Physiological regulation of kidney epithelial cell function is achieved by the regulated recycling of vesicles that carry proteins between the cytoplasm and distinct plasma membrane domains. The cellular control of this cycling process is poorly understood. This proposal aims to unravel the mechanisms that are involved in the polarized insertion of transport proteins into epithelial cell membranes. We will concentrate on intracellular pathways that are of particular importance to kidney function, i.e., post-Golgi vesicles that deliver proteins to either apical or basolateral plasma membranes, and endocytotic vesicles that are involved in the recycling of membrane proteins.
Our specific aims are: 1) to examine selected cellular pathways and mechanisms involved in the sorting and the selective insertion of apical and basolateral plasma membrane proteins. We postulate that apical and basolateral plasma membrane proteins are segregated after leaving the Golgi apparatus into discrete vesicles that interact differently with the microtubular apparatus of epithelial cells, and with different plasma membrane domains. We will examine physiologically important molecules such as proton pumps, anion exchangers, and glucose transporters under conditions that alter intracellular trafficking, including microtubule disruption, acid-base manipulations and manoeuvres that block protein export from the Golgi to the cell surface. We will ask whether membrane and secreted proteins ride in the same vesicles, and whether lipid-anchored membrane proteins are handled differently by epithelial cells. Finally, we will examine the subcellular distribution and assembly of the proton pump using specific antibodies. 2) to examine the role of GTP-binding proteins in transport vesicle function. We postulate that the apically and basolaterally-directed vesicles whose pathways will be mapped, contain or can interact with specific G-proteins and/or other proteins that are involved in vesicle function and targeting. Using available antibody and cDNA probes for specific G-proteins, we will examine the role of G-proteins in vesicle trafficking using high-resolution morphological techniques combined with biochemical assays and functional studies in intact kidney and on isolated vesicles. Much of the published work on epithelial polarity has been obtained using virally-infected cultured cells by following expression and membrane insertion of exogenous viral proteins. An important and unique aspect of the proposed studies is that they will mostly be carried out in situ using markers of endogenous proteins that are of functional importance in kidney physiology.
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