Vesicles formed in the transport reaction have an electron-dense, 10-nm coat structure that consists of the Sec proteins required in budding. This coat (COPII) resembles another coat complex (COPI) that creates transport vesicles within the Golgi apparatus. Our working model is that the Sec protein subunits of the COPII coat bind to the ER membrane and recruit cargo molecules into a cluster that then dimples the membrane to form a bud. A direct interaction between one of the COPII subunits, Sec24p, and membrane proteins is implicated in the capture of cargo proteins. This capture results in the concentrative sorting of membrane and secretory proteins, the latter being selected by an indirect interaction mediated by various membrane receptor proteins that link the coat to soluble cargo proteins. Fission of the bud from the membrane separates transported from resident proteins.
In addition to a role in cargo selection, the COPII coat is responsible for the membrane shape change that accompanies vesicle budding. Liposomes formulated with phospholipids representative of a yeast ER membrane fraction bind the COPII proteins in the same sequence of events and with the same nucleotide dependence as observed with native ER membrane. Furthermore, COPII buds and vesicles form on the surface of the liposome and capture solute from the interior of the liposome. Other coat protein complexes (clathrin and COPI) display similar budding activity on synthetic membrane liposomes.
We have used dynamic biophysical measurements and thin-section electron microscopy to explore stages in the assembly of the COPII coat on the surface of liposomes. The membrane recruitment of individual subunits of the coat is detected by light scattering, which measures the increment of mass associated with the formation of a coated surface. Coat assembly and disassembly is a dynamic process that is governed by the recruitment of a GTP-binding protein, Sar1, and the hydrolysis of GTP stimulated by a GTPase-activating protein (GAP) coat subunit, Sec23. Membrane bending in preparation for the formation of a bud appears to be initiated by the insertion of an amino-terminal amphipathic helical peptide of Sar1p into the bilayer. Local expansion of the cytoplasmically exposed phospholipid layer may induce the membrane to curve into a nascent vesicle.
Conditions that lead to the formation of the COPII coat produce an intrinsically unstable complex. Coat disassembly promoted by GTP hydrolysis is accelerated by a scaffold complex, Sec13/31, which assembles onto the inner coat of Sar1p and Sec23/24p and enhances the GAP activity of Sec23 by 10-fold. This rate enhancement removes the coat before budding on a synthetic membrane is completed. We have identified two regulatory components that stabilize the coat in the synthetic budding reaction. Sec12p, the Sar1p guanine nucleotide exchange factor (GEF), promotes coat stability by recharging Sar1p as GTP is consumed during coat polymerization. In addition, Sec16p, which organizes the ER exit site, interferes with Sec13/31p-mediated activation of the Sec23 GAP. These two proteins may suffice to recreate the complete and physiologic budding reaction.
Traffic in Human Genetic Diseases