TOPOGENESIS OF NA+,K+- ATPASE IN POLARIZED MDCK EPITHELI

肾和肠的转运上皮细胞高度分化， 作为选择性渗透屏障的专用单分子层 外部介质和循环系统之间的联系。 这个过程 取决于内部高度的结构和功能极性， 细胞，这反映在酶的不对称分布， 顶和基底外侧域之间的运输活动， 质膜 这些细胞功能中的一种必需酶是 Na+，K+-ATP酶。 Na ~+-K ~+-ATP酶定位于基底侧血浆 膜，从那里它产生显著的Na+跨细胞通量 代谢物的主动摄取、水的运输和 细胞体积的控制。 Na ~+-K ~+-ATP酶的生物学意义 反映在酶活性水平的改变 在细胞和生物体水平上产生深远的影响， 患有高血压、肾小管转运缺陷和尿毒症。 的 这里提出的研究试图阐明所涉及的调节机制 在活性α β-单元复合物的组装中，以及 参与建立和维持这种两极化的分布， 培养中分化上皮细胞层中的重要蛋白质。 实验计划定量分析翻译后 新生物聚集和周转时空规律 合成的亚基及其在质膜上的出现。 实验也计划选择性地干扰细胞内的 运输的亚单位，以确定组装的亚细胞位点， 阿尔法贝塔复合体 参与建立的分子机制 维持Na ~+，K ~+-ATP酶的极化分布， 在酶从非极化到极化的转变过程中， 极化分布，并通过观察天然Na+，K+-ATP酶的命运 直接植入极化上皮细胞的顶端质膜 细胞 此外，实验计划确定蛋白质结合 特异性地结合到Na+，K+-ATP酶的细胞质结构域， 的功能，以维持蛋白质的极化分布， cell. 总之，这项研究的结果将提供，为第一， 时间，全面了解所涉及的监管机制 在Na+，K+-ATP酶的组装和拓扑发生中，从而提供 也是对非病毒多亚基复合物组装的新见解， 在质膜上形成不同的蛋白质结构域。

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.