Cholesterol-rich membrane domains (e.g., lipid rafts) are thought to act as molecular sorting machines, capable of coordinating the organization of signal transduction pathways within limited regions of the plasma membrane and organelles. The significance of these domains in polarized postendocytic sorting is currently not understood. We show that dimeric IgA stimulates the incorporation of its receptor into cholesterol-sensitive detergent-resistant membranes confined to the basolateral surface/basolateral endosomes. A fraction of human transferrin receptor was also found in basolateral detergent-resistant membranes. Disrupting these membrane domains by cholesterol depletion (using methyl-␤-cyclodextrin) before ligand-receptor internalization caused depolarization of traffic from endosomes, suggesting that cholesterol in basolateral lipid rafts plays a role in polarized sorting after endocytosis. In contrast, cholesterol depletion performed after ligand internalization stimulated cargo transcytosis. It also stimulated caveolin-1 phosphorylation on tyrosine 14 and the appearance of the activated protein in dimeric IgA-containing apical organelles. We propose that cholesterol depletion stimulates the coupling of transcytotic and caveolin-1 signaling pathways, consequently prompting the membranes to shuttle from endosomes to the plasma membrane. This process may represent a unique compensatory mechanism required to maintain cholesterol balance on the cell surface of polarized epithelia. INTRODUCTIONIn humans, the major antibody that mediates immunological defense against mucosal infections is dimeric IgA (dIgA). This antibody is produced by plasma cells in the lamina propria located underneath the mucosal surface (reviewed in Lamm et al., 1995;Rojas and Apodaca, 2002) and is carried from the blood to mucosal secretions by the polymeric immunoglobulin receptor (pIgR). The itinerary of pIgR and its ligand was intensively studied in the polarized MadinDarby canine kidney (MDCK) cell line that heterologously expresses rabbit pIgR (for a recent review, see Rojas and Apodaca, 2002). The receptor is initially delivered from the trans-Golgi network (TGN) to the basolateral plasma membrane, where it encounters and binds dIgA. The complex is subsequently internalized and transcytosed to the apical plasma membrane of the cell. At that surface, the ectodomain of pIgR is proteolytically cleaved off to yield the secretory component (SC), which is released together with dIgA into mucosal secretions. In the course of transcytosis, pIgRdIgA complexes traverse various endosomal compartments, among which is an endocytic compartment located immediately underneath the apical surface (Gibson et al., 1998). This apical compartment, defined as apical recycling endosomes (AREs), is enriched in dIgA and is relatively deficient in recycling transferrin (Apodaca et al., 1994;Barroso and Sztul, 1994;Brown et al., 2000). AREs are thought to play a unique role in transcytosis, possibly by exploiting apical recycling mechanisms to shuttle transcy...

The classical view suggests that adaptor proteins of the clathrin coat mediate the sorting of cargo protein passengers into clathrin-coated pits and the recruitment of clathrin into budding areas in the donor membrane. In the present study, we provide biochemical and morphological evidence that the adaptor protein 1 (AP-1) adaptor of the trans-Golgi network clathrin interacts with microtubules. AP-1 in cytosolic extracts interacted with in vitro assembled microtubules, and these interactions were inhibited by ATP depletion of the extracts or in the presence of 5-adenylylimidodiphosphate. An overexpressed ␥-subunit of the AP-1 complex associated with microtubules, suggesting that this subunit may mediate the interaction of AP-1 with the cytoskeleton. Purified AP-1 did not interact with purified microtubules, but interaction occurred when an isolated microtubule-associated protein fraction was added to the reaction mix. The ␥-adaptin subunit of AP-1 specifically co-immunoprecipitated with a microtubuleassociated protein of type 1a from rat brain cytosol. This suggests that type 1a microtubule-associated protein may mediate the association of AP-1 with microtubules in the cytoplasm. The microtubule binding activity of AP-1 was markedly inhibited in cytosol of mitotic cells. By means of its interaction with microtubule-associated proteins, we propose novel roles for AP-1 adaptors in modulating the dynamics of the cytoskeleton, the stability and shape of coated organelles, and the loading of nascent AP-1-coated vesicles onto appropriate microtubular tracks.

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