Clathrins are self-assembling cytoplasmic proteins that serve to mediate membrane trafficking. At intracellular membranes, individual clathrin subunits surround the invaginating membrane to form a protein coat that assists in cargo capture and vesicle formation. The protein coat is a polyhedral, multimeric assembly of clathrin triskelia (3-legged structures partly composed of 3 clathrin heavy chains (CHC)). There are two forms of CHC in vertebrates CHC17 and CHC22 which have distinct cellular functions. CHC17 is implicated in receptor-mediated endocytosis at the plasma membrane and organelle biogenesis at the trans-Golgi network. During clathrin-mediated endocytosis, CHC17 cannot recognise membrane or cargo and so an adaptor protein binds the membrane, selects the cargo, and associates with clathrin leading to pit formation. Several adaptor proteins have clathrin binding sites and colocalize with clathrin structures in cells. Our structural knowledge of these adaptor-clathrin interactions, and their functional importance, is unclear. We analysed the cryo-EM structure of CHC17 cages assembled in the presence of the clathrin-binding subunit (β2-appendage) of assembly polypeptide-2 (AP2) (the adaptor protein that is thought to primarily initiate clathrin recruitment). We found that the β2-appendage binds in at least two positions in the cage. We propose that β2-appendage binding to more than one triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2. These data then led us to ask: is multi-modal binding a fundamental property of clathrin-adaptor interactions? CHC22 acts to sequester the GLUT4 glucose transporter in an insulin-responsive compartment - a behaviour critical to controlling blood sugar levels. Understanding how clathrin self-assembles into basket-like structures to facilitate such cellular functions will significantly advance our understanding of clathrin biology. To this end, we endeavor to map the molecular structure of CHC22.

Clathrin is best known for its contribution to clathrin-mediated endocytosis yet it also participates to a diverse range of cellular functions. Key to this is clathrin's ability to assemble into polyhedral lattices that include curved football or basket shapes, flat lattices or even tubular structures. In this review, we discuss clathrin structure and coated vesicle formation, how clathrin is utilised within different cellular processes including synaptic vesicle recycling, hormone desensitisation, spermiogenesis, cell migration and mitosis, and how clathrin's remarkable 'shapeshifting' ability to form diverse lattice structures might contribute to its multiple cellular functions.

Clathrin-coated pits are formed by the recognition of membrane and cargo by the heterotetrameric AP2 complex and the subsequent recruitment of clathrin triskelia. A potential role for AP2 in coated-pit assembly beyond initial clathrin recruitment has not been explored. Clathrin binds the β2 subunit of AP2, and several binding sites on β2 and on the clathrin heavy chain have been identified, but our structural knowledge of these interactions is incomplete and their functional importance during endocytosis is unclear. Here, we analysed the cryo-EM structure of clathrin cages assembled in the presence of β2 hinge and appendage (β2HA) domains. We find that the β2-appendage binds in at least two positions in the cage, demonstrating that multi-modal binding is a fundamental property of clathrin-AP2 interactions. In one position, β2-appendage cross-links two adjacent terminal domains from different triskelia below the vertex. Functional analysis of β2HA-clathrin interactions reveals that endocytosis requires two clathrin interaction sites: a clathrin-box motif on the hinge and the “sandwich site” on the appendage, with the appendage “platform site” having less importance. From these studies and the work of others, we propose that β2-appendage binding to more than one clathrin triskelion is a key feature of the system and likely explains why clathrin assembly is driven by AP2.