The Clathrin Coated Vesicle Cycle

描述(摘自申请者的摘要)：笼状蛋白包被的囊泡 周期驱动突触小泡的循环，对维持 反复的神经传递。的许多结构组件 分子筛的内吞机制已经确定，但我们对 细胞胞吞作用的分子机制及其调控 它还处于初级阶段。此外，只有一种酶，GTP酶动力蛋白，已经被 被发现在胞内蛋白介导的内吞作用中发挥作用，即使这些 反应是依赖于三磷酸腺苷的。我的实验室率先开发了一种 一组共同、忠实和高效地重建的无细胞分析 包被蛋白囊泡循环中的每一步，包括包被小孔组件， 涂层囊泡的形成和循环的顺序去涂层反应 外壳蛋白，释放封闭的小泡。使用这些 具有良好特征的分析，我们处于独特的地位来阐明 支配突触小泡循环的分子机制。长期目标 这一建议的目的是确定胞浆和外周的最小集合 重建细胞骨架蛋白介导的内吞作用所需的膜蛋白 完成笼状蛋白包裹的囊泡循环。为了达到这一目标，我们建议 具体目标如下：1)建立一种新的包衣囊泡检测方法 从高度浓缩的质膜中萌发，从而能够鉴定 内吞机械的外周和整体膜部件；2)至 确定导致涂层小孔组装和内吞的事件的等级 笼状蛋白包被囊泡的形成；3)鉴定最小的胞浆成分 内吞被囊泡形成所必需的；4)鉴定可 调节去涂层反应并测试hsc70的作用，去涂层 体内的ATPase；5)建立原代瞬时表达系统 神经元证明蛋白质的相互作用和机制在 体外培养是体内突触小泡循环所必需的。

DESCRIPTION (Adapted from applicant's abstract): The clathrin coated vesicle cycle drives synaptic vesicle recycling and is essential for the maintenance of repeated rounds of neurotransmission. Many of the structural components of the clathrin endocytic machinery have been identified, but our understanding of the molecular mechanisms underlying and regulating clathrin-mediated endocytosis is still in its infancy. Moreover, only one enzyme, the GTPase dynamin, has been identified to play a role in clathrin-mediated endocytosis even though these reactions are ATP-dependent. My laboratory has pioneered the development of a set of cell-free assays that together, faithfully and efficiently reconstitute each step in the clathrin-coated vesicle cycle, including coated pit assembly, coated vesicle formation and the sequential uncoating reactions that recycle the coat proteins, freeing the enclosed vesicles. Using these well-characterized assays, we are in a unique position to elucidate the molecular mechanisms that govern synaptic vesicle recycling. The long-term goal of this proposal is to identify the minimal set of cytosolic and peripheral membrane proteins required to reconstitute clathrin-mediated endocytosis and to complete the clathrin-coated vesicle cycle. To meet this objective, we propose the following Specific Aims: 1) To develop a novel assay for coated vesicle budding from highly enriched plasma membranes to enable identification of peripheral and integral membrane components of the endocytic machinery; 2) To determine the hierarchy of events leading to coated pit assembly and endocytic clathrin coated vesicle formation; 3) To identify minimal cytosolic components required for endocytic coated vesicle formation; 4) To identify proteins that regulate the uncoating reaction and to test the role of hsc70, the uncoating ATPase in vivo; and, 5) To develop transient expression systems in primary neurons to demonstrate that protein interactions and mechanisms identified in vitro are required for synaptic vesicle recycling in vivo.