MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT

描述：真核细胞的隔室组织依赖于 蛋白质准确地分布到不同的隔室等 蛋白质在每个隔室内的滞留。对蛋白质进行分类的能力 不同的目的地是运输机械的一个主要特点 它控制着蛋白质的分布和保留。的分子基础 这种选择性是本提案的重点。 胞质蛋白重链突变酵母菌的研究 亚基研究表明，网状蛋白参与选择性蛋白质转运 质膜和高尔基复合体的突起：内吞作用 交配信息素受体，高尔基体液泡前体的分离 对空泡的复杂性，以及以前未被认识到的在定位中的作用 膜蛋白到高尔基体的晚期隔室。其他蛋白质有 已确定在高尔基体膜蛋白定位和一种 在这一过程中存在缺陷的突变体已经被分离出来。 这些研究形成了遗传、细胞生物学结合的基础 和生化方法研究选择性蛋白质的三个方面 转运：1)高尔基体膜蛋白定位机制；2)高尔基体膜蛋白定位 网状蛋白相关蛋白在网状蛋白依赖中的作用 转运过程；3)受体内化的机制 受体介导的交配肽信息素的内吞作用。 高尔基体膜蛋白定位存在缺陷的突变体(TC)将被 用于克隆蛋白质产物参与的野生型基因 在本地化进程中。TCS编码蛋白的定位将 在野生型和突变型菌株中进行测定以探索相互关系 天冬氨酸天冬氨酸蛋白与包被蛋白囊泡相关蛋白之间的关系 队形。确定高尔基体膜上的笼状蛋白涂层的功能 蛋白质定位，基因中断将产生消除所有 推测的网状蛋白相关蛋白(AP-L)复合体的亚基 在囊蛋白包裹的囊泡的形成过程中发挥中心作用 高尔基情结。将使用遗传策略来识别功能 多余的AP亚基和其他与AP-L相互作用的蛋白质。最后， 一类新型的内吞作用靶向信号将被用作探针 分离负责选择性包装受体的蛋白质 新生的内吞小泡。总之，这些方法将确定 以前未被识别的选择性蛋白质运输的成分 机制并解决每个确定的角色所扮演的特定角色 参与者。

DESCRIPTION: The compartmental organization of eukaryotic cells relies on the accurate distribution of proteins to different compartments and on retention of proteins within each compartment. The ability to sort proteins with different destinations is a key feature of the transport machinery which governs protein distribution and retention. The molecular basis of this selectivity is the focus of this proposal. Studies of yeast strains carrying mutations in the clathrin heavy chain subunit revealed that clathrin participates in selective protein transport processes at the plasma membrane and the Golgi complex: endocytosis of the mating pheromone receptors, sorting of vacuolar precursors from the Golgi complex to vacuoles, and a previously unrecognized role in localization of membrane proteins to a late Golgi compartment. Additional proteins have been identified which function in Golgi membrane protein localization and a collection of mutants with defects in this process have been isolated. These studies form the foundation for a combination of genetic, cell biology and biochemical approaches to investigate three aspects of selective protein transport: 1) the mechanism of Golgi membrane protein localization; 2) the role played by clathrin-associated proteins in clathrin-dependent protein transport processes; 3) the mechanism of receptor internalization during receptor-mediated endocytosis of mating peptide pheromones. Mutants (tcs) with defects in Golgi membrane protein localization will be used to clone wild-type versions of genes whose protein products participate in the localization process. The localization of TCS-encoded proteins will be determined in wild-type and mutant strains to explore interrelationships between the Tcs proteins and proteins involved in clathrin-coated vesicle formation. To define the function of clathrin coats in Golgi membrane protein localization, gene disruptions will be generated to eliminate all subunits of a clathrin-associated protein (AP-l) complex which is postulated to play a central role in the formation of clathrin-coated vesicles at the Golgi complex. Genetic strategies will be employed to identify functionally redundant AP subunits and other proteins which interact with AP-l. Finally, a novel class of endocytosis targeting signals will be used as probes to isolate proteins responsible for selective packaging of receptors into nascent endocytic vesicles. Together, these approaches will identify previously unrecognized components of the selective protein transport machinery and address the specific roles played by each identified participant.