Eukaryotic cells contain numerous membrane-enclosed organelles, each with a distinct function and characteristic protein composition. The transfer of cargo proteins and lipids between organelles is mediated by protein-coated vesicles that capture cargo, bud from a donor membrane, and then deliver their contents by fusing with a recipient organelle. Our research addresses the molecular-level mechanisms that enable vesicles to package the correct cargo proteins and to bud from and fuse with the appropriate compartments. We use x-ray crystallography to determine the atomic structures of the component proteins, and we relate structure to function through biochemical experiments that reconstitute aspects of the vesicle-budding process.
Structural Biology of Vesicle Formation
Transport vesicles form when coat subunits are recruited from the cytoplasm to specified sites on a membrane surface. Budding begins when an ARF-family G protein is converted to the GTP-bound state by its cognate guanine nucleotide exchange factor (GEF), causing it to bind to the membrane and recruit coat proteins. To provide a structural framework for understanding coat assembly, we solved a series of crystal structures representing different stages of this process.
In early work we focused on the G protein and its conversion to the GTP-bound form. A comparison of ARF-GDP and ARF bound to a nonhydrolyzable GTP analog revealed a unique conformational switching mechanism in which GTP binding triggers the exposure of the protein's membrane anchor—a myristoylated amino-terminal α helix. The elements of polypeptide sequence that participate in the structural transition are highly conserved among ARF proteins, implying that the GTP-myristoyl switch mechanism is used for all ARF-mediated budding events throughout the eukaryotic secretory pathway.
Subsequent work revealed the structural basis for ARF activation by ARF-specific GEFs: the crystal structure of the complex of ARF and GEF showed that an extensive hydrophobic interface drives the two proteins together and forces key amino acid side chains of the GEF directly into the GTPase active site to expel the GDP molecule.
More recently we have focused on the large coat subunits, in particular the prebudding complex of the COPII coat—a 220-kDa particle composed of Sec23, Sec24, and Sar1-GTP subunits—that is responsible for cargo selection in the first step of the secretory pathway. Following a biochemical characterization, we completed a crystallographic analysis of the prebudding complex from Saccharomyces cerevisiae. This revealed a bow tie–shaped structure, 15 nm long, with a membrane-proximal surface that is concave and positively charged to conform to the surface of the COPII vesicle. The inner surface of the complex, which is formed from all three subunits, covers a large membrane surface area. This work advanced our understanding of the topology of a prebudding complex, the orientation of coat and ARF molecules in a membrane-bound particle, and the nature of the GTP-dependent coat assembly reaction.
Following the formation of the Sec23/24-Sar1 prebudding complex, the Sec13/31 component of the COPII coat is recruited to the membrane; 24 copies of Sec13/31 self-assemble into a spherical cage that deforms the membrane into a bud. Recently, we determined the atomic structure of the Sec13/31 particle, the assembly unit of the cage, which we found to be a 28-nm-long rod. We have constructed a molecular model of the complete COPII cage to explain the functional organization of coat proteins on COPII vesicles.
Biochemistry of Cargo Selection
Classical studies of endocytosis led to the discovery of transport signals—short cytoplasmic sequences—on select cargo proteins that confer transport activity through a direct interaction between the signal and coat protein machinery. Significant progress has been made toward identifying signals for other vesicular transport steps in cells. Nevertheless, it is likely that the majority of signals conferring movement between intracellular membranes remain to be discovered. We have focused on identifying novel signals that allow export from the endoplasmic reticulum (ER) via interaction with the COPII coat proteins.
In a series of biochemical experiments, we found a set of signals on ER-to-Golgi SNARE proteins, and we carried out a crystallographic analysis to identify how each signal is recognized by the COPII prebudding complex. The signal sequences bind to distinct sites on the equatorial surface of the Sec24 component of COPII. Some of the transport signals are short peptide sequences; others appear to be conformational epitopes, suggesting a mechanism by which the coat complex could discriminate among cargo proteins according to their folding status.