The Role of AP180 and CALM in the Mechanism of Clathrin Coated Vesicle Assembly during Clathrin Mediated Endocytosis

Abstract

Synaptic vesicles (SVs) are formed in the nerve terminal by synaptic vesicle endocytosis (SVE), a specialised form of clathrin mediated endocytosis (CME). CME & SVE are characterised by the formation of a clathrin coat around the budding SV. The CME protein machinery has neuronal homologues that perform SVE. Clathrin assembly lymphoid myeloid leukemia protein (CALM) & assembly protein 180 kDa (AP180) have a crucial role in coat assembly. CALM performs this role in non-neuronal cells, AP180 in neurons. The C-terminal assembly domains (ADs) of AP180/CALM function in clathrin assembly, but a detailed mechanism is lacking. The AP180/CALM ADs contain a clathrin & adapter protein (CLAP) binding sub-domain. A second sub-domain with no motifs appears to be crucial for clathrin binding (ADΔCLAP, the AD minus the CLAP). AP180 also has 14 in vivo phosphorylation sites, phosphorylated when the nerve terminal is at rest. AP180 fusion proteins were designed, truncated & mutated to elucidate AP180-AP2, AP180-clathrin & CALM-clathrin interaction mechanisms. In vitro pull-downs indicated that different clusters of phosphosites regulate AP2 binding & that the phosphosites are involved in direct AP2 binding, or mediate AP2 binding when they are not phosphorylated. All 14 phosphosites prevented clathrin binding in vitro, clathrin assembly activity in vitro & affected clathrin interaction in transferrin uptake assays ex vivo. The ADΔCLAP was essential in vitro for efficient clathrin binding; this observation was extended to the specific clathrin binding sequences homologous in both the AP180/CALM ADΔCLAP. One of 2 binding sequences, Site 1, was conserved & the most important for clathrin binding in vitro, assembly in vitro & clathrin interaction ex vivo. These findings change the prevailing model of phospho-dependent AP180 interaction & AP180/CALM-clathrin interaction, paving the way for structural studies of potential new AP2 & clathrin sites which mediate these novel interactions.