Regulatory mechanisms of clathrin dependant traffic at the TGN and endosomes

描述（由申请人提供）：细胞行为的差异是正常人类发育的核心，以及损伤、病原体和突变如何导致功能障碍。细胞行为差异的许多方面可归因于蛋白质定位的差异，如粘附分子减少或酶分泌增加。反式高尔基网络（TGN）和核内体之间的网格蛋白依赖交通在细胞迁移，细胞信号传导和存活的重要蛋白质定位中起重要作用。本提案中的实验符合一个长期目标，即了解发育程序、突变和细胞信号传导如何作用于交通机制，从而导致不同的细胞行为。这是了解导致TGN和核内体高效和准确运输的分子机制的第一步。目前还不清楚网格蛋白和它的许多接头和辅助蛋白是如何结合在一起，为运输过程中涉及的复杂步骤提供所需的所有活动。这些步骤包括选择和集中货物，在体内产生和瞄准运输载体。先前鉴定的在TGN和核内体上相互作用的网格蛋白接头网络为理解网格蛋白依赖运输的分子机制开辟了新的途径。在本提案中，导致膜传输保真度的分子机制和调节酵母膜传输的信号通路将被研究。在目的1中，特定的假设将被测试，关于网格蛋白和接头如何确保外壳在正确的膜上组装，事件以正确的顺序发生，以及正确的蛋白质运输。有待验证的假设是：1)合作结合和2)紧密排列的网格蛋白外壳空间竞争决定了接头何时起作用。测试这些假设的综合方法将使用体外生物化学、细胞分离、膜交通测定和接合蛋白特定突变的活细胞显微镜。在ai2中，新发现的网格蛋白接头在低营养条件下的调节将被研究。有待验证的假设有：1)低营养物质抑制膜运输；2)抑制作用在适配器修饰水平上起作用；3)保守的信号通路与其他细胞对低营养物质的反应协调运输。

DESCRIPTION (provided by applicant): Differences in cellular behavior are central to normal human development and how injury, pathogens and mutations cause dysfunction. Many aspects of differential cellular behavior can be attributed to differences in protein localization such as reduced adhesion molecules or increased secretion of enzymes. Clathrin dependent traffic between the trans-Golgi network (TGN) and endosomes plays an important role in localization of proteins important for cell migration, cell signaling and survival. The experiments in this proposal fit into a long term goal to understand how developmental programs, mutations and cell signaling act on the traffic machinery to cause differential cellular behavior. This is the first step will be understand the molecular mechanisms leading to efficient and accurate traffic at the TGN and endosomes. It is still unclear how clathrin and its many adaptor and accessory proteins combine to provide all activities required for the complicated steps involved in traffic. These steps include selecting and concentrating cargo, generating and targeting a transport carrier in vivo. Previous identification of a network of interacting clathrin adaptors that act at TGN and endosomes has opened new avenues for understanding the molecular mechanism of clathrin dependent traffic. In this proposal, molecular mechanisms leading to fidelity in membrane traffic and a signaling pathway regulating membrane traffic in yeast will be examined. In aim1, specific hypotheses will be tested about how clathrin and adaptors ensure coats assemble at the correct membrane, that events occur in the correct order and the correct proteins are transported. Hypotheses to be tested are that 1) cooperative binding and 2) competition for space in the tightly packed clathrin coat determine when an adaptor functions. A combined approach to test these hypotheses will use in vitro biochemistry, cell fractionation, assays of membrane traffic and live-cell microscopy of specific mutations in adaptor proteins. In Aim2, a newly identified regulation of clathrin adaptors in low nutrient conditions will be investigated. Hypotheses to be tested are that 1) low nutrients inhibits membrane traffic, 2) inhibition acts at the level of adaptor modification and 3) conserved signaling pathways coordinate traffic with other cellular responses to low nutrients.