Regulation of post-Golgi traffic by Arf and Arf-dependent adaptors

描述（由申请人提供）：膜运输在真核生物中是必不可少的，因为它是蛋白质从细胞分泌、细胞器完整性得以维持以及跨膜蛋白和脂质在细胞隔室内运输的过程。许多流行的现代人类疾病现在被理解为受到膜运输缺陷或变化的影响或甚至完全由膜运输缺陷或变化引起;包括囊性纤维化、阿尔茨海默病、帕金森病和癌症。在过去的25年里，我们对特定蛋白质和脂质在膜运输中所起作用的理解取得了重大进展。然而，我们的分子模型在几个领域仍然相当粗糙，随着每一步确定的组分数量激增，对交通特异性和调节来源的清晰理解变得混乱。该应用的重点是通过特定的蛋白质运载物、Arf家族调节性GTPases以及Arf依赖性衔接子、Mint和GGA来调节一个位点（晚期高尔基体/反式高尔基体网络（TGN））的膜交通。至少有七种不同的Arf依赖性接头或复合物直接参与在TGN包装货物和分拣到不同目的地。我们已经开发出能够特异性检测和消除每个人的Arf亚型的试剂，并使用这些来确定一些新的Arf行动的网站。在这里，我们建议使用这些和相关的试剂，专注于一个单一的步骤顺行膜交通，以解决交通日益复杂，需要更详细的分子模型。这项建议有三个具体目标。在第一，我们将确定在Arf调节交通从晚期高尔基体/TGN的特异性的来源进行系统的测试，在培养的哺乳动物细胞的Arf亚型所需的招聘和后高尔基体交通的薄荷，GGA，和AP-1依赖载体。我们将测试的假设，不同的Arf亚型（Arf 1，3-5）的行为对直接招募不同的外壳蛋白或复合物的晚期高尔基体/TGN。在第二个目标中，我们将确定Mint 3和Mint 2的磷酸化位点以及对货物结合和定位到TGN的影响。此外，我们将测试GGA 1仅与GGA 2和GGA 3结合使用的模型。我们将测试的假设，即蛋白质磷酸化的衔接子是一个广泛传播的调控事件，在载体的生物合成和同源衔接子的调节差异导致功能的差异。在目标3中，我们将测试至少有一些货物，例如，APP可以使用分拣机退出TGN，这样做会导致路由和处理方面的重要差异。我们将研究APP和LR 11在TGN的流量，并确定参与替代APP出口的序列基序。膜运输在真核生物中是必不可少的，因为它是蛋白质从细胞分泌、细胞器完整性得以维持以及跨膜蛋白和脂质在细胞隔室内运输的过程。 公共卫生相关性：该应用的重点是通过特定的蛋白质运载物、Arf家族调节性GTPases以及Arf依赖性衔接子、Mint和GGA来调节一个位点（晚期高尔基体/反式高尔基体网络（TGN））的膜交通。虽然对我们理解细胞生物学和细胞信号传导至关重要，但我们的研究结果有可能影响几种最常见的人类疾病，包括所有现在被认为是由膜交通缺陷或变化影响甚至完全引起的疾病;包括囊性纤维化，阿尔茨海默病，帕金森病和癌症。

DESCRIPTION (provided by applicant): Membrane traffic is essential in eukaryotes as it is the process by which proteins are secreted from cells, organelle integrity is maintained, and both transmembrane proteins and lipids are transported within compartments of the cell. Many of the prevalent modern human diseases are now understood to be impacted or even wholly caused by defects or changes in membrane traffic; including cystic fibrosis, Alzheimer's disease, Parkinson's disease, and cancers. Substantial progress has been made in the last 25 years in our understanding of the roles played by specific proteins and lipids in membrane traffic. However, our molecular models remain rather crude in several areas and as the number of components identified at each step has soared a clear understanding of the sources of specificity and regulation in traffic has become muddled. This application focuses on the regulation of membrane traffic at one site, the late Golgi/trans-Golgi network (TGN), by specific protein cargos, Arf family regulatory GTPases, and the Arf dependent adaptors, Mints and GGAs. At least seven different Arf-dependent adaptors or complexes are directly involved in packaging cargo at the TGN and sorting to different destinations. We have developed reagents capable of the specific detection and depletion of each human Arf isoform and have used these to identify a number of novel sites of Arf action. Here we propose to use these and related reagents to focus on a single step of anterograde membrane traffic to address the growing complexity in traffic and the need for more detailed molecular models. This proposal has three specific aims. In the first we will determine the sources of specificity in Arf regulated traffic from the late Golgi/TGN by performing systematic tests in cultured mammalian cells of Arf isoforms required for recruitment and post-Golgi traffic of Mint, GGA, and AP-1 dependent carriers. We will test the hypothesis that the different Arf isoforms (Arf1, 3-5) act in pairs to directly recruit the different coat proteins or complexes to the late Golgi/TGN. In the second aim we will determine the sites of phosphorylation of Mint3 and Mint2 and the impact on cargo binding and localization to the TGN. In addition, we will test the model that GGA1 only work in combination with GGA2 and GGA3. We will test the hypotheses that protein phosphorylation of adaptors is a wide spread regulatory event in carrier biogenesis and that differences in regulation of homologous adaptors lead to differences in functions. And in aim #3 we will test the hypothesis that at least some cargo, e.g., APP, can exit the TGN using sorting machineries and that in doing so it leads to important differences in routing and processing. We will examine the traffic of APP and LR11 at the TGN and determine the sequence motifs involved in alternative APP exit. Membrane traffic is essential in eukaryotes as it is the process by which proteins are secreted from cells, organelle integrity is maintained, and both transmembrane proteins and lipids are transported within compartments of the cell. Public Health Relevance: This application focuses on the regulation of membrane traffic at one site, the late Golgi/trans-Golgi network (TGN), by specific protein cargos, Arf family regulatory GTPases, and the Arf dependent adaptors, Mints and GGAs. While of fundamental importance to our understanding of cell biology and cell signaling, our results have the potential to impact several of the most prevalent human diseases, including all that are now understood to be impacted or even wholly caused by defects or changes in membrane traffic; including cystic fibrosis, Alzheimer's disease, Parkinson's disease, and cancers.