AP180 is a brain enriched protein essential for the recycling of synaptic vesicles after exocytosis and CALM is a ubiquitous homologue. AP180 co-purifies with clathrin-coated vesicle (see purification) and we have shown that it functions to tether clathrin to phosphatidylinositol(4,5)bisphosphate (PtdIns(4,5)P2) containing membranes. Thus in the domain structure above there is a membrane binding domain (ANTH domain) and a clathrin/adaptor binding domain. The adaptor binding is discussed further in other parts of these Web pages but it is not as strong as the clathrin binding, and the dominant phenotype of overexpression is a disruption of clathrin-assembly.

Of all clathrin-binding proteins we have tested, AP180 is the most efficient at recruiting clathrin to membranes.

AP180 recruits clathrin to membranes very efficiently

The stars (*) in the picture above point to the fact that all the clathrin is recruited to the membrane and in experiments where we titrate in more clathrin then it continues to be all recruited until there is a 1:1 stoichiometry between AP180 and clathrin on the membrane. This is because AP180 promotes the polymerisation of clathrin on the membrane. We visualise this by electron microscopy below:

CAPs of clathrin assembled on lipid monolayers
Does this mean that AP180 not only recruits clathrin to PtdIns(4,5)P2 zones in the membrane, and promotes its polymerisation, but that it also defines the size of a coated vesicle? More experiments need to be done to test this.
Platinum shadowed clathrin assemblies
The platinum shadowing (from a single angle) shows that AP180 does not induce curvature in clathrin assemblies (left panel). However the additional presence of AP2 (right panel) does help to induce curvature, (this is better seen in the rotary shadowing in the bottom right corner). A similar effect is seen with amphiphysin and much more extensive invagination is seen in the presence of epsin1. Thus AP180 promotes the polymerisation of clathrin on membranes while playing a role in defining the vesicle size.

In COS cells, overexpression of the C-terminus of AP180 (lipid binding domain is missing) leads to complete inhibition o f transferrin uptake. As clathrin-polymerisation is so important in budding from endosomes and from the TGN, this construct can be used as a general inhibitor for clathrin pathways (browse through our experiments ). In these Web pages we see the disruption of AP1, AP2, EEA1, mannose-6-phosphate receptor and TGN46. Note the complete loss of the M6P receptor implies that there is an upregulation or redirecting of pathways to the lysosome. Thus AP180 C-terminus is 'a real killer' for clathrin budding pathways.