Biogenesis of transport vesicles coated by COPI

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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.