PROJECT SUMMARY Small GTPases of the Arf and Rab families are master regulators of intracellular trafficking in eukaryotic cells that direct the key trafficking events of vesicle formation, cargo recognition, and transport. Cells exert tight spatiotemporal control of these GTPases through a network of GTPase effector proteins (GEFs) and GTPase activating proteins (GAPs). Perturbations to these key regulators give rise to numerous human diseases. Despite their importance, many GEFs and GAPs have yet to be identified — meaning we lack a comprehensive understanding of how cells manage the flow of materials between different membrane- bound compartments. There is a critical need, then, to study the modulators of GTPase activity and determine their roles in membrane homeostasis. The overarching goal of this proposal is to identify and characterize novel regulators of GTPase activity. I have recently identified a novel family of GAPs using structural prediction approaches. The specific aims of this proposal are to characterize the physiological role of these new GAPs. Using a combination of genetic and live-cell imagining techniques, Specific Aim 1 will focus on identifying the target substrate for each of the GAPs. I will knock out and overexpress the novel GAPs and subsequently monitor fluorescently-tagged GTPases. The rationale being that improper regulation of GTPases will cause their mislocalization. Aim 1 will also determine how these proteins localize to distinct subcellular locations and how this ultimately drives their regulation of GTPases. Together, this specific aim will elucidate the biological niches in which each of these proteins function. Specific Aim 2 will investigate the structure and function of these novel GAPs to better understand how they act to regulate GTPase activity. Using in vitro GAP assays, the enzymatic activity of purified GAPs will be reconstituted on synthetic membranes — quantifying their activity and substrate specificity in vitro. In order to validate my structural predictions, the structure of GAPs in complex with their substrate GTPase will be determined using cryogenic electron microscopy. Crucially, these structural studies will involve proteins bound to synthetic membranes, providing unique insights into the molecular architecture of membrane-bound GTPase inactivation. Completion of the research in this fellowship proposal will provide valuable insight into the regulation of the secretory pathway and, by extension, will have broad impact in cell and membrane biology.