Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat.

Vesicle budding from the endoplasmic reticulum (ER) employs a cycle of GTP binding and hydrolysis to regulate assembly of the COPII coat. We have identified a novel mutation (sec24-m11) in the cargo-binding subunit, Sec24p, that specifically impacts the GTP-dependent generation of vesicles in vitro. Using a high-throughput approach, we defined genetic interactions between sec24-m11 and a variety of trafficking components of the early secretory pathway, including the candidate COPII regulators, Sed4p and Sec16p. We defined a fragment of Sec16p that markedly inhibits the Sec23p- and Sec31p-stimulated GTPase activity of Sar1p, and demonstrated that the Sec24p-m11 mutation diminished this inhibitory activity, likely by perturbing the interaction of Sec24p with Sec16p. The consequence of the heightened GTPase activity when Sec24p-m11 is present is the generation of smaller vesicles, leading to accumulation of ER membranes and more stable ER exit sites. We propose that association of Sec24p with Sec16p creates a novel regulatory complex that retards the GTPase activity of the COPII coat to prevent premature vesicle scission, pointing to a fundamental role for GTP hydrolysis in vesicle release rather than in coat assembly/disassembly.

Vesicle-mediated export from the ER: COPII coat function and regulation.

Vesicle trafficking from the endoplasmic reticulum (ER) is a vital cellular process in all eukaryotes responsible for moving secretory cargoes from the ER to the Golgi apparatus. To accomplish this feat, the cell employs a set of conserved cytoplasmic coat proteins - the coat protein II (COPII) complex - that recruit cargo into nascent buds and deform the ER membrane to drive vesicle formation. While our understanding of COPII coat mechanics has developed substantially since its discovery, we have only recently begun to appreciate the factors that regulate this complex and, in turn, ER-to-Golgi trafficking. Here, we describe these factors and their influences on COPII vesicle formation. Properties intrinsic to the GTP cycle of the coat, as well as coat structure, have critical implications for COPII vesicle trafficking. Extrinsic factors in the cytosol can modulate COPII activity through direct interaction with the coat or with scaffolding components, or by changing composition of the ER membrane. Further, lumenal and membrane-bound cargoes and cargo receptors can influence COPII-mediated trafficking in equally profound ways. Together, these factors work in concert to ensure proper cargo movement in this first step of the secretory pathway. This article is part of a Special Issue entitled: Functional and structural diversity of endoplasmic reticulum.

Traffic of p24 Proteins and COPII Coat Composition Mutually Influence Membrane Scaffolding.

Eukaryotic protein secretion requires efficient and accurate delivery of diverse secretory and membrane proteins. This process initiates in the ER, where vesicles are sculpted by the essential COPII coat. The Sec13p subunit of the COPII coat contributes to membrane scaffolding, which enforces curvature on the nascent vesicle. A requirement for Sec13p can be bypassed when traffic of lumenally oriented membrane proteins is abrogated. Here we sought to further explore the impact of cargo proteins on vesicle formation. We show that efficient ER export of the p24 family of proteins is a major driver of the requirement for Sec13p. The scaffolding burden presented by the p24 complex is met in part by the cargo adaptor Lst1p, which binds to a subset of cargo, including the p24 proteins. We propose that the scaffolding function of Lst1p is required to generate vesicles that can accommodate difficult cargo proteins that include large oligomeric assemblies and asymmetrically distributed membrane proteins. Vesicles that contain such cargoes are also more dependent on scaffolding by Sec13p, and may serve as a model for large carrier formation in other systems.

ER cargo properties specify a requirement for COPII coat rigidity mediated by Sec13p.

Eukaryotic secretory proteins exit the endoplasmic reticulum (ER) via transport vesicles generated by the essential coat protein complex II (COPII) proteins. The outer coat complex, Sec13-Sec31, forms a scaffold that is thought to enforce curvature. By exploiting yeast bypass-of-sec-thirteen (bst) mutants, where Sec13p is dispensable, we probed the relationship between a compromised COPII coat and the cellular context in which it could still function. Genetic and biochemical analyses suggested that Sec13p was required to generate vesicles from membranes that contained asymmetrically distributed cargoes that were likely to confer opposing curvature. Thus, Sec13p may rigidify the COPII cage and increase its membrane-bending capacity; this function could be bypassed when a bst mutation renders the membrane more deformable.