UC San Diego

The COPII coat is essential for ER-Golgi transport and consists of four subunits: the inner adaptor proteins Sec23 and Sec24, and the outer shell proteins Sec13 and Sec31. Transport is initiated when activated Sar1-GTP in the ER membrane recruits the COPII coat by directly binding the Sec23 subunit; together these proteins catalyze vesicle formation. Vesicles are then tethered to the Golgi by TRAPPI, Ypt1, and Uso1, allowing SNAREs to subsequently catalyze fusion of the vesicle and Golgi membranes. These basic concepts of ER-Golgi transport are introduced and further detailed in chapter one. Recently an interaction between Sec23 and TRAPPI was shown to be required for vesicle tethering to the Golgi, implying the COPII coat regulates trafficking events downstream of budding. It was unclear how long the coat remained bound to vesicles and how this prolonged association might influence tethering and fusion events. Experiments in this dissertation delineate numerous roles of the COPII coat in ER-Golgi transport. Chapter two demonstrates COPII vesicles remain coated after they form and until they are tethered to the Golgi, but appear to uncoat prior to fusion. It also shows the conserved kinase Hrr25 binds and phosphorylates Sec23 and Sec24 at the Golgi after displacing TRAPPI. Hrr25 indirectly inhibits vesicle budding by phosphorylating Sec23, which disrupts its ability to bind Sar1-GTP. Finally, we describe how sequential interactions of Sar1-GTP, TRAPPI, and Hrr25 with Sec23 spatially and temporally regulate vesicle traffic. Chapter three examines the role of Hrr25 in vesicle fusion. Hrr25 appears to partially stimulate the essential process of uncoating, likely by decreasing the affinity of phosphorylated Sec24 with vesicle cargo. Casein kinase II (CKII) is also shown to regulate ER-Golgi transport, potentially by phosphorylating and inhibiting Hrr25. The fourth and final chapter examines the significance of these findings in relation to other trafficking events and proposes experiments to better understand the roles of COPII phosphorylation, Hrr25, and CKII in ER-Golgi traffic