Regulatory mechanisms of clathrin dependant traffic at the TGN and endosomes

描述(由申请人提供)：细胞行为的差异是人类正常发育以及损伤、病原体和突变如何导致功能障碍的核心。细胞行为差异的许多方面可以归因于蛋白质定位的不同，如黏附分子减少或酶分泌增加。依赖于笼蛋白的跨高尔基体网络(TGN)和内体之间的交通在定位对细胞迁移、细胞信号和生存至关重要的蛋白质方面发挥着重要作用。这项提议中的实验符合一个长期目标，即了解发育程序、突变和细胞信号如何作用于交通机械，从而导致不同的细胞行为。这是了解导致TGN和内小体高效和准确交通的分子机制的第一步。目前尚不清楚笼蛋白及其许多接头和辅助蛋白是如何结合在一起，为涉及交通的复杂步骤提供所需的所有活动。这些步骤包括选择和浓缩货物，在体内产生和靶向运输载体。以前发现的作用于TGN和内体的相互作用的网状蛋白适配器网络为理解网状蛋白依赖的交通的分子机制开辟了新的途径。在这项提案中，将研究导致膜运输保真度的分子机制和调节酵母膜运输的信号通路。在目标1中，将测试特定的假设，关于笼蛋白和适配器如何确保涂层在正确的膜上组装，事件以正确的顺序发生，正确的蛋白质被运输。需要检验的假设是1)协同结合和2)在紧密堆积的网状蛋白被层中竞争空间决定了适配器何时起作用。验证这些假说的一种综合方法将使用体外生物化学、细胞分离、膜运输分析和活细胞显微镜对接头蛋白的特定突变进行检测。在AIM2中，将研究一种新发现的在低营养条件下对网状蛋白适配器的调节。有待检验的假设是：1)低营养抑制膜运输，2)在接头修饰水平上的抑制作用，3)保守的信号通路协调运输与其他细胞对低营养的反应。

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.