Cells of transporting epithelia in the kidney and intestine form highly specialized monolayers which function as selective permeability barriers between the external medium and the circulatory system. This process depends upon a high degree of structural and functional polarity within the cells, which is reflected in the asymmetric distribution of enzymes and transport activities between the apical and basolateral domains of the plasma membrane. An essential enzyme in the function of these cells is the Na+,K+-ATPase. The Na+K+-ATPase is localized to the basolateral plasma membrane from where it generates significant transcellular fluxes of Na+ required for the active uptake of metabolites, the transport of water and the control of cell volume. The biological importance of the Na+K+-ATPase is reflected in the fact that altered levels of enzyme activity have profound effects at the cellular and organismal level, and are associated with hypertension, renal tubular transport defects, and uremia. The studies proposed here seek to elucidate the regulatory mechanisms involved in the assembly of the active AlphaBeta-unit complex, and the mechanism(s) involved in establishing and maintaining the polarized distribution of this important protein in differentiated epithelial cell layers in culture. Experiments are planned to analyze quantitatively the post-translational spatial and temporal regulation of assembly and turnover of newly synthesized subunits and their appearance at the plasma membrane. Experiments are planned also to selectively perturb the intracellular transport of the subunits to determine the subcellular site of assembly of the AlphaBeta-complex. The molecular mechanisms involved in establishing and maintaining the polarized distribution of the Na+,K+-ATPase will be investigated during transition of the enzyme from a nonpolarized to a polarized distribution, and by observing the fate of native Na+,K+-ATPase implanted directly into the apical plasma membrane of polarized epithelial cells. In addition, experiments are planned to identify proteins that bind specifically to the cytoplasmic domain of the Na+,K+-ATPase which may function to maintain the polarized distribution of the protein in the cell. Together, the results of this study will provide, for the first time, a comprehensive understanding of the regulatory mechanisms involved in the assembly and topogenesis of the Na+,K+-ATPase and, thereby, provide also a new insight into the assembly of nonviral multisubunit complexes and the formation of distinct protein domains on the plasma membrane.
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