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循环，对维持神经传递的重复循环至关重要。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节机制的理解仍处于起步阶段。此外，只有一种酶，即GTPase动力蛋白，已被确定在网格蛋白介导的内吞作用中起作用，尽管这些反应依赖于atp。我的实验室率先开发了一套无细胞检测方法，这些方法忠实而有效地重建了网格蛋白包被囊泡循环的每一步，包括包被坑组装、包被囊泡形成和连续的脱包反应，这些脱包反应回收了包被蛋白，释放了封闭的囊泡。利用这些表征良好的分析，我们处于一个独特的位置来阐明控制突触囊泡循环的分子机制。本提案的长期目标是确定重建网格蛋白介导的内吞作用和完成网格蛋白包被囊泡循环所需的最小胞质和外周膜蛋白集。为了实现这一目标，我们提出了以下具体目标：1)开发一种新的检测方法，用于从高度富集的质膜中萌发包膜囊泡，以鉴定内吞机制的外周和整体膜组分；2)确定导致包覆坑组装和内吞网格蛋白包覆囊泡形成的事件层次；3)鉴定内吞包被囊泡形成所需的最小胞质成分；4)鉴定脱衣反应的调控蛋白，并在体内检测脱衣atp酶hsc70的作用；5)在原代神经元中建立瞬时表达系统，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循环所必需的。网站性能 ======================================== 节结束 ===========================================

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===========================================