COPII Coat

Transport of proteins in eukaryotic cells from the endoplasmic reticulum (ER) to the Golgi complex proceeds by deformation of specialized portions of the donor membrane to form carrier vesicles. A group of cytosolic proteins collectively known as COPII carry out a programmed set of sequential interactions, leading to cargo sorting and vesicle budding. Vesicular transport can be reconstituted by using three cytosolic components containing five proteins: the small GTPase Sar1p, the Sec23p/24p complex, and the Sec13p/Sec31p complex. These proteins will support a cargo-carrying budding reaction from isolated ER membranes. Sar1p, a GTP-binding protein, initiates coat formation. The GDP-bound form of Sar1p is normally cytosolic. It is recruited to the ER membrane by interaction with Sec12p, an ER-bound membrane protein that serves as its guanine exchange factor. Sar1p-GTP then recruits cytosolic Sec23p/24p complex, most likely through its interaction with Sec23p. In addition to recruiting Sec23p/24p, the GTP-bound form of Sar1p stabilizes Sec23p and binds to certain ER/Golgi SNARE proteins involved in the specificity of targeting and in the fusion reaction of vesicles with acceptor membranes. The interaction of Sar1p-GTP with Sec23p also facilitates the association of the Sec23p/24p complex with cargo proteins; Sec24p is probably the component responsible for cargo recognition. ER membranes with Sec23p/24p and Sar1p can then recruit Sec13p/31p, a complex that is likely to act as a scaffold, like clathrin, to effect membrane deformation and vesicle budding. Completing the cycle, Sec23p acts as a GTPase activating protein for Sar1p. It is thought that on GTP hydrolysis, Sar1p-GDP is released, leading to uncoating before fusion of the vesicle to the target membrane and recycling of COPII components. In collaboration with Tomas Kirchhausen (The Center for Blood Research) and Randy Scheckman (UC Berkeley) we investigated the molecular organization and structure of Sec23p/24p and Sec13p/31p complexes isolated from yeast cells. The results - obtained from a combination of biochemical and biophysical methods - reveal distinct shapes and quaternary structures for each type of complex. These results led us to propose a model for assembly of a COPII coat.

We calculated a 3D map for Sec23p/24p at a resolution of about 2.5 nm by single particle averaging techniques. The complex consists of two triangularly shaped halves, each containing three approximately globular domains. The shapes of the two halves are similar, and their positions in the complex are related by a pseudo-2-fold axis. The most salient difference between the two halves is that one of them contains a relatively enlarged domain. This half was therefore assigned to the larger Sec24p, whereas the second half was assigned to the smaller Sec23p.
 
The Sec13p/31p complex is a heterotetramer composed of two copies of Sec13p and two copies of Sec31p. Micrographs of the negatively stained Sec13p/31p complex revealed elongated flexible molecules of variable length. The longest molecules were about 28–30 nm in length and consisted of five linearly arranged globular domains linked by relatively flexible joints. A gallery of such molecules illustrates the variety of shapes that Sec13p/31p can adopt. The simplest organization that was consistent with the elongated shape of Sec13p/31p was a side-by-side arrangement of its subunits, such that the globular domain at one end of the complex corresponds to two Sec13p subunits, and the globular domain at the opposite end contains the carboxylterminal part of Sec31p.
 
The outer dimensions and structural features of Sec23p/24p and Sec13p/31p, together with the known details of the interactions between Sec23p/24p and Sec13p/31p and the observation that equivalent amounts of these complexes constitute the coat in COPII-coated vesicles, lead to significant constraints in their possible dispositions within a coat. The model we proposed involves simultaneous interactions between the opposite ends of two Sec23p/24p complexes and two antiparallel Sec31p molecules, themselves contributed by two different Sec13p/31p complexes. The relatively fixed concave shape of Sec23p/24p can impart local curvature to the lattice. Lateral growth is provided by the molecular crosslinks between Sec23p/24p and the adjacent Sec13p/31p complexes, whereas the head-to-head associations of two Sec13p/31p complexes provide longitudinal growth mediated by Sec13p contacts.


» Lederkremer et al. (2001) Proc. Natl. Acad. Sci. USA 98: 10704-10709.