Product(s) used in this publication: PepSpots™ Peptides on Cellulose
The clathrin heavy chain N-terminal domain interacts with endocytic adapter proteins via clathrinbinding motifs to assemble clathrin triskelia into cages. However, the precise mechanism of clathrinassembly is not yet known. Clathrinassembly protein AP180 has more clathrinbinding motifs than any other endocytic protein and has a major role in the assembly of the clathrin coat during synaptic vesicle biogenesis. We now demonstrate that some of the previously identified binding motifs in AP180 may be non-functional and that a non-conventional clathrinbindingsequence has a major influence on AP180 function. The related protein, clathrinassembly lymphoid myeloid leukemia protein (CALM), has fewer clathrinbinding motifs and functions ubiquitously in clathrin-mediated endocytosis. The C-terminal ~16 kDa sub-domain in AP180, which has relatively high similarity with CALM, was shown in earlier work to have an unexplained role in clathrinbinding. We identified the specific sequences in this sub-domain that bind to clathrin. Evidence for a role for these sequences in promoting clathrinbinding was examined using in vitro and ex vivo experiments that compared the clathrinbinding ability of site mutants with the wild type sequence. A sequence conserved in both AP180 and CALM (LDSSLA[S/N]LVGNLGI) was found to be the major interaction site and mutation caused a deficit in clathrinassembly, which is the first example of a mutation having this effect. In contrast, single or double mutation of DL(L/F) motifs in full length AP180 had no significant effect on clathrinbinding, despite higher clathrin affinity for isolated peptides containing these motifs. We conclude that the novelclathrin interaction sites identified here in CALM and AP180 have a major role in how these proteins interface with clathrin. This work advances the case that AP180 and CALM are required to use a combination of standard clathrin N-terminal domain binding motifs and the sequence identified here for optimal binding and assembling clathrin.