Clathrin-coated vesicles mediate trafficking of proteins and nutrients in the cell and between organelles. Proteins included in the clathrin-coated vesicles (CCVs) category include clathrin heavy chain (CHC), clathrin light chain (CLC), and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here, we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2 appendages crosslink adjacent CHC β-propellers and that the appendage densities are enriched in CCV hexagonal faces. We resolve how adaptor protein 2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

Vesicle trafficking by clathrin coated vesicles (CCVs) is one of the major mechanisms by which proteins and nutrients are absorbed by the cell and transported between organelles. The individual proteins comprising the coated vesicles include clathrin heavy chain, clathrin light chain, and a variety of adaptor protein complexes. Much is known about the structures of the individual CCV components, but data are lacking about the structures of the fully assembled complexes together with membrane and in complex with cargo. Here we determined the structures of natively assembled CCVs in a variety of geometries. We show that the adaptor β2appendages crosslink adjacent CHC β-propellers and that the appendage densities reside almost exclusively in CCV hexagonal faces. We resolve how AP2 and other associated factors in hexagonal faces form an assembly hub with an extensive web of interactions between neighboring β-propellers and propose a structural model that explains how adaptor binding can direct the formation of pentagonal and hexagonal faces.

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In COPII mediated vesicle formation, Sec13/Sec31 heterotetramers play a role in organizing the membranes into a spherical vesicle. There they oligomerize into a cage that interacts with the other COPII proteins to direct vesicle formation and concentrate cargo into a bud. In this role they must be flexible to accommodate different sizes and shapes of cargo, but also have elements that provide rigidity to help deform the membrane. Here we characterize the influence the C-terminal disordered region of Sec31 has on cage flexibility and rigidity. After deleting this region (residues 820-1220), we characterized Sec13/Sec31ΔC heterotetramers biophysically and structurally through cryo-EM. Our results show that Sec13/31ΔC self-assembles into canonical cuboctahedral cages in vitro at buffer conditions similar to wild type. The distribution of cage sizes indicated that unlike the wild type, Sec13/31ΔC cages have a more homogeneous geometry. However, the structure of cuboctahedrons exhibited more conformational heterogeneity than wild type. Through localized reconstruction of cage vertices and molecular dynamics flexible fitting we found a new hinge for the flexing of Sec31 β-propeller domain and more flexibility of the previously known hinge. Together, these results show that the C-terminal region of Sec31 regulates the flexing of other domains such that flexibility and rigidity are not compromised during transport of large and/or asymmetric cargo.

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