Clathrins are cytoplasmic proteins that play essential roles in endocytosis and other membrane traffic pathways. Upon recruitment to intracellular membranes, the canonical clathrin triskelion assembles into a polyhedral protein coat that facilitates vesicle formation and captures cargo molecules for transport. The triskelion is formed by trimerization of three clathrin heavy-chain subunits. Most vertebrates have two isoforms of clathrin heavy chains, CHC17 and CHC22, generating two clathrins with distinct cellular functions. CHC17 forms vesicles at the plasma membrane for receptor-mediated endocytosis and at the trans-Golgi network for organelle biogenesis. CHC22 plays a key role in intracellular targeting of the insulin-regulated glucose transporter 4 (GLUT4), accumulates at the site of GLUT4 sequestration during insulin resistance, and has also been implicated in neuronal development. Here, we demonstrate that CHC22 and CHC17 share morphological features, in that CHC22 forms a triskelion and latticed vesicle coats. However, cellular CHC22-coated vesicles were distinct from those formed by CHC17. The CHC22 coat was more stable to pH change and was not removed by the enzyme complex that disassembles the CHC17 coat. Moreover, the two clathrins were differentially recruited to membranes by adaptors, and CHC22 did not support vesicle formation or transferrin endocytosis at the plasma membrane in the presence or absence of CHC17. Our findings provide biochemical evidence for separate regulation and distinct functional niches for CHC17 and CHC22 in human cells. Furthermore, the greater stability of the CHC22 coat relative to the CHC17 coat may be relevant to its excessive accumulation with GLUT4 during insulin resistance.
Bibliographical note© 2017 by The American Society for Biochemistry and Molecular Biology, Inc. Author's Choice—Final version free via Creative Commons CC-BY license.
Funding: National Institutes of Health Grants R56-DK083589, P30-DK063720 (a Pilot and Feasibility Study from the UCSF DERC), and R01-DK095663, Wellcome Trust Investigator Award 107858/Z/15/Z (to F. M. B.), a grant from the American Heart Association (to S. M. C.), Japan Society for the Promotion of Science KAKENHI Grant 25460338 (to K. S.), an International Exchange Award by the Royal Society (to C. J. S. and F. M. B.), Medical Research Council Grant G0601125 (to C. J. S. and A. R.), and a University College London Excellence Fellowship (to P. N. D.)