ISOLATION OF CELLS DEFECTIVE IN SORTING GLYCOPROTEINS

所有真核细胞的一个中心特征是能够 维持和控制复杂的蛋白质流量 细胞器。这种控制的机制是 所取得的成就目前尚不清楚。本研究的目标是提出 在这方面的应用是为了确定分子机制 负责几件与生活息息相关的分类事件 一间牢房。为了实现这一点，实验被设计成 鉴定三个温度条件突变的细胞 不同的分类事件： (1)内吞作用的内化阶段 营养素和激素的受体选自另一个 膜上的蛋白质，用于结合到被涂覆的坑中； (2)胞吐过程中质膜糖蛋白的分选 在肾上皮细胞中，定义和维持的事件 这些细胞的表面极性是一个基本的 对其功能的要求； (3)细胞吞噬后细胞表面受体的分选 肾上皮细胞，用于将这些受体循环到 浆膜表面，并因此维持蛋白质的组成 那个膜域。 一旦鉴定出具有所需表型的细胞， 突变株将接受互补分析， 免疫细胞化学和生化分析对突变体进行分组 常见缺陷。有趣的突变体的复制体将被分离 与之互补的野生型DNA转染后。穿过 系列DNA转移技术，能够逆转的基因 将隔离每个分类事件中的突变，并且 基因产品的特征。

A central feature of all eukaryotic cells is the ability to maintain and control a complex traffic of proteins moving between cellular organelles. The mechanisms by which this control is achieved are currently unknown. The goal of the research proposed in this application is to identify the molecular mechanisms responsible for several of the sorting events integral to the life of a cell. To accomplish this, experiments have been designed to identify cells with temperature-conditional mutations in three different sorting events: (1) the internalization phase of endocytosis' the event by which receptors for nutrients and hormones are selected from other proteins of the membrane for incorporation into coated pits; (2) the sorting of plasma membrane glycoproteins during exocytosis in kidney epithelial cells, the event that defines and maintains the surface polarity of these cells that is a fundamental requirement for their function; (3) the sorting of cell surface receptors after endocytosis in kidney epithelial cells that serves to recycle those receptors to the serosal surface, and thus maintains the protein composition of that membrane domain. Once cells with the desired phenotype have been identified, the mutants will be subjected to complementation analysis, immunocytochemical and biochemical assays to group mutants with common defects. Revertants of interesting mutants will be isolated after transfection with complementing, wild-type DNA. Through serial DNA transfer techniques, genes capable of reverting mutations in each of the sorting events will be isolated and the gene products characterized.