MOLECULAR STUDIES OF CLATHRIN-COATED MEMBRANE FUNCTION

笼状蛋白涂层膜与多种疾病有关。 真核细胞中的细胞内蛋白质运输过程。 酵母同时含有重链和轻链网状蛋白亚基和 显示与在动物中发现的类似的分泌途径 细胞。在这两种细胞类型中，新合成的细胞表面 糖蛋白在运输过程中通过 内质网，高尔基体则分泌囊泡。 为了研究笼状蛋白在细胞内蛋白质运输中的作用， 酵母笼状蛋白重链基因(CHC1)已被克隆并 用来干扰体内的染色体基因。笼状蛋白缺乏症 酵母是有活力的，但生长速度比野生型细胞慢。 突变细胞中两种分泌蛋白的转运异常 似乎反映了高尔基体或新生的异常 分泌囊泡运输步骤。 更准确地定义笼状蛋白的功能将由 利用克隆DNA的体外诱变产生 CHC1的温度或冷敏感突变。有条件地- 有缺陷的等位基因将被插入到染色体CHC1中 轨迹。条件性突变的直接影响将是 通过在不允许的温度下孵化细胞来确定 并监测分泌物和空泡的比率和保真度 蛋白质运输和速率内吞作用。 提出了另外三种方法来识别其基因 产品影响与笼状蛋白涂层有关的运输阶段 膜。首先，交配过程中的一种笼状蛋白缺陷细胞 信息素前体的处理形成了筛选的基础 获得导致相同缺陷的其他突变。第二, 笼状蛋白包裹的囊泡将被广泛提纯到 确定相关蛋白质的特征。从蛋白质，探针 相应的基因将被生成并用于获得 分子克隆。第三，能够抑制生长的基因 我们将寻找缺乏笼蛋白的细胞的特性。基因 通过这些方法分离出来的基因将被用于组合遗传， 笼状蛋白的分子克隆及生化研究 涂膜功能。

Clathrin-coated membranes have been implicated in a variety of intracellular protein transport processes in eukaryotic cells. Yeast contain both heavy and light chain clathrin subunits and display a secretory pathway analogous to that found in animal cells. In both cell types, newly synthesized cell surface glycoproteins are processed during transport through the endoplasmic reticulum, the Golgi body then secretory vesicles. To study the role of clathrin during intracellular protein traffic, the yeast clathrin heavy chain gene (CHC1) has been cloned and used to disrupt the chromosomal gene in vivo. Clathrin-deficient yeast are viable but grow more slowly than wild-type cells. Transport of two secreted proteins is anomalous in mutant cells and appears to reflect abnormalities in Golgi body or nascent secretory vesicle transport steps. A more precise definition of clathrin's function will be pursued by using in vitro mutagenesis of cloned DNA to generate temperature or cold-sensitive mutations of CHC1. Conditionally- defective alleles will be inserted into the chromosomal CHC1 locus. The immediate effects of conditional mutations will be determined by incubating cells at the nonpermissive temperature and monitoring the rate and fidelity of secreted and vacuolar protein transport and rates endocytosis. Three additional approaches are proposed to identify genes whose products affect stages of transport involving clathrin-coated membranes. First, a clathrin-deficient cell defect in mating pheromone precursors processing forms the basis for a screen to obtain other mutations which cause the same defect. Second, clathrin-coated vesicles will be extensively purified to characterize associated proteins. From the proteins, probes for the respective genes will be generated and used to obtain molecular clones. Third, genes capable of suppressing the growth properties of clathrin-deficient cells will be sought. Genes isolated by these approaches will be used in a combined genetic, molecular cloning and biochemical investigation of clathrin- coated membrane function.