their further plasma membrane organization and activity; however, the specific machinery involved in this Golgi event is still poorly understood. In this study, we show that the formation of GPI-AP homoclusters (made of single GPI-AP species) in the Golgi relies directly on the levels of calcium within cisternae. We further demonstrate that the TGN calcium/manganese pump, SPCA1, which regulates the calcium concentration within the Golgi, and Cab45, a calcium-binding luminal Golgi resident protein, are essential for the formation of GPI-AP homoclusters in the Golgi and for their subsequent apical sorting. Down-regulation of SPCA1 or Cab45 in polarized epithelial cells impairs the oligomerization of GPI-APs in the Golgi complex and leads to their missorting to the basolateral surface. Overall, our data reveal an unexpected role for calcium in the mechanism of GPI-AP apical sorting in polarized epithelial cells and identify the molecular machinery involved in the clustering of GPI-APs in the Golgi.

Clathrin and clathrin adaptor AP-1 control apical trafficking of megalin in the biosynthetic and recycling routes

Megalin (gp330, LRP-2) is a protein structurally related to the low-density lipoprotein receptor family that displays a large luminal domain with multiligand binding properties. Megalin localizes to the apical surface of multiple epithelia, where it participates in endocytosis of a variety of ligands performing roles important for development or homeostasis. We recently described the apical recycling pathway of megalin in Madin–Darby canine kidney (MDCK) cells and found that it is a long-lived, fast recycling receptor with a recycling turnover of 15 min and a half-life of 4.8 h. Previous work implicated clathrin and clathrin adaptors in the polarized trafficking of fast recycling basolateral receptors. Hence, here we study the role of clathrin and clathrin adaptors in megalin’s apical localization and trafficking. Targeted silencing of clathrin or the γ1 subunit of clathrin adaptor AP-1 by RNA interference in MDCK cells disrupted apical localization of megalin, causing its redistribution to the basolateral membrane. In contrast, silencing of the γ2 subunit of AP-1 had no effect on megalin polarity. Trafficking assays we developed using FM4-HA-miniMegalin-GFP, a reversible conditional endoplasmic reticulum–retained chimera, revealed that clathrin and AP-1 silencing disrupted apical sorting of megalin in both biosynthetic and recycling routes. Our experiments demonstrate that clathrin and AP-1 control the sorting of an apical transmembrane protein.

Multiple motifs regulate apical sorting of p75 via a mechanism that involves dimerization and higher-order oligomerization

The sorting signals that direct proteins to the apical surface of polarized epithelial cells are complex and can include posttranslational modifications, such as N- and O-linked glycosylation. Efficient apical sorting of the neurotrophin receptor p75 is dependent on its O-glycosylated membrane proximal stalk, but how this domain mediates targeting is unknown. Protein oligomerization or clustering has been suggested as a common step in the segregation of all apical proteins. Like many apical proteins, p75 forms dimers, and we hypothesized that formation of higher-order clusters mediated by p75 dimerization and interactions of the stalk facilitate its apical sorting. Using fluorescence fluctuation techniques (photon-counting histogram and number and brightness analyses) to study p75 oligomerization status in vivo, we found that wild-type p75–green fluorescent protein forms clusters in the trans-Golgi network (TGN) but not at the plasma membrane. Disruption of either the dimerization motif or the stalk domain impaired both clustering and polarized delivery. Manipulation of O-glycan processing or depletion of multiple galectins expressed in Madin-Darby canine kidney cells had no effect on p75 sorting, suggesting that the stalk domain functions as a structural prop to position other determinants in the lumenal domain of p75 for oligomerization. Additionally, a p75 mutant with intact dimerization and stalk motifs but with a dominant basolateral sorting determinant (Δ250 mutant) did not form oligomers, consistent with a requirement for clustering in apical sorting. Artificially enhancing dimerization restored clustering to the Δ250 mutant but was insufficient to reroute this mutant to the apical surface. Together these studies demonstrate that clustering in the TGN is required for normal biosynthetic apical sorting of p75 but is not by itself sufficient to reroute a protein to the apical surface in the presence of a strong basolateral sorting determinant. Our studies shed new light on the hierarchy of polarized sorting signals and on the mechanisms by which newly synthesized proteins are segregated in the TGN for eventual apical delivery.

Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin–Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

Apical targeting of the P2Y4 receptor is directed by hydrophobic and basic residues in the cytoplasmic tail

The P2Y4 receptor is selectively targeted to the apical membrane in polarized epithelial cell lines and has been shown to play a key role in intestinal chloride secretion. In this study, we delimit a 23 amino acid sequence within the P2Y4 receptor C-tail that directs its apical targeting. Using a mutagenesis approach, we found that four hydrophobic residues near the COOH-terminal end of the signal are necessary for apical sorting, whereas two basic residues near the NH2-terminal end of the signal are involved to a lesser extent. Interestingly, mutation of the key hydrophobic residues results in a basolateral enrichment of the receptor construct, suggesting that the apical targeting sequence may prevent insertion or disrupt stability of the receptor at the basolateral membrane. The signal is not sequence specific, as an inversion of the 23 amino acid sequence does not disrupt apical targeting. We also show that the apical targeting sequence is an autonomous signal and is capable of redistributing the normally basolateral P2Y12 receptor, suggesting that the apical signal is dominant over the basolateral signal in the main body of the P2Y12 receptor. The targeting sequence is unique to the P2Y4 receptor, and sequence alignments of the COOH-terminal tail of mammalian orthologs reveal that the hydrophobic residues in the targeting signal are highly conserved. These data define the novel apical sorting signal of the P2Y4 receptor, which may represent a common mechanism for trafficking of epithelial transmembrane proteins.