Insight into the intra- and inter-molecular interactions of clathrin isoforms CHC17 and CHC22.

Abstract

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.