The epithelial-specific adaptor AP1B sorts basolateral proteins, but the trafficking routes where it performs its sorting role remain controversial. Here, we used an RNAi approach to knock down the medium subunit of AP1B (1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). 1B-knocked down MDCK cells displayed loss of polarity of several endogenous and exogenous basolateral markers transduced via adenovirus vectors, but exhibited normal polarity of apical markers. We chose two well characterized basolateral protein markers, the transferrin receptor (TfR) and the vesicular stomatitis virus G protein, to study the sorting role of AP1B. A surface-capture assay introduced here showed that 1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.

Clathrin-coated vesicles are vehicles for intracellular trafficking in all nucleated cells, from yeasts to humans. Many studies have demonstrated their essential roles in endocytosis and cellular signalling processes at the plasma membrane. By contrast, very few of their non-endocytic trafficking roles are known, the best characterized being the transport of hydrolases from the Golgi complex to the lysosome. Here we show that clathrin is required for polarity of the basolateral plasma membrane proteins in the epithelial cell line MDCK. Clathrin knockdown depolarized most basolateral proteins, by interfering with their biosynthetic delivery and recycling, but did not affect the polarity of apical proteins. Quantitative live imaging showed that chronic and acute clathrin knockdown selectively slowed down the exit of basolateral proteins from the Golgi complex, and promoted their mis-sorting into apical carrier vesicles. Our results demonstrate a broad requirement for clathrin in basolateral protein trafficking in epithelial cells.

Proton-coupled monocarboxylate transporters (MCT) MCT1, MCT3, and MCT4 form heterodimeric complexes with the cell surface glycoprotein CD147 and exhibit tissue-specific polarized distributions that are essential for maintaining lactate and pH homeostasis. In the parenchymal epithelia of kidney, thyroid, and liver, MCT͞CD147 heterocomplexes are localized in the basolateral membrane where they transport lactate out of or into the cell depending on metabolic conditions. A unique distribution of lactate transporters is found in the retinal pigment epithelium (RPE), which regulates lactate levels of the outer retina. In RPE, MCT1͞ CD147 is polarized to the apical membrane and MCT3͞CD147 to the basolateral membrane. The mechanisms responsible for tissuespecific polarized distribution of MCTs are unknown. Here, we demonstrate that CD147 carries sorting information for polarized targeting of the MCT1͞CD147 hetero-complexes in kidney and RPE cells. In contrast, MCT3 and MCT4 harbor dominant sorting information that cotargets CD147 to the basolateral membrane in both epithelia. RNA interference experiments show that MCT1 promotes CD147 maturation. Our results open a unique paradigm to study the molecular basis of tissue-specific polarity.apical ͉ basolateral ͉ polarized epithelium

CD147, a type I integral membrane protein of the immunoglobulin superfamily, exhibits reversed polarity in retinal pigment epithelium (RPE). CD147 is apical in RPE in contrast to its basolateral localization in extraocular epithelia. This elicited our interest in understanding the basolateral sorting signals of CD147 in prototypic Madin-Darby canine kidney (MDCK) cells. The cytoplasmic domain of CD147 has basolateral sorting information but is devoid of well-characterized basolateral signals, such as tyrosine and di-leucine motifs. Hence, we carried out systematic site-directed mutagenesis to delineate basolateral targeting information in CD147. Our detailed analysis identified a single leucine (252) as the basolateral targeting motif in the cytoplasmic tail of CD147. Four amino acids (243-246) N-terminal to leucine 252 are also critical basolateral determinants of CD147, because deletion of these amino acids leads to mistargeting of CD147 to the apical membranes. We ruled out the involvement of adaptor complex 1B (AP1B) in the basolateral trafficking of CD147, because LLC-PK1 cells lacking AP1B, target CD147 basolaterally. At variance with MDCK cells, the human RPE cell line ARPE-19 does not distinguish between CD147 (WT) and CD147 with leucine 252 mutated to alanine and targets both proteins apically. Thus, our study identifies an atypical basolateral motif of CD147, which comprises a single leucine and is not recognized by RPE cells. This unusual basolateral sorting signal will be useful in unraveling the specialized sorting machinery of RPE cells. INTRODUCTIONEpithelial cells have distinct apical and basolateral membrane domains with different protein and lipid compositions. The asymmetry is essential for the multiple vectorial functions they perform (Rodriguez-Boulan and Nelson, 1989;Yeaman et al., 1999). The polarized protein distribution results from sorting mechanisms operating in the biosynthetic and recycling pathways that recognize specific sorting signals in plasma membrane proteins. Structural features believed to operate as apical sorting signals include glycosylphophatidylinositol anchors (Lisanti et al., 1989), N-glycans (Scheiffele et al., 1995), O-glycans (Yeaman et al., 1997;Jacob et al., 2000), and protein sequences in the transmembrane (Kundu et al., 1996;Lin et al., 1998), or cytoplasmic domains (Chuang and Sung, 1998;Rodriguez-Boulan and Gonzalez, 1999;Nelson and Yeaman, 2001). On the other hand, basolateral signals are formed by short peptide sequences usually found in the protein domain facing the cytosol (Le Gall et al., 1995;Yeaman et al., 1999;Mostov et al., 2000). They mainly include tyrosine motifs (consensus motif NPXY or YXXF) as well as di-leucine and di-hydrophobic residues (Matter et al., 1992;Aroeti et al., 1993;Hunziker and Fumey, 1994;Simonsen et al., 1997;Bonifacino and Dell'Angelica, 1999;Rodionov et al., 2000).Different types of epithelial cells vary widely in the final distribution of plasma membrane proteins or in the pathways that these proteins follow to the cell surf...