We have developed biochemical assays that measure the early events of polypeptide translocation into the endoplasmic reticulum (ER) and of vesicle-mediated protein transport from the ER to the Golgi apparatus. Vesicles formed in the transport reaction have an electron-dense, 10-nm coat structure that consists of the Sec proteins (the GTP binding protein Sar1p, Sec23/24p, and Sec13/31p) 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 (1).

A model for the binding of coat proteins and capture of membrane molecules in COPII vesicles

Traffic of large and unusually shaped cargo complexes such as procollagen (PC1, 300 nm rod-shaped oligomeric protein) and lipoprotein particles (carrying apolipoprotein B and cholesterol) display special requirements to form larger-than-normal COPII coat complexes. Work in collaboration with the Rape lab led to the discovery of a role for ubiquitylation of the Sec31 subunit of the COPII coat in the capture of procollagen into large COPII vesicles (2). These large, PC1-containing vesicles have been observed in cultured human cells, and in a cell-free COPII vesicle budding reaction (3). The capture of PC1 into larger-than-normal COPII vesicles may be directly influenced by the co-packaging of the Sar1 guanine exchange factor (GEF), Sec12, bound to the ER membrane receptor for PC1, TANGO1 (4). In addition, lipoprotein sorting from the ER appears to require the intervention of a fatty acid binding protein, FABP5, as well as the COPII machinery for particle packaging into transport vesicles (5).

We have also reported a connection between the COPII coat and the formation of the autophagosome, an organelle responsible for the capture of protein aggregates and organelles that are delivered to and degraded in the lysosome. By following the covalent attachment of a lipid group, phosphatidylethanolamine, to the C-terminus of a cytoplasmic protein, LC3, we found that an organelle that mediates vesicular flow between the ER and the Golgi apparatus, the ERGIC, is the membrane that gives rise to an autophagosome precursor organelle (6). Further, we found that the ERGIC forms novel COPII vesicles that build the autophagosome membrane (7).