Assembly and regulation of the transitional endoplasmic reticulum in budding yeasts

In most eukaryotes, COPII transport vesicles bud from specialized regions of the ER membrane termed transitional ER (tER) sites, but the mechanism that creates tER sites is unknown. Our working model is that tER sites are self-organizing ER domains whose morphology and dynamics reflect the kinetics of their components. In the budding yeast Pichia pastoris, all of the COPII coat proteins localize to the tER, as does the transmembrane protein Sec12 and the peripheral membrane protein Sec16. Previous work identified a temperaturesensitive sec16 mutant that has a dispersed tER phenotype at the restrictive temperature, indicating that Sec16 plays an important role in maintaining tER structure.My interest has focused on studying how Sec16 regulates tER sites, as interpreted within the framework of the self-organization model. I identified two essential regions within P. pastoris Sec16, one of which is necessary and partly sufficient for tER localization, and the other of which is responsible for localizing Sec12 to tER sites. Surprisingly, the central conserved domain, which contains the temperature-sensitive sec16 mutation, is not essential for cell viability or tER organization. I also obtained evidence that Sec16 can exist in a tER-associated form that binds the COPII coat protein Sec13, as well as a cytosolic form that is favored by the sec16 mutation. The tER-associated form of Sec16 apparently restrains COPII vesicle budding, thereby stabilizing tER sites. The sec16 mutation displaces the protein from tER sites, thereby accelerating vesicle budding and causing faster shrinkage of tER sites. These results suggest that Sec16 is a central regulator of tER dynamics.In P. pastoris, Sec16 also functions to recruit Sec12 to tER sites. My contribution to this story was the discovery that when Sec16 and Sec12 from P. pastoris are co-expressed in S. cerevisiae, the heterologous Sec16 localizes to tER sites and recruits the heterologous Sec12. These results pave the way for exploring how the Sec16-Sec12 interaction regulates the COPII reaction cycle.Two additional movies are submitted as supplementary files. The first one shows wild-type P. pastoris cells, and the second shows P. pastoris cells with sec16-P1092L mutation. In both ones tER sites are labeled with Sec13-GFP.