The spatiotemporal basis for the function and regulation of Arf1 at the Golgi
The Golgi complex serves as the cellular hub for intracellular membrane trafficking. Golgi trafficking is orchestrated by the small GTPase Arf1, which recruits several effector proteins to the Golgi membrane at precise points in cisternal maturation to facilitate vesicle formation. To achieve this, Arf1 is tightly controlled by GEFs and GAPs, which reside on specific compartments to activate and inactivate Arf1, respectively. Once active, Arf1 becomes stabilized on the membrane where it can then carry out its functions. Despite the essential role of Arf1 in properly orchestrating Golgi trafficking, the localization of Arf1 and its regulating GAPs throughout maturation were unknown. Here, I utilized time-lapse fluorescence microscopy and novel approaches for Arf1 visualization to establish the maturation dynamics of Arf1 and its GAPs. Arf1 was found to predominantly localize to later Golgi compartments, likely to support the increase in Arf1-dependent trafficking at the late Golgi. Additionally, as with the GEFs, each GAP was found to regulate Arf1 at different stages of maturation, with Glo3 mainly functioning at the early Golgi, Gcs1 at the medial Golgi, and Age2 at the late Golgi. To further understand how the differential localization of the GAPs is achieved, I investigated the mechanism of localization for the particularly unstudied Arf GAP, Age2. I determined that Arf1 localizes to the late Golgi through interactions with Arf1 and the membrane via an amphipathic helix. Overall, this work establishes a comprehensive timeline for Arf1 function and regulation at the Golgi and provides mechanistic insight into how this timeline is achieved.