Biogenesis of transport vesicles coated by COPI

描述（由申请人提供）：我们正在向母R01申请（GM058615）提交修订，以响应NIH公告（NOT-OD-09-058: NIH宣布竞争性修订申请的恢复法案资金可用性）。母体资助研究由外壳蛋白I （COPI）复合物形成的运输囊泡。这次修订研究了一个新的主题，超出了父母资助的范围，研究了一个关键的COPI成分，被称为ARFGAP1，如何在网格蛋白介导的内吞作用中发挥新的作用。网格蛋白AP2复合物参与质膜的内吞作用，而COPI参与从高尔基体到内质网以及在高尔基体堆栈之间的运输。这两种包被复合物是第一个被发现的，尽管经过多年的深入研究，但尚未显示出它们具有共同的成分。我们现在已经收集了证据，表明ARFGAP1在转铁蛋白受体（TfR）内吞作用定义的网格蛋白ap2依赖性转运的一个子集中起作用，并提出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耳结构域结合的最小部分来深入了解ARFGAP1如何与AP2相互作用，并绘制ARFGAP1如何与1耳结构域结合的图谱。其次，我们将采用先进的成像方法来探究GAP活性如何影响囊泡形成和货物分类，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水泡运输机制的一般理解。此外，由于TfR内吞作用对铁摄取至关重要，预期的结果也将有助于从分子上理解铁摄取是如何实现的，以及这个过程是如何成为病理的。

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.