Vesicular traffic in polarized epithelial cells : identification and characterization of general and epithelial specific factors

Polarized epithelial cells are differentiated into apical and basolateral plasma membrane domains separated by tight junctions. The apical cell surface usually faces the external milieu, the basolateral domain provides cell-cell and cell-substratum contact and is surrounded by body fluids. In Madin-Darby canine kidney (MDCK) cells the unique protein and lipid composition of the two surface domains is generated by sorting in the trans-Golgi network (TGN) and selective delivery to the cell surface by a vesicular carrier mechanism. Sorting to the apical plasma membrane has been proposed to be mediated by a co-clustering of proteins and glycosphingolipids in the TGN. In order to identify the molecular machinery involved in sorting and transport of apically destined cargo I have characterized glycolipid-enriched, detergent-insoluble complexes from MDCK cells. Several proteins of the complexes were found to be components of immunoisolated apical and basolateral exocytic carrier vesicles thus representing prime candidates for the sorting machinery. By using preparative two-dimensional (2-D) gel electrophoresis and the information of 2-D gel databases I was able to identify and to purify several of these proteins for peptide microsequencing. Subsequently, I isolated the cDNAs encoding VIP36 (VesicularIntegral Membrane Protein of 36 kDa) and annexin XIIIb. VIP36 is a glycolipid raft component present in apical and basolateral vesicular carriers. The protein had a significant sequence similarity to leguminous plant lectins. VIP36 was shown to be an integral membrane protein localized to the Golgi apparatus and the cell surface, presumably recycling between them. The protein might bind to sugar residues of glycoproteins, glycolipids or glycosylphosphatidyl inositol-anchored proteins and provide a link between the luminal face of glycolipid rafts and the cytosolic vesiculation machinery. A mammalian homologue of VIP36 is localized to the early secretory pathway. This suggests that a new family of hitherto unknown animal lectins may be involved in the sorting of saccharide bearing molecules throughout the biosynthetic pathway. Annexin XIIIb is a component enriched in apical exocytic carrier vesicles and is a new member of an epithelial specific sub-family of annexins. Annexin XIIIb was localized to the apical cell surface and vesicular structures in MDCK cells. Annexins are implicated in membrane-membrane interactions and annexin XIIIb may be involved in delivery to the apical cell surface in MDCK cells. With the identification of these new, general and epithelial specific factors it will be possible to gain access to additional components of the epithelial sorting and targeting machinery to elucidate the molecular mechanisms responsible for protein and lipid sorting and vesicular transport.