Biogenesis of transport vesicles coated by COPI

描述(由申请人提供)：我们正在提交对父R01申请(GM058615)的修订，以回应NIH的公告(NOT-OD-09-058：NIH宣布可为竞争性修订申请提供恢复法案资金)。家长基金研究由Coat Protein I(COPI)复合体形成的运输囊泡。这次修订研究了父母资助范围之外的一个新课题，研究了一个关键的COPI成分，即ARFGAP1，如何在分子筛蛋白介导的内吞作用中发挥新的作用。胞膜蛋白AP2复合体参与质膜的内吞作用，而COPI参与高尔基体到内质网的运输，也参与高尔基体之间的转运。这两种包衣复合体是第一个被鉴定的化合物，尽管经过多年的深入研究，但尚未证明它们具有共同的成分。我们现在收集的证据表明，ARFGAP1在转铁蛋白受体(TFR)内吞作用定义的依赖于笼蛋白AP2的运输中发挥作用，并建议通过两种主要方法进一步阐明这一过程。首先，使用生化方法，我们将检查AP2和ARFGAP1之间的相互作用是否调节任一组分与TFR结合的能力。我们还通过定义ARFGAP1与1-EAR域结合的最小部分来深入了解ARFGAP1与AP2是如何相互作用的，并还映射了ARFGAP1如何与1-EAR域结合。其次，我们将寻求先进的成像方法来询问间隙活动如何影响囊泡形成和货物分拣，以及ARFGAP1和AP2之间的相互作用是否也影响这两个事件。我们预计，潜在的结果将有助于对囊泡运输机制的总体理解。此外，由于TFR内吞作用对铁摄取是必不可少的，预期的结果也将有助于分子理解铁摄取是如何实现的，以及这一过程可能如何成为病理过程。 与公共健康相关：蛋白质的功能受到它们的定位的严格调控。这种定位部分是通过在细胞内充当高速公路的运输路径来实现的。我们建议理解这一过程的调节者如何调节一种被称为转铁蛋白受体的表面蛋白的分布。我们的结果可能有助于对细胞内运输机制的基本了解，也有助于洞察铁代谢的调节。

DESCRIPTION (provided by applicant): We are submitting a revision to the parent R01 application (GM058615) in response to an NIH announcement (NOT-OD-09-058: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications). The parent grant studies transport vesicles formed by the Coat Protein I (COPI) complex. This revision studies a new topic outside the scope of the parent grant, examining how a key COPI component, known as ARFGAP1, has a novel role in clathrin-mediated endocytosis. The clathrin AP2 complex participates in endocytosis from the plasma membrane, while COPI acts in transport from the Golgi to the ER and also among the Golgi stacks. These two coat complexes are the first ones identified, and have not been shown to share a common component despite having been intensely investigated for many years. We have now gathered evidence that ARFGAP1 acts in a subset of clathrin AP2-dependent transport, as defined by the endocytosis of transferrin receptor (TfR), and propose to further elucidate this process through two major approaches. First, using biochemical approaches, we will examine whether the interaction between AP2 and ARFGAP1 regulates the ability of either component in binding to TfR. We also seek insight into how ARFGAP1 interacts with AP2 by defining a minimal portion of ARFGAP1 that binds to the 1-ear domain, and also map how ARFGAP1 binds to the 1-ear domain. Second, we will pursue advanced imaging approaches to interrogate how the GAP activity affects vesicle formation and cargo sorting, and whether the interaction between ARFGAP1 and AP2 also affect these two events. We anticipate that potential results will contribute to a general understanding of mechanisms in vesicular transport. Moreover, because TfR endocytosis is essential for iron uptake, anticipated results will also contribute to a molecular understanding of how iron uptake is achieved, and how this process may become pathologic. PUBLIC HEALTH RELEVANCE: The function of proteins is critically regulated by their localization. This localization is achieved in part by transport pathways that act as highways within the cell. We propose to understand how a regulator of this process modulates the distribution of a surface protein known as the transferrin receptor. Our results will likely contribute to a basic understanding of transport mechanisms within the cell, and also shed insight into the regulation of iron metabolism.