MOLECULAR STUDIES OF SELECTIVE PROTEIN TRANSPORT

描述（改编自申请人的摘要）：分泌和分泌的能力 内吞途径来分选和分配蛋白质到适当的 车厢是必不可少的，以保持功能和结构 真核细胞的组织。该项目的总体目标是 了解控制特定运输的选择性的分子基础 内吞和分泌途径中的步骤。这些途径的缺陷是 可能导致癌症等多基因疾病的病因， 心脏病 网格蛋白介导的酵母蛋白转运分析表明，网格蛋白 参与高尔基体和质膜的转运途径， 最终形成溶酶体样空泡。在过去的融资中 在此期间，获得了一些见解，可以采用独特的方法来解决三个问题， 这些途径中重要的转运步骤：1）网格蛋白包被的囊泡（CCV） 未包被; 2）肌动蛋白基细胞内吞作用期间的货物识别; 3）受体 在核内体中分类。基因生物化学和细胞 生物学战略将被应用于解决集中在每一个特定目标 这些过程。首先，将ccv在体内脱膜的机制 通过表征DNA J-结构域家族的一个成员， Hsc 70共伴侣。将分析C11 p和网格蛋白之间的相互作用 并测定相互作用突变体对体内CCV脱壳的影响。 将评估Hsc 70家族成员的脱膜作用，并额外 将通过蛋白质相互作用和遗传学方法来寻找与脱膜有关的因素。 战略布局第二，肌动蛋白相关蛋白Sla 1 p在连接 将表征肌动蛋白基内吞作用机制的货物。Sla1p 参与识别不依赖于泛素的内吞蛋白的序列 靶向信号将被定位、突变并在体内测试突变体。的 Sla 1 p结构域在内吞作用中的功能将被表征， 将研究磷酸化在调节Sla 1 p内吞作用活性中的作用。 第三，泛素在核内体内分选中的潜在新作用将 被解决。泛素依赖的分选步骤和泛素的类型 将定义排序所需的修改。货物序列的作用 在排序过程中将进行分析。泛素修饰的组分 分选装置将通过蛋白质相互作用、遗传和 生化策略这些研究预计将提供 在理解细胞内蛋白质的关键方面的重大进展 运输：货物的选择，囊泡外套动力学，和排序内 核内体

DESCRIPTION (Adapted from applicant's abstract): The ability of secretory and endocytic pathways to sort and distribute proteins to the appropriate compartments is essential to maintain the functional and structural organization of eukaryotic cells. The overall goal of this project is to understand the molecular basis of selectivity that governs specific transport steps in the endocytic and secretory pathways. Defects in these pathways are likely to contribute to the etiology of multigenic diseases such as cancer and heart disease. Analysis of clathrin-mediated protein transport in yeast suggests that clathrin participates in transport pathways from the Golgi and the plasma membrane that ultimately leads to the lysosome-like vacuole. During the previous funding period, insights were achieved that allow unique approaches to address three important transport steps in these pathways: 1) clathrin coated vesicle (ccv) uncoating; 2) cargo recognition during actin-based edocytosis; 3) receptor sorting in endosomes. A combination of genetic, biochemical, and cell biological strategies will be applied to address specific aims focused on each of these processes. First, the mechanism of ccv uncoating in vivo will be determined by characterization of Aux1p, a member of the DNA J-domain family of Hsc70 co-chaperones. Interactions between Aux1p and clathrin will be analyzed and effects of interaction mutants on ccv uncoating in vivo will be determined. The uncoating roles of Hsc70 family members will be evaluated and additional factors involved in uncoating will be sought by protein-interaction and genetic strategies. Second, the role of the actin-associated protein Sla1p in linking cargo to the actin-based endocytosis machinery will be characterized. Sla1p sequences involved in recognition of an ubiquitin-independent endocytic targeting signal will be mapped, mutated and mutants tested in vivo. The function of Sla1p domains in endocytosis will be characterized, and a role for phosphorylation in regulating Sla1p endocytosis activity will be investigated. Third, a potentially novel role for ubiquitin in sorting within endosomes will be addressed. The ubiquitin-dependent sorting step and the type of ubiquitin modification necessary for sorting will be defined. The role of cargo sequences in the sorting process will be analyzed. Ubiquitin-modified components of the sorting apparatus will be sought by protein interaction, genetic and biochemical strategies. Together these studies are expected to provide significant advances in understanding key aspects of intracellular protein transport: cargo selection, vesicle coat dynamics, and sorting within endosomes.