Structural Characterization of Protein Coats in Vesicle Trafficking

Cargo trafficking using vesicles has been studied in eukaryotic cells for more than half a century. Three conventional and evolutionarily archaic pathways have since been identified. These pathways include: Coat Protein II (COPII), Coat Protein I (COPI), and Clathrin-Mediated Endocytosis (CME). In each case, more than a dozen players are involved in a convoluted protein-protein interaction network. These interactions lead to the recruitment of cargo, deformation of the donor membrane, budding of the vesicle, scission of the vesicle, and anchoring to the receiving membrane. Successful implementation of these steps requires temporal and spatial coordination; two aspects of coat assembly that are intertwined. Ideally, with high temporal and spatial resolution, and multiple recording channels for different proteins, an understanding of the exact mechanisms of coat assembly could be delineated. Realistically, researchers have studied the temporal and spatial coordination of coat assembly independently and only recently have they been able to achieve molecular and sub-nanometer resolutions. In this dissertation the spatial organization of COPII and clathrin coats are investigated and the results are presented in chapters 2 (COPII), 3 and 4 (clathrin). Chapters 2 and 3 were published in peer reviewed journals. The COPII pathway is involved in cargo transport from the endoplasmic reticulum (ER) to the cis-Golgi apparatus. Clathrin is involved in multiple pathways including: endocytosis, trans-Golgi to endosomes, and endosomes to plasma membrane. In both pathways, the resulting coated vesicle has multiple layers of coat protein with the outermost layer resembling a cage which is made of repeating subunits. Cages are dynamic assemblies of Sec13/31 heterotetramer in COPII cages, and clathrin trimer (triskelion) in clathrin cages. These cages can range in diameter between 60-120 nm and can be highly symmetric. In chapter 1, the properties of these coated vesicles and their cages will be introduced along with the principles of electron microscopy which is the technique of choice for studying these ultra-structures. Sec13/31 heterotetramer is the quaternary structure of the proteins Sec13 and Sec31 in cells. The heterotetramer looks like a dumbbell with WD40 domains at each end and an α-helical central rod. In vitro, the heterotetramer can self-assemble at certain nonphysiological and rather high ionic strengths. Nonetheless, the same cage structures have been observed in situ. By reconstituting Sec13/31 cages in vitro and studying them with cryo-EM, researchers have identified the WD40 domains as sites of protein-protein interaction which, when repeated, assemble the heterotetramers into cages. The size of these cages should fit the size of the cargo they carry, but it is not clear how cage size is regulated by Sec23/24 which recruits cargo and has no direct interaction with the WD40 domains. In chapter 2, the role of the unstructured C-terminal region of Sec31 is investigated in regulating cage size. This region consists of a linear peptide motif that is buried in Sec23 and Sar1 and therefore acts as a mediator between the cage and the cargo-recruiting components. We discovered that this disordered region can play a role in the flexibility of the cages. Clathrin triskelion is the quaternary structure of the clathrin heavy chain in cells. Like COPII vesicles, clathrin coated vesicles (CCVs) form cages of different sizes in coordination with their cargo size. Similarly, the cargo-recruiting proteins in CCVs, such as adaptor protein 2 (AP2), have no direct influence on the known elements of clathrin cage assembly. In chapter 3, natively assembled CCVs are studied using cryo-EM. An atomic model for clathrin is presented along with clathrin cages of different sizes and a novel cage geometry that was predicted to not exist. The first structural evidence for the interaction of AP2 with the N-terminal domain of clathrin is presented along with the nonrandom distribution of AP2 around CCVs. In chapter 4, CCVs are studied using cryo-electron tomography (cryo-ET). CCVs are inherently heterogeneous and tomography allows for higher resolution reconstructions of heterogeneous structures. A workflow for the reconstruction of the clathrin layer and the adaptor layer is presented. Finally, more venues for studying CCV structures are discussed.