RECOGNITION AND SORTING OF GLYCOPROTEINS

质膜的组成和功能是由一种 复杂的细胞内运输移动细胞表面糖蛋白之间 细胞器 这就需要对不同的类进行识别和排序 不仅在生物合成过程中，而且在再分配过程中， 过程，如受体的内化和再循环， 受体介导的内吞作用，或在激素的下调过程中 受体。 任何一个环节的失败都可能导致疾病 过程，或从它的颠覆为使用的病原体，如病毒。 在本申请中描述的实验被设计为揭示分子 控制细胞内转运的细胞机制的细节 一种主要的细胞表面糖蛋白。 通过基因 工程，功能获得突变体已经产生， 进入他们以前被排除在外的途径。 这允许 对细胞内分选事件重要的蛋白质的特征被 区别于那些重要的蛋白质结构， 整体 提出了六个实验：（1）确定数量， 识别蛋白质的独立机制之间的关系 内吞过程中的被膜孔;（2）鉴定控制内吞的蛋白质 内吞途径中的分选;（3）鉴定负责的蛋白质 用于分选极化上皮细胞中的跨膜糖蛋白;（4） 为了确定跨膜结构域在细胞内 糖蛋白的运输。 先前制备的突变体将用于建立体外测定， 内部化和循环利用，以确定 这些事件的细胞成分。 表达这些的永久细胞系 突变体将用于确定哪些蛋白质竞争识别， 涂层凹坑的组成部分。 基因工程将被用来引入 逐渐变小的糖蛋白突变，以确定氨基 负责细胞内有趣变化的酸性序列 交通 特征的二级、三级和四级结构 重要的排序将进行调查。 合成肽， 运输信号将被用来识别细胞蛋白质， 与他们在实验中采用化学交联，直接亲和 层析或抗独特型抗体。

The composition and function of the plasma membrane is maintained by a complex intracellular traffic moving cell surface glycoproteins between organelles. This requires the recognition and sorting of different classes of proteins, not only during biosynthesis, but also during redistributive processes, such as the internalization and recycling of receptors during receptor-mediated endocytosis, or during the down-regulation of hormone receptors. Disease can result form the failure of any one of these processes, or from its subversion for use by a pathogen such as a virus. Experiments described in this application are designed to reveal molecular details of the cellular mechanisms that control the intracellular transport of a major class of cell surface glycoproteins. Through genetic engineering, gain-of-function mutants have been produced that are sorted into pathways from which they were previously excluded. This allows features of proteins important for intracellular sorting events to be distinguished from those important for the structure of the protein as a whole. Six experiments are proposed (1) to determine the number and relationship of independent mechanisms for recognition of proteins by coated pits during endocytosis; (2) to identify the proteins controlling sorting in the endocytic pathway; (3) to identify the proteins responsible for sorting transmembrane glycoproteins in polarized epithelial cells; (4) to determine the role of the transmembrane domain in the intracellular transport of glycoproteins. Mutants made previously will be used to establish in vitro assays for internalization and recycling for the purpose of identifying essential cellular components of these events. Permanent cell lines expressing these mutants will be used to determine which proteins compete for recognition by components of coated pits. Genetic engineering will be used to introduce progressively smaller mutations into glycoproteins to identify the amino acid sequences responsible for interesting changes in their intracellular traffic. The secondary, tertiary and quarternary structure of features important for sorting will be investigated. Synthetic peptides that mimic transport signals will be used to identify cellular proteins that interact with them in experiments employing chemical cross-linking, direct affinity chromatography, or anti-idiotype antibodies.