Endocytic membrane transport is critical to many facets of renal function. Vectorial transport of ions, solutes and proteins is regulated by the polarized expression of transporters and receptors, which is determined by rates of endocytic insertion and retrieval. The plasma membrane expression of transporters is frequently regulated by signaling receptors, whose expression is likewise regulated by endocytosis. As many diseases, such as insulin-dependent diabetes, cystic fibrosis and Liddle's syndrome are associated with disruptions of endocytic membrane transport, it is clearly important to understand the molecular mechanisms regulating endocytosis. Combined molecular and cell biological studies of fibroblasts have elucidate the molecular regulation of many endocytic transport steps. However, comparable studies have not been conducted in polarized epithelial and it is clear that the studies of fibroblasts do not apply to epithelia, whose endocytic pathways are regulated by epithelial-specific proteins, and by ubiquitous proteins that acquire new functions within the more complex membrane pathways of epithelia. To better understand molecular regulations of epithelial endocytosis we propose to address the epithelial function of proteins known to participate in membrane recycling, both those common to epithelia and fibroblasts, Rab4a, Rab11a and Rme-1, as well as those specific to epithelial cells, Rab17 and Rab25. We will also identify proteins that regulate polar sorting in endosomes by characterizing how the protein constitution of endosomes is altered when polar sorting is disrupted. Endosome protein analysis will be assayed using subcellular fractionation, 2-dimensional gel electrophoresis and mass spectroscopy. The cellular effects of transfected proteins will be evaluated through the combined use of quantitative confocal microscopy of polarized cells expressing GFP chimeras of mutant and wildtype proteins and biochemical analysis of stable cell lines expressing transfected proteins. The dissection of the molecular mechanisms of endocytosis provided here will illuminate the processes that regulate membrane recycling, transcytosis, transport to the trans-Golgi network and plasma membrane polarity. In so doing, they will provide the framework for understanding diseases, and developing therapeutics for transporter disorders, immune disorders, toxin-mediated pathologies and polarity disorders, such as polycystic kidney disease.
