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之间移动的复杂交通。 实现这一控制的机制目前尚不清楚。 本申请中提出的研究的目标是确定 几个分拣事件的主要分子机制 一个细胞的生命。为了实现这一点，已经设计了实验 要在四个不同的排序事件中标识具有突变的单元格，请执行以下操作： (1)内吞作用的内化阶段，在这个阶段，受体 营养素和激素选自血浆中的其他蛋白质 用于结合到涂覆坑中的膜； (2)内吞作用的循环阶段，在这个阶段，受体 构成内化的营养物质与注定要 对于溶酶体； (3)胞吐过程中质膜糖蛋白的分选 上皮细胞； (4)肾脏内吞作用后细胞表面受体的分选 负责将这些受体再循环到浆膜的上皮细胞 表面，从而维持膜的蛋白质组成 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 对有常见缺陷的突变体进行生化分析。翻译者们 经过基因导入和互补后，将分离出有趣的突变体， 野生型DNA。通过系列DNA转移技术，能够 将隔离每个分类事件中的还原突变，并且 基因产品的特征。针对特定目标3和4，极化细胞系 表达突变的流感病毒血凝素或人类低密度脂蛋白 受体已被构建，并将用于研究该途径 突变蛋白通过这个途径到达基侧质膜。

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.