title: AP1B sorts basolateral proteins in recycling and biosynthetic routes of MDCK cells.
abstract: 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 (mu1B) in the prototype epithelial cell line Madin-Darby canine kidney (MDCK). Mu1B-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 mu1B-knocked down MDCK cells plated on filters at confluency and cultured for 4.5 d, sorted TfR correctly in the biosynthetic route but incorrectly in the recycling route. In contrast, these same cells missorted vesicular stomatitis virus G apically in the biosynthetic route. Strikingly, recently confluent MDCK cells (1-3 d) displayed AP1B-dependence in the biosynthetic route of TfR, which decreased with additional days in culture. Sucrose density gradient analysis detected AP1B predominantly in TfR-rich endosomal fractions in MDCK cells confluent for 1 and 4 d. Our results are consistent with the following model: AP1B sorts basolateral proteins in both biosynthetic and recycling routes of MDCK cells, as a result of its predominant functional localization in recycling endosomes, which constitute a post-Golgi station in the biosynthetic route of some plasma membrane proteins. TfR utilizes a direct route from Golgi to basolateral membrane that is established as the epithelial monolayer matures.
authors.affiliation: Margaret Dyson Vision Research Institute, Weill Medical College of Cornell University, 1300 York Avenue, New York, NY 10021, USA.
authors.forename: Diego
authors.initials: D
authors.lastname: Gravotta
authors.forename: Ami
authors.initials: A
authors.lastname: Deora
authors.forename: Emilie
authors.initials: E
authors.lastname: Perret
authors.forename: Claudia
authors.initials: C
authors.lastname: Oyanadel
authors.forename: Andrea
authors.initials: A
authors.lastname: Soza
authors.forename: Ryan
authors.initials: R
authors.lastname: Schreiner
authors.forename: Alfonso
authors.initials: A
authors.lastname: Gonzalez
authors.forename: Enrique
authors.initials: E
authors.lastname: Rodriguez-Boulan
mesh_terms: D033966:Adaptor Protein Complex beta Subunits / Q000737:chemistry / Q000502:physiology*; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008954:Models, Biological; D010455:Peptides / Q000737:chemistry; D010641:Phenotype; D021381:Protein Transport; D034741:RNA, Small Interfering / Q000378:metabolism; D011990:Receptors, Transferrin / Q000378:metabolism / Q000502:physiology; D013997:Time Factors
chemical_list: D033966:Adaptor Protein Complex beta Subunits; D010455:Peptides; D034741:RNA, Small Interfering; D011990:Receptors, Transferrin