Filling in the GAPs‐ Mapping the Spatiotemporal Kinetics of Arf1 and its Regulators

Abstract

The Golgi serves as a hub for intracellular membrane trafficking. The small GTPase Arf1 coordinates several Golgi trafficking pathways by directly recruiting many effectors, such as cargo adaptors and lipid regulators, to the Golgi at precise and distinct points of cisternal maturation. To accomplish this, Arf1 distribution at the Golgi is tightly controlled by GEFs and GAPs, which activate and inactivate Arf1, respectively. Once active, Arf1 becomes stabilized on the membrane where it can then carryout its functions. Despite the significance of Arf1 Golgi localization in properly orchestrating Golgi trafficking, its maturation kinetics remain largely unknown due to its sensitivity to tagging. Furthermore, while the maturation kinetics of the Arf‐GEFs are well‐characterized, they are yet to be determined for the ArfGAPs. Here, I have utilized time‐lapse fluorescence microscopy and novel approaches for Arf1 visualization to begin to map the spatiotemporal dynamics of Arf1 and its GAPs. I have made a breakthrough in developing a method for directly visualizing the location of Arf1 in living cells that does not interfere with Arf1 function. Additionally, I have established the timing of each of the yeast Golgi‐localized ArfGAPs relative to the major Golgi trafficking pathways. Further, I am investigating the role of a highly conserved sequence in the TGN‐localized ArfGAP, Age2. This work will ultimately provide important insights into fundamental questions about Golgi trafficking and function.