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.

Cytotoxic T cells recognize viral proteins as peptide fragments which are produced in the cytosol and transported on major histocompatibility complex (MHC) class I proteins to the cell surface. Viral peptides that meet the stringent binding characteristics of class I proteins are generated by the 20S proteasome. The interferon (IFN)-gamma-inducible activator of the 20S proteasome, PA28, strongly influences the proteasomal cleavage pattern in vitro. This led us to investigate whether changes in cellular levels of PA28 affect the efficiency of viral antigen processing. A mouse fibroblast line expressing the murine cytomegalovirus pp89 protein was transfected with either the human or murine gene encoding the PA28alpha subunit, which is sufficient to activate the peptide-hydrolysing activity of the 20S proteasome in vitro. Here we report that enhanced expression of PA28alpha at a level similar to that obtained after IFN-gamma induction resulted in a marked enhancement of recognition by pp89-specific cytotoxic T cells; the presentation of influenza nucleoprotein was also significantly improved. These results demonstrate a fundamental in vivo function for PA28alpha in antigen processing.

The epithelial-specific adaptor AP1B sorts basolateral plasma membrane (PM) proteins in both biosynthetic and recycling routes, but the site where it carries out this function remains incompletely defined. Here, we have investigated this topic in Fischer rat thyroid (FRT) epithelial cells using an antibody against the medium subunit 1〉. This antibody was suitable for immunofluorescence and blocked the function of AP1B in these cells. The antibody blocked the basolateral recycling of two basolateral PM markers, Transferrin receptor (TfR) and LDL receptor (LDLR), in a perinuclear compartment with marker and functional characteristics of recycling endosomes (RE). Live imaging experiments demonstrated that in the presence of the antibody two newly synthesized GFP-tagged basolateral proteins (vesicular stomatitis virus G [VSVG] protein and TfR) exited the trans-Golgi network (TGN) normally but became blocked at the RE within 3-5 min.By contrast, the antibody did not block trafficking of green fluorescent protein (GFP)-LDLR from the TGN to the PM but stopped its recycling after internalization into RE in ϳ45 min. Our experiments conclusively demonstrate that 1) AP1B functions exclusively at RE; 2) TGN-to-RE transport is very fast and selective and is mediated by adaptors different from AP1B; and 3) the TGN and AP1B-containing RE cooperate in biosynthetic basolateral sorting. INTRODUCTIONEpithelial cells display an asymmetric distribution of plasma membrane (PM) proteins into apical and basolateral PM domains (Yeaman et al., 1999;Mostov et al., 2003; RodriguezBoulan et al., 2005). Early studies demonstrated that this polarity was achieved by sorting of newly synthesized proteins at the level of the trans-Golgi network (TGN; Rindler et al., 1984;Fuller et al., 1985;Griffiths and Simons, 1986) and of endocytosed proteins at the level of recycling endosomes (RE;Mostov and Cardone, 1995;Odorizzi and Trowbridge, 1997). Trafficking in both biosynthetic and recycling routes is controlled by apical sorting signals (e.g., N-and O-glycans, lipid anchors, and protein domains with affinity for lipid rafts), by microtubule motor determinants (Rodriguez-Boulan and Gonzalez, 1999;Schuck and Simons, 2004;Rodriguez-Boulan et al., 2005) and by basolateral sorting signals (e.g., tyrosine, dileucine, and monoleucine motifs, some similar to endocytic determinants, and noncanonic motifs not yet matching any consensus sequence; Rodriguez-Boulan et al., 2005). Tyrosine-based signals are recognized by a family of organelle-specific tetrameric AP (adaptor protein) adaptors via their subunit, whereas dileucine motifs may be recognized via large (␥ and ␦) and small ( 1, 3) subunits of AP adaptors acting together (Bonifacino and Lippincott-Schwartz, 2003;Janvier et al., 2003).The early paradigm of two separate sorting sites for proteins in biosynthetic or recycling pathways has, however, progressively shifted over the past decade to a different paradigm in which both TGN and RE share a sorting role in the biosynthetic route. This new paradigm emer...