The biological functions of epithelial cells are intimately linked to their ability to form distinct apical and basolateral plasma membrane domains. To a large extent, the existence of these distinct domains reflects the selective sorting of membrane components on both the secretory and endocytic pathways. Polarized sorting is often controlled by discrete targeting signals often found in a protein's cytoplasmic domain. Recently, others and we have begun to characterize the machinery responsible for decoding these signals. During the previous grant period, we identified an epithelial cell-specific clathrin adapter complex, AP-1B, which plays a pivotal role in polarized protein sorting and targeting. How the AP-1B complexes actually accomplishes this function, however, remains poorly understood. Furthermore, it is clear that this single adapter complex is but one element of the complex machinery responsible for facilitating the generation and maintenance of plasma membrane polarity. We plan to build upon our recent findings not only to better understand the features of AP-1B, but also to elucidate other mechanisms responsible for polarized sorting and epithelial cell morphogenesis, both fundamental problems in cell biology and pathology. The approach will remain interdisciplinary, making use of cell biological, biochemical, and genetic approaches.
Our Specific Aims i nclude: 1) to establish the site or sites at which AP-1B mediates polarized sorting and to define the pathway taken by AP-1B-dependent cargo to the basolateral plasma membrane. Of interest will be to evaluate the suggestion that sorting on both the endocytic and secretory pathways may occur at a common site. 2) To identify and characterize the functionally important components involved in AP-1B-dependent basolateral transport. For example, subunits of the mammalian exocyst complex and the Rab8 GTPase may he directly or indirectly recruited by AP-1B. 3) To reconstitute the assembly and function of AP-1B-containing clathrin-coated vesicles in vitro. Using isolated Golgi-endosome membranes, we will apply strategies developed for the analysis of COPI and COPII vesicles to the analysis of AP-1B vesicles. 4) To elucidate the role of PDZ domain proteins in intracellular sorting. While PDZ proteins often create scaffolds at the plasma membrane, our recent results demonstrate that they also control sorting on the secretory pathway. 5) To identify and characterize novel molecules that function during polarized sorting. Cell biological approaches will be applied together with mutant screens and expression analysis in Drosophila.
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