The Clathrin Coated Vesicle Cycle

超出所提供的空间。 网格蛋白包被的囊泡循环驱动突触囊泡再循环，并且对于维持重复的神经传递循环是必不可少的。网格蛋白内吞机制的许多结构成分已经被确定，但我们对网格蛋白介导的内吞作用的分子机制和调控机制的理解仍处于起步阶段。此外，只有一种酶，即GT3发动蛋白，已被确定在网格蛋白介导的树突状细胞增多中发挥作用，尽管这些反应是ATP依赖性的。我的实验室率先开发了一套无细胞检测方法，这些方法可以忠实而有效地重建网格蛋白包被囊泡周期中的每一步，包括包被小凹组装、包被囊泡形成以及循环利用外壳蛋白的顺序脱膜反应，从而释放封闭的囊泡。使用这些良好的特性分析，我们在一个独特的位置，以阐明突触囊泡回收的分子机制。这个建议的长期目标是确定所需的细胞溶质和外周膜蛋白的最小集合，以重建网格蛋白介导的内吞作用，并完成网格蛋白包被的囊泡周期。为了达到这一目的，我们提出了以下具体目标：1）开发一种用于从高度富集的质膜出芽的包被囊泡的新测定法，以能够鉴定内吞机制的外周和整体膜组分; 2）确定导致包被凹坑组装和内吞网格蛋白包被囊泡形成的事件的层次;（3）确定胞浆内吞囊泡形成所需的最小组分：（4）确定调节去包被反应的蛋白质，并检测体内去包被ATP酶hsc 70的作用;以及，5）在原代神经元中开发瞬时表达系统，以证明体外鉴定的蛋白质相互作用和机制是体内突触囊泡再循环所需的。性能现场=

EXCEED THE SPACE PROVIDED. The clathrin coated vesicle cycle drives synapticvesicle 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 understandingof 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-mediatedendocytosis 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 uncoatingreactions 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-coatedvesicle 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 uncoatingreaction 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. PERFORMANCE SITE ========================================Section End===========================================