A Genetic and Biochemical Study of Sec24p Functions in COPII Biogenesis

The traffic of proteins and lipids from the ER to Golgi in ER-derived, or COPII, vesicles is a critical process for forward transport through the secretory pathway. Proteins enter the secretory pathway when they are synthesized by ER-bound ribosomes and folded into their native conformation by chaperones and other proteins. Quality control mechanisms ensure that misfolded proteins are diverted from forward transport and are instead degraded by ER-associated degradation (ERAD). Properly folded proteins that have been deemed transport competent are packaged as cargo into COPII vesicles. The formation of COPII vesicles is initiated by the insertion of an amphipathic helix of the small GTPase, Sar1p, into the ER-membrane, which deforms the membrane. The inner adaptor layer of the COPII coat, Sec23/24p is recruited; Sec23p functions as the GAP for Sar1p whereas Sec24p selects and binds cargo proteins. The outer layer of the coat, Sec13/31p, is then recruited and propagates membrane curvature through polymerization into a cage-like lattice around the vesicle. Though the assembly of both the inner and outer layers of the coat is necessary for vesicle formation, it also promotes GTP hydrolysis by Sar1p, which triggers coat disassembly. To overcome this paradox, the COPII components likely employ a mechanism to prevent premature GTP hydrolysis. Recent studies have suggested that the scaffold-like ER-membrane protein, Sec16p, may have a role in organizing and stabilizing COPII coat assembly. Therefore, successful export of cargo proteins out of the ER is a highly regulated process that involves coordinated efforts among chaperones, quality control, and coat assembly components.;In this thesis, I employed genetic and biochemical approaches to describe two functions of Sec24p that both foster productive generation of COPII vesicles. In the first study, I identified the ER-export motif of the yeast ABC transporter, Yor1p, and its corresponding binding site on Sec24p. Disruption of the diacidic export signal of Yor1p, as well as mutation of the Sec24p B-site pocket in which it binds, prevents its ER-export despite being properly folded. I attempted to use Yor1p as a model to address whether a destabilized and ER-retained mutant version of the protein forms an interaction with Sec24p. The dissection of how forward transport components, such as Sec24p, and quality control processes interface to prevent the packaging and ER-export of misfolded Yor1p will be beneficial in understanding how similar processes occur for other ABC transporters. In the second study, I identified a novel function of Sec24p that aids in stabilizing the assembly of the COPII coat. A mutation in Sec24p that disrupts the interaction with a truncated form of Sec16p causes accelerated GTP hydrolysis by Sar1p, thereby triggering premature coat disassembly. I propose that the stability of the coat machinery during vesicle formation is in part provided by the Sec24p/Sec16p interaction, which imparts a delay on GTP hydrolysis and allows sufficient opportunity for cargo recruitment into the vesicle. Since Sec24p is also the cargo adaptor, I speculate that these two functions of Sec24p may be related to couple cargo capture to coat stability. Together the work discussed in this thesis provides a greater understanding of the events that occur during COPII biogenesis.