The plasma membranes of polarized epithelial cells are divided into two domains. The protein compositions of these domains are different, reflecting their physiologic functions. This compositional heterogeneity underlies the epithelial capacity to mediate vectorial solute transport. In order to catalyze transcellular fluxes against steep concentration gradients, epithelia must populate their apical and basolateral surfaces with distinct classes of transport proteins. The ability to spatially segregate transport activities is required for an enormous variety of homeostatic functions, an its impairment is implicated in numerous disease processes. The research described in this proposal will examine the mechanisms through which transport proteins are sorted to their appropriate domains. The studies outlined in this proposal are designed to identify epithelial sorting signals and to investigate their interaction with components of the sorting apparatus. Our strategy will take advantage of a family of ion transport proteins whose closely related members are localized in distinct sub-cellular compartments. The Na,K-ATPase and the H,K-ATPase belong to the E1-E2 class of ion pumps. The protein subunits which comprise these enzymes are highly homologous, yet the pump complexes are concentrated on different surfaces of epithelial plasma membranes and manifest different cell biologic properties. The two ATPases also differ in a number of interesting catalytic parameters. Both of these pumps subserve vital cellular and organismic functions, and both are involved in a number of clinically relevant conditions. Previous work in our laboratory has demonstrated that sorting mechanisms and structure-function relationships can be elucidated by studying the behavior of novel chimeric transport proteins. We will continue with this approach in order to: 1) identify narrowly defined domains of the H,K and Na,K-ATPase alpha- subunits which determine their parent pumps' subcellular distributions and enzymatic functions; and 2) identify narrowly defined sequence domains of the H,K and Na,K-Atpase beta-subunits which are important for the maturation, sorting and recycling of their parent pumps.Transporter chimeras will be expressed in polarized LLC-PK1 cells and their steady state distributions, dynamic properties and functional characteristics will be established. In this manner, it will be possible to identify sequence domains which determine the parent transport proteins' cell biologic and physiologic traits. Identification of these domains will provide insight into the mechanisms through which these proteins carry out their transport functions and will provide tools with which to probe the cellular machinery that governs their distribution and regulation.
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