MULTIPLE ROLES FOR ARF1 IN THE FINAL STAGES OF GOLGI MATURATION

Abstract

Eukaryotic cells employ intricate networks of membrane-dependent protein and lipid transport pathways among organelles. Many of these so-called ‘membrane trafficking’ pathways converge at the Golgi complex, a dynamic organelle at which lipids and protein cargo are processed, sorted, and prepared for delivery to the appropriate intracellular destination. Virtually every step of membrane trafficking is controlled by Arf and Rab small GTPases, which function as molecular switches to define when and where trafficking events are to occur. These events take place within the context of Golgi maturation, a process in which the functional identities of individual Golgi cisternae evolve over time. Proper Arf and Rab function is key to maintaining the fidelity of Golgi maturation, but many of the precise mechanisms by which they regulate this process are unclear. This dissertation describes new insights into the specific contributions of the Arf GTPase Arf1 to the late stages of Golgi maturation. Using a combination of live-cell imaging and in vitro biochemical assays, I have found that Arf1 controls an essential Golgi pool of the signaling lipid phosphatidylinositol 4-phosphate (PI4P) by directly recruiting the Pik1-Frq1 (PI4KIIIβ) phosphatidylinositol 4-kinase complex. PI4P marks a transition to the final stages of Golgi maturation: a last round of intra-Golgi recycling, followed by terminal vesiculation of the compartment into secretory vesicles. Arf1 also oversees a sharp, timed shift from enrichment of Ypt1 (Rab1) and Ypt6 (Rab6) at maturing cisternae to enrichment of Ypt31/32 (Rab11) just prior to terminal vesiculation. These findings reveal important roles for Arf1 in establishing two essential hallmarks of mature Golgi compartments—PI4P enrichment and a Ypt31/32 Rab domain—and provide new insights into the Golgi maturation timeline.