CELL BIOLOGY OF ION PUMPS--SORTING AND FUNCTION

极化上皮细胞的质膜分为两层 域名。这些结构域的蛋白质组成不同， 反映了它们的生理功能。这种成分的异质性 上皮细胞具有介导载体溶质运输的能力。 为了在浓度过高的情况下催化跨细胞的通量 梯度，上皮必须填充在其顶端和基底外侧表面 具有不同类别的运输蛋白。在空间上的能力 各种不同的运输活动都需要隔离运输活动 动态平衡功能，其功能受损与多种疾病有关 流程。本提案中描述的研究将检查 运输蛋白被分选到其各自的 适当的域。本提案中概述的研究旨在 识别上皮分选信号并研究它们之间的相互作用 具有分拣装置的部件。我们的战略将利用 属于离子转运蛋白家族，其密切相关的成员是 定位于不同的亚细胞隔间。Na，K-ATPase与 H，K-ATPase属于E_1-E_2类离子泵。蛋白质亚基 组成这些酶的分子高度同源，然而泵复合体 集中在上皮质膜的不同表面和 表现出不同的细胞生物学特性。这两种ATPase也不同 在许多有趣的催化参数中。这两台泵都是 削弱重要的细胞和组织功能，两者都参与了 一些临床上相关的疾病。我们之前的工作 实验室已经证明了分选机制和结构功能 通过研究新型嵌合体的行为可以阐明它们之间的关系 运输蛋白。我们将继续采用这种方法，以便：1) 确定H，K和Na，K-ATPaseα的狭义结构域- 决定其亲本泵的亚细胞分布的亚基 酶功能；以及2)鉴定狭义的序列结构域 H，K和Na，K-ATPaseβ亚基对 其亲本泵的成熟、分类和回收。运输机 嵌合体将在极化的LLC-PK1细胞中表达，并稳定表达 状态分布、动态特性和功能特性 将会建立起来。以这种方式，就有可能确定 决定亲本转运蛋白细胞的序列结构域 生物学和生理学特征。这些域的标识将 洞察这些蛋白质执行的机制 它们的传输功能，并将提供工具来探测 管理它们的分布和调节的细胞机制。

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.