ISOLATION OF CELLS DEFECTIVE IN SORTING GLYCOPROTEINS

所有真核细胞的一个中心特征是能够 维持和控制蛋白质在 细胞器 这种控制的机制是 目前实现的是未知的。 提出的研究目标 在这个应用中是为了确定分子机制， 负责生活中不可或缺的几个分类事件 一个细胞。 为了实现这一点，实验已经被设计成 鉴定具有三种温度条件突变的细胞， 不同的排序事件： (1)内吞作用的内化阶段， 营养素和激素的受体选自其它受体， 用于掺入被膜小凹的膜蛋白; (2)胞吐过程中质膜糖蛋白的分选 在肾脏上皮细胞中， 这些细胞的表面极性 功能要求; (3)细胞内吞后细胞表面受体的分选 肾脏上皮细胞负责回收这些受体， 蛋白质表面，从而保持蛋白质的组成 这个膜域。 一旦鉴定出具有所需表型的细胞， 突变体将接受互补分析， 免疫细胞化学和生物化学测定来分组具有 常见缺陷 感兴趣的突变体的回复突变体将被分离 在用互补的野生型DNA转染后。 通过 系列DNA转移技术，基因能够恢复 每个分选事件中的突变将被分离， 基因产物特征。

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.