THE CLATHRIN COATED VESICLE CYCLE

描述（改编自申请人摘要）：网格蛋白包被囊泡 循环驱动突触囊泡再循环，对于维持 神经传递的循环的许多结构组件 网格蛋白内吞机制已经确定，但我们的理解， 网格蛋白介导的内吞作用的分子机制是 还处于初期阶段此外，只有一种酶，即GT3发动蛋白， 被鉴定在网格蛋白介导的内吞作用中起作用， 反应是ATP依赖性的。我的实验室率先开发了一种 一套无细胞检测，一起，忠实和有效地重建 网格蛋白包被的囊泡循环中的每一步，包括包被的小窝组装， 包被囊泡的形成和随后的去包被反应 外壳蛋白释放出封闭的囊泡使用这些 良好的特点分析，我们在一个独特的位置，以阐明 控制突触囊泡循环的分子机制。远景目标 这个建议的一个重要方面是确定胞质和外周的最小集合， 重组网格蛋白介导的内吞作用和 完成网格蛋白包被的囊泡循环。为了实现这一目标，我们建议 具体目的如下：1）建立一种新的包被囊泡的检测方法 从高度富集的质膜出芽，以能够鉴定 内吞机制的外周和整体膜组件; 2） 确定导致被膜小核组装和内吞的事件的层次结构 网格蛋白包被的囊泡形成; 3）鉴定最小的胞质组分 内吞包被囊泡形成所需的蛋白质; 4）鉴定 调节脱膜反应，检测hsc 70的作用， ATP酶在体内;和，5）开发瞬时表达系统在原代 神经元，以证明蛋白质的相互作用和机制，确定在 突触囊泡在体内的再循环需要体外。

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.