Calcyon, a novel partner of clathrin light chain, stimulatesclathrin-mediated endocytosis

Abstract

In the central nervous system, clathrin-meriate,d endocytosis (CME) is crucial for ' I • efficient synaptic transmission as CME regulates both presynaptic release of neurotransmitter and postsynaptic responses to .transmitter. Clathrin-coated vesicle assembly and disassembly are regulated by som~ 30 adaptor and accessory proteins, most I of which interact with clathrin heavy chain. Calcy_on, which is a single transmembrane protein predominantly expressed in brain, is localized to vesicular compartments within pre and postsynaptic structures. Calcyon has been implicated as a candidate gene for schizophrenia, but the function of calcyon is not yet described. Using the calcyon cytosolic domain as bait, we isolated clathrin light chain (LC) in a yeast two-hybrid screen. The interaction domains were mapped to the heavy chain binding domain and Cterminal regions in LC. In calcyon, residues123 to 155 of calcyon mediated the interaction with LC. Addition of a purified fusion protein containing the calcyon C terminus stimulated clathrin self-assembly in vitro in a dose-dependent fashion. There was a high degree of overlap in the distribution of LC and-calcyon in neuronal processes and cell bodies. Co-immunoprecipitation studies further suggested an association of calcyon with adaptor proteins that play a role in clathrin coated vesicle formation at the plasma membrane, trans Golgi Network and endosomes. Compared to control, HEK293 cells overexpressing calcyon exhibited significantly enhanced transferring (Tfn) uptake but equivalent levels.ofTfn recycli1:1g. Copversely, transferrin uptake was largely abolished in neocortical neurons obta;ined from mice homozygous for a calcyon null allele, whereas recycling proceeded at wild type levels. Deletion of the calcyon gene in mice also inhibited agonist-sti~ulated endocytosis of the GluRl and GluR2 subunits of the ligand gated AMP A type glutamate receptor in primary neurons in cultures. Collectively, these data indicate a role for calcyon in clathrin-mediated endocytosis in brain.