SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

这项建议的一个主要目标是阐明生物发生机制 其在极化的上皮细胞中起作用，以确保 特异性质膜蛋白质的管腔或基底外侧 细胞表面的结构域。 利用培养的肾脏模型系统 上皮细胞（MDCK）感染包膜病毒， 仅来自一个或另一个质膜结构域，或转染 用含有天然或修饰的 病毒包膜蛋白，我们将确定遵循的亚细胞途径 通过来自高尔基体的不同极性的病毒糖蛋白， 每个质膜结构域识别膜内的分选信号 蛋白质并表征分选装置的细胞元件。 细胞膜Na ~+，K ~+ ATP酶的生物合成和结构 酶位于上皮细胞的基底外侧表面，将 在培养的MDCK细胞和大鼠脑中进行了研究，使用生物化学，细胞 分级分离和重组DNA方法。 我们亦会研究 上皮细胞极化分泌的决定因素， 含有编码天然或修饰的内分泌的基因的表达载体 或外分泌蛋白和膜蛋白的截短片段， 通常分离为一个质膜域。 的程度 分泌、膜生物发生和内吞途径的汇合 将进行调查。 紧密连接的主要蛋白质成分 分离和表征它们的跨膜 的过程，以及它们的组合成节点的过程。 cDNA克隆 将获得并用于确定是否 不同组织的连接渗透性的差异由以下原因引起： 连接组件的组织或性质的变化。

A main goal of this proposal is an elucidation of the biogenetic mechanisms which operate in polarized epithelial cells to ensure the segregation of specific plasma membrane proteins to either the luminal or basolateral domains of the cell surface. Using model systems of cultured kidney epithelial cells (MDCK) infected with enveloped viruses which bud exclusively from one or the other plasma membrane domain, or transfected with suitable expression vectors containing genes for natural or modified viral envelope proteins, we will determine the subcellular route followed by viral glycoproteins of different polarity from the Golgi apparatus to each plasma membrane domain, identify sorting signals within the membrane proteins and characterize cellular elements of the sorting apparatus. Aspects of the biosynthesis and structure of the Na+,K+ ATPase, a cellular enzyme located in the basolateral surface of epithelial cells, will be studied in cultured MDCK cells and in rat brain, using biochemical, cell fractionation and recombinant DNA methods. We will also study the determinants of polarized secretion in epithelial cells employing expression vectors containing genes encoding natural or modified endocrine or exocrine proteins and truncated fragments of membrane proteins which are normally segregated to one plasma membrane domain. The extent of convergence of the secretory, membrane biogenetic and endocytic pathways will be investigated. The major protein components of tight junctions will be isolated and characterized with respect to their transmembrane disposition, and the process of their assembly into junctions. cDNA clones for the junction proteins(s) will be obtained and used to determine if differences in junction permeability of different tissues result from variations in the organization or nature of junctional components.