MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT

真核细胞的隔室组织依赖于准确的 蛋白质在不同隔室的分布和对蛋白质保留的影响 每个隔间内的驻留蛋白质。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。分子基础 这种选择性是我们建议的重点。 胞内蛋白重链突变(Chc1)酵母菌的研究 链亚单位揭示了笼状蛋白在几个 选择性蛋白质转运步骤，包括选择性保留高尔基体 膜蛋白，受体介导的交配肽的内吞作用 α-因子，以及蛋白质从高尔基体到 溶酶体样液泡。基于clathrin在高尔基体膜中的作用 蛋白质保留，利用遗传筛选分离出两个 高尔基体膜缺陷的附加突变体(LAM突变体) 蛋白质滞留。这些研究为联合研究提供了基础 遗传和生化方法研究三个方面的问题 蛋白质选择性转运：1)高尔基体膜蛋白的作用机制 2)笼蛋白相关蛋白在每一种蛋白中的作用 依赖于笼状蛋白的转运过程；3)笼状蛋白的转运机制。 介导的α-因子内吞作用。 将研究笼状蛋白与高尔基体膜蛋白的相互作用 生物化学，通过分级的笼状蛋白包裹的小泡，和 通过确定突变型高尔基体膜的定位进行遗传学研究 在chc1突变体中缺乏细胞质保留信号的蛋白质。 更多高尔基膜蛋白保留缺陷的LAM突变体将 被鉴定并用于克隆LAM基因的野生型。 针对LAM基因产物产生的抗体将用于表征 LAM蛋白。为了进一步定义笼形蛋白涂层的功能， 选择性转运，编码笼状蛋白相关蛋白(AP)的基因 将被克隆并用于产生基因中断。依赖于笼状蛋白 将对突变株的运输途径进行监测。不同的AP和 我们将结合chc1突变来探索基因间的相互关系。 生化分级将被用来评估物理相互作用 在不同的AP之间以及AP和clathrin之间。最后，体外试验 依赖于笼状蛋白选择性转运的分析将由 在通透性细胞中重建α-因子内吞作用。这个 将确定体外反应的功能成分，并 以渗透性突变细胞或 胞浆的分级。总而言之，这些研究将确定 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。

The compartmental organization of eukaryotic cells relies on accurate distribution of proteins to different compartments and on retention of resident 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 our proposal. Studies of yeast strains carrying mutations (chc1) in the clathrin heavy chain subunit revealed that clathrin plays an important role in several selective protein transport steps, including selective retention of Golgi membrane proteins, receptor-mediated endocytosis of the mating peptide alpha-factor, and sorting of proteins from the Golgi apparatus to the lysosome-like vacuole. Based on clathrin's function in Golgi membrane protein retention, a genetic screen was employed to isolate two additional mutants (lam mutants) which are defective in Golgi membrane protein retention. These studies provide the basis for a combined genetic and biochemical approach to investigate three aspects of selective protein transport: 1) the mechanism of Golgi membrane protein retention; 2) the role played by clathrin-associated proteins in each clathrin-dependent transport process; 3) the mechanism of clathrin- mediated alpha-factor endocytosis. Clathrin's interaction with Golgi membrane proteins will be investigated biochemically by fractionation of clathrin-coated vesicles, and genetically by determining the localization of mutant Golgi membrane proteins lacking cytoplasmic retention signals in chc1 mutants. Additional lam mutants defective in Golgi membrane protein retention will be identified and used to clone wild-type versions of LAM genes. Antibodies raised against LAM gene products will be used to characterize the Lam proteins. To further define the function of clathrin coats in selective transport, genes encoding clathrin-associated proteins (APs) will be cloned and used to generate gene disruptions. Clathrin-dependent transport pathways will be monitored in mutant strains. Different ap and chc1 mutations will be combined to explore genetic interrelationships. Biochemical fractionations will be used to assess physical interactions between different APs and between APs and clathrin. Finally, an in vitro assay for clathrin-dependent selective transport will be developed by reconstituting alpha-factor endocytosis in permeabilized cells. The functional components of the in vitro reaction will be identified and characterized by complementation of permeabilized mutant cells or fractionation of cytosol. Together, these studies will identify previously unrecognized components of the selective protein transport machinery and address the specific roles played by each identified participant.