title: Sec24p and Sec16p cooperate to regulate the GTP cycle of the COPII coat.
abstract: 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.
authors.affiliation: Department of Biological Sciences, Columbia University, New York, NY, USA.
authors.forename: Leslie F
authors.initials: LF
authors.lastname: Kung
authors.forename: Silvere
authors.initials: S
authors.lastname: Pagant
authors.forename: Eugene
authors.initials: E
authors.lastname: Futai
authors.forename: Jennifer G
authors.initials: JG
authors.lastname: D'Arcangelo
authors.forename: Roy
authors.initials: R
authors.lastname: Buchanan
authors.forename: John C
authors.initials: JC
authors.lastname: Dittmar
authors.forename: Robert J D
authors.initials: RJ
authors.lastname: Reid
authors.forename: Rodney
authors.initials: R
authors.lastname: Rothstein
authors.forename: Susan
authors.initials: S
authors.lastname: Hamamoto
authors.forename: Erik L
authors.initials: EL
authors.lastname: Snapp
authors.forename: Randy
authors.initials: R
authors.lastname: Schekman
authors.forename: Elizabeth A
authors.initials: EA
authors.lastname: Miller
mesh_terms: D022181:COP-Coated Vesicles / Q000502:physiology*; D006160:Guanosine Triphosphate / Q000378:metabolism* / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000502:physiology*; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D008958:Models, Molecular; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000502:physiology*; D020798:Two-Hybrid System Techniques
chemical_list: D008565:Membrane Proteins; C096367:SEC16 protein, S cerevisiae; C118289:SEC24 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D006160:Guanosine Triphosphate