The central aspect of epithelial cells is their polarized structure, characterized by two distinct domains of the plasma membrane, the apical and the basolateral membrane. Apical protein sorting requires various signals and different intracellular routes to the cell surface. The first apical targeting motif identified is the membrane anchoring of a polypeptide by glycosyl-phosphatidyl-inositol (GPI). A second group of apical signals involves N- and O-glycans, which are exposed to the luminal side of the sorting organelle. Sucrase-isomaltase (SI) and lactase-phlorizin hydrolase (LPH), which use separate transport platforms for trafficking, are two model proteins for the study of apical protein sorting. In contrast to LPH, SI associates with sphingolipid/cholesterol-enriched membrane microdomains or "lipid rafts". After exit form the trans-Golgi network (TGN), the two proteins travel in distinct vesicle populations, SAVs (SI-associated vesicles) and LAVs (LPH-associated vesicles) . Here, we report the identification of the lectin galectin-3 delivering non-raft-dependent glycoproteins in the lumen of LAVs in a carbohydrate-dependent manner. Depletion of galectin-3 from MDCK cells results in missorting of non-raft-dependent apical membrane proteins to the basolateral cell pole. This suggests a direct role of galectin-3 in apical sorting as a sorting receptor.

A key aspect in the structure of epithelial cells is the maintenance of a polarized organization based on a highly specific sorting machinery for cargo destined for the apical or the basolateral membrane domain at the exit site of the trans-Golgi network. We could recently identify two distinct post-trans-Golgi network vesicle populations that travel along separate routes to the plasma membrane, a lipid raft-dependent and a lipid raft-independent pathway. A new component of raftcarrying apical vesicles is ␣-kinase 1 (ALPK1), which was identified in immunoisolated vesicles carrying raftassociated sucrase-isomaltase (SI). This kinase was absent from vesicles carrying raft-non-associated lactasephlorizin hydrolase. The expression of ALPK1 increases by the time of epithelial cell differentiation, whereas the intracellular localization of ALPK1 on apical transport vesicles was confirmed by confocal analysis. A phosphorylation assay on isolated SI-carrying vesicles revealed the phosphorylation of a protein band of about 105 kDa, which could be identified as the motor protein myosin I. Finally, a specific reduction of ALPK1-expression by RNA interference results in a significant decrease in the apical delivery of SI. Taken together, our data suggest that the phosphorylation of myosin I by ALPK1 is an essential process in the apical trafficking of raft-associated SI.Epithelial cell polarity is the result of a domain-specific protein sorting process. On the cellular and molecular level, it is manifested by differences in the protein and lipid content of apical or basolateral membrane domains and a polarized organization of the cytoskeleton (for a review, see Ref. 1). These phenomena are closely connected by a sorting machinery as a central element that is responsible for the specific sorting and a directed transport of protein and lipid components to the apical or basolateral membrane compartment of epithelial cells. This machinery operates at the level of the TGN 1 by the discrimination of apical determinants from basolateral sorting signals, which are located in the cytosolic domains of transmembrane proteins (2-5). The formation of vesicles targeted to the basolateral membrane is catalyzed by adaptor coat proteins (6, 7), whereas the components required for the formation of apical transport carriers are less well defined. One characteristic feature of a wide variety of apical trans-membrane proteins is their association with sphingolipid-and cholesterolrich lipid rafts, which serve as platforms for apical targeting (8). Raft association in the cell is mediated by glycosylphosphatidylinositol anchoring of polypeptides (9), determinants present in trans-membrane domains (10), or carbohydrate chains in close proximity to the membrane (11, 12). Glycosylphosphatidylinositol-anchored proteins associate in the TGN with lipid rafts, and recent data suggest that these platforms are first delivered to the basolateral membrane followed by transcytosis to the apical cell surface (13). This indirect transport pathway of epithelial...

Epithelial polarity is based on intracellular sorting machinery that maintains the asymmetric distribution of lipids and proteins to the cell surface. Dependent on their lipid raft affinity, newly synthesized apical polypeptides are segregated into distinct vesicle populations subsequent to the passage through the Golgi apparatus. Using a combined fluorescence microscopic and biochemical approach, we found that lipid raft-associated sucrase-isomaltase (SI) as well as non-raft-associated lactase-phlorizin hydrolase (LPH) traverse endosomal compartments before entering the apical membrane. Fluorescent fusion proteins of both hydrolases were costained with Rab4-, Rab8-and Rab11-positive endosomes in polarized Madin-Darby canine kidney and non-polarized COS-1 cells. Immunoisolation of post-Golgi vesicles subsequent to different times of TGN release revealed that LPH and SI navigate in chronological order through Rab4-, Rab8-and Rab11-positive endosomes. Thereafter, the two hydrolases are segregated into distinct vesicle populations. In addition, apical membrane traffic could be significantly inhibited by RNA interference-mediated depletion of these guanosine triphosphatases. These results suggest that in epithelial cells, lipid raft-dependent and -independent apical cargo follow a transendosomal route.