SYNTHESIS AND DISTRIBUTION OF PROTEINS IN MEMBRANES

这项提议的一个主要目标是阐明生物遗传机制。 它们在极化的上皮细胞中运行，以确保分离 腔内或基侧的特定质膜蛋白 细胞表面的区域。利用培养肾脏的模型系统 上皮细胞(MDCK)感染发芽的包膜病毒 仅来自一个或另一个质膜结构域，或被转染 具有合适的含有天然或修饰基因的表达载体 病毒包膜蛋白，我们将确定所遵循的亚细胞路线 由不同极性的病毒糖蛋白从高尔基体到 每个质膜结构域，识别膜内的分选信号 蛋白质，并表征分拣装置的细胞元素。 细胞Na~+，K~+-ATPase的生物合成和结构 位于上皮细胞基底外侧表面的酶，将被 在培养的MDCK细胞和大鼠脑中进行研究，使用生化细胞 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内分泌基因的表达载体 或外分泌蛋白和膜蛋白的截短片段 通常分离到一个质膜结构域。其影响范围 分泌、膜生物发生和内吞途径的融合 将会被调查。紧密连接的主要蛋白质成分是 对其跨膜进行分离和鉴定 处理，以及它们组装成连接点的过程。CDNA克隆 将获得的连接蛋白(S)用于确定是否 不同组织的连接渗透率的差异是由于 连接成分在组织或性质上的变化。

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.