Abstract
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.
Highlights
Clathrin-coated vesicles mediate protein and lipid transport between intracellular compartments in the regulated secretory and endocytic pathways of all eukaryotic cells
We found that M11 was an effective inhibitor of clathrin assembly by the AP180/AP-2 combination, suggesting that AP180-AP-2 complex formation is required for cooperative assembly (Fig. 8C)
We found that 56% less AP-2 was bound to the phosphorylated GST-AP180 resin than to the unphosphorylated GST-AP180 resin, that 48% less AP-2 was bound to the phosphorylated GST-M42 resin than to the unphosphorylated GST-M42 resin, and that 57% less AP-2 was bound to the phosphorylated GST-C58 resin than to the unphosphorylated GST-C58 resin (Fig. 11)
Summary
Clathrin-coated vesicles mediate protein and lipid transport between intracellular compartments in the regulated secretory and endocytic pathways of all eukaryotic cells (reviewed in Ref. 1). AP180 localizes to clathrin-coated vesicles budding from presynaptic plasma membranes [28]. Both AP-2 and AP180 contain high affinity binding sites for inositides, the binding of which inhibits their ability to promote clathrin assembly (29 –35). We show that disruption of the AP180/AP-2 interaction by a recombinant fragment of AP180 containing the AP-2 binding site inhibits the enhanced assembly activity of the AP180 plus AP-2 combination. This indicates that formation of the AP180-AP-2 com-. 901 of AP180; GST-M42, GST fused with amino acids 305–744 of AP180; GST-N33, GST fused with amino acids 1–304 of AP180; MES, 2-(N-morpholino)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PKA, cyclic AMP-dependent protein kinase; PKC, protein kinase C; CCV, clathrin-coated vesicle
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