The interaction of assembly protein AP180 and clathrin is inhibited by multi-site phospho-mimetics.

Clathrin-mediated endocytosis at the nerve terminal is dependent on assembly protein 180 (AP180) and adapter protein complex 2 (AP2). Both membrane adapter proteins bind to each other and to clathrin, to drive assembly of the clathrin coat over nascent synaptic vesicles. Using knowledge of in vivo phosphorylation sites, AP180 was mutated to determine the effect on binding. N-terminally truncated AP180 exhibited phospho-mimetic (Ser/Thr to Glu)-dependent interaction with AP2, but not clathrin. C-terminally truncated and full length phospho-mutant AP180 bound less AP2 than wild type. However, there was no difference in AP2 binding for the phospho-mimetic or phospho-deficient (Ser/Thr to Ala) AP180 mutants. Thus, the phospho-mutant approach did not provide clarity for the role of phosphorylation in AP180-AP2 binding. Clathrin exhibited a phospho-mimetic-dependent interaction with full-length AP180. Furthermore, phospho-mimetic AP180 was deficient at assembling clathrin cages. These latter discoveries support a model where AP180 phosphorylation inhibits clathrin binding and assembly.