ISOLATION OF CELLS DEFECTIVE IN SORTING GLYCOPROTEINS

所有真核细胞的一个中心特征是维持和 控制着蛋白质在细胞器之间的复杂运输。 实现这种控制的机制目前尚不清楚。 本申请中提出的研究目标是识别 分子机制负责的几个排序事件的核心 细胞的生命。 为了实现这一点，设计了实验， 以在四种不同的分选事件中鉴定具有突变的细胞： （1）内吞作用的内化阶段，其中， 营养素和激素选自血浆的其它蛋白质 用于结合到涂覆的凹坑中的膜; （2）内吞作用的再循环阶段，其中 组成性内化营养素与蛋白质分离， 对于溶酶体; （3）胞吐过程中质膜糖蛋白的分选 上皮细胞; （4）肾脏内吞后细胞表面受体的分选 上皮细胞负责将这些受体再循环到血清中， 表面，从而保持该膜的蛋白质组成 域 一旦鉴定出具有所需表型的细胞， 将进行互补分析，免疫细胞化学和 生化分析对具有常见缺陷的突变体进行分组。 回复突变体 感兴趣的突变体将在用互补基因转染后分离， 野生型DNA 通过一系列DNA转移技术， 每个分选事件中的回复突变将被分离， 基因产物特征。 对于特定目标3和4，极化细胞系 表达突变流感病毒血凝素或人LDL 受体的构建，并将用于研究该途径 突变蛋白通过该通道到达基底外侧质膜。

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 central to the life of a cell. To accomplish this, experiments have been designed to identify cells with mutations in four different sorting events: (1)the internalization phase of endocytosis, in which receptors for nutrients and hormones are selected from other proteins of the plasma membrane for incorporation into coated pits; (2)the recycling phase of endocytosis, in which receptors that constitutively internalize nutrients are separated from proteins destined for lysosomes; (3)the sorting of plasma membrane glycoproteins during exocytosis in epithelial cells; (4)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. For specific aim 3 and 4, polarized cell line expressing either a mutant influenza virus hemagglutinin or the human LDL receptor have been constructed and will be used to investigate the pathway by which the mutant protein reaches the basolateral plasma membrane.