MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT

描述：真核细胞的区室组织依赖于 蛋白质在不同区域的精确分布 蛋白质保留在每个隔室中。 蛋白质的分类能力 具有不同目的地是运输机械的关键特征 其控制蛋白质分布和保留。 的分子基础 这种选择性是本建议的重点。 携带网格蛋白重链突变的酵母菌株的研究 亚基揭示网格蛋白参与选择性蛋白质转运 在质膜和高尔基复合体的过程： 交配信息素受体，高尔基体液泡前体的分选 复杂的空泡，以及以前未被认识到的作用，在本地化的 膜蛋白到晚期高尔基体隔室。 其他蛋白质有 已经鉴定出在高尔基体膜蛋白定位中起作用， 已经分离出在该过程中具有缺陷的突变体的集合。 这些研究为基因、细胞生物学 和生物化学方法来研究选择性蛋白质的三个方面 运输：1）高尔基体膜蛋白定位的机制; 2） 网格蛋白相关蛋白在网格蛋白依赖性蛋白中的作用 转运过程; 3）受体内化机制， 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缺陷的突变体（tcs）将被 用于克隆野生型基因，其蛋白质产物参与 在本地化过程中。 TCS编码蛋白的定位将 在野生型和突变株中测定，以探索相互关系 Tcs蛋白和网格蛋白包被的囊泡中的蛋白质之间 阵 明确高尔基体膜网格蛋白衣的功能 蛋白质定位，基因破坏将产生消除所有 网格蛋白相关蛋白（AP-1）复合物的亚基， 在网格蛋白包被的囊泡的形成中发挥核心作用， 高尔基复合体。 将采用遗传策略从功能上鉴定 冗余AP亚基和其它与AP-1相互作用的蛋白质。 最后， 一类新的胞吞靶向信号将被用作探针， 分离负责将受体选择性包装成 新生的内吞小泡。 这些方法将共同确定 以前未识别的选择性蛋白质转运组分 机制，并解决每个确定的机构发挥的具体作用， 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.