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Clathrin-coated vesicles are involved in protein and lipid trafficking between intracellular compartments in eukaryotic cells. AP-2 and AP180 are the resident coat proteins of clathrin-coated vesicles in nerve terminals, and interactions between these proteins could be important in vesicle dynamics. AP180 and AP-2 each assemble clathrin efficiently under acidic conditions, but neither protein will assemble clathrin efficiently at physiological pH. We find that there is a direct, clathrin-independent interaction between AP180 and AP-2 and that the AP180-AP-2 complex is more efficient at assembling clathrin under physiological conditions than is either protein alone. AP180 is phosphorylated in vivo, and in crude vesicle extracts its phosphorylation is enhanced by stimulation of casein kinase II, which is known to be present in coated vesicles. We find that recombinant AP180 is a substrate for casein kinase II in vitro and that its phosphorylation weakens both the binding of AP-2 by AP180 and the cooperative clathrin assembly activity of these proteins. We have localized the binding site for AP-2 to amino acids 623-680 of AP180. The AP180/AP-2 interaction can be disrupted by a recombinant AP180 fragment containing the AP-2 binding site, and this fragment also disrupts the cooperative clathrin assembly activity of the AP180-AP-2 complex. These results indicate that AP180 and AP-2 interact directly to form a complex that assembles clathrin more efficiently than either protein alone. Phosphorylation of AP180, by modulating the affinity of AP180 for AP-2, may contribute to the regulation of clathrin assembly in vivo.

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.

F1-20/AP-3 is a synapse-specific phosphoprotein. In this study we characterize the ability of bacterially expressed F1-20/AP-3 to bind and assemble clathrin cages. We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble clathrin as efficiently as preparations of F1-20/AP-3 from bovine brain. This establishes that the clathrin assembly activity found in F1-20/AP-3 preparations from brain extracts is indeed encoded by the cloned gene for F1-20/AP-3. It also demonstrates that post-translation modification is not required for activation of the clathrin binding or assembly function of F1-20/AP-3. Ultrastructural analyses of the clathrin cages assembled by bacterially expressed F1-20/AP-3 reveals a strikingly narrow size distribution. This may be important for the regulation of quantal size during neurotransmission. We also express the 33 kD NH2-terminus of F1-20/AP-3 in E. coli, and measure its ability to bind to clathrin triskelia, to bind to clathrin cages, and to assemble clathrin triskelia into clathrin cages. It has been suggested that the 33 kD NH2-terminus of F1-20/AP-3 constitutes a clathrin binding domain. We find that the bacterially expressed 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia, fails to bind to preassembled clathrin cages, and is not sufficient for clathrin assembly. The finding that the 33 kD NH2-terminus of F1-20/AP-3 binds to clathrin triskelia but fails to assemble clathrin triskelia into clathrin cages is consistent with the published proteolysis studies. The finding that the 33 kD NH2-terminus of F1-20/AP-3 fails to bind to clathrin cages is novel and potentially important. It is clear from these experiments that the 33 kD NH2-terminus of F1-20/AP-3 is sufficient to carry out some aspects of clathrin binding; however it appears that defining the regions of the protein involved in clathrin binding and assembly may be more complex than originally anticipated.

Bacterially expressed synapse-specific clathrin assembly protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 microM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 microM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 microM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 microM. Moreover, 10 microM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of clathrin assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit clathrin assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific clathrin assembly protein AP-3.