Clathrin binding and assembly activities of expressed domains of the synapse-specific clathrin assembly protein AP-3.

We separately expressed the 58-kDa C-terminal, 42-kDa middle, 16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called F1-20, AP180, NP185, and pp155), and determined their clathrin binding and assembly properties. The 58-kDa C-terminal region of AP-3 is able to bind to clathrin triskelia and assemble them into a homogeneous population of clathrin cages and will also bind to preassembled clathrin cages. The 42-kDa central region of AP-3 can bind to both clathrin triskelia and to clathrin cages, but cannot assemble clathrin triskelia into clathrin cages. The 16-kDa C-terminal region of AP-3 can bind to clathrin cages, but cannot bind to clathrin triskelia or assemble clathrin triskelia into clathrin cages. The clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of AP-3 is able to bind to clathrin triskelia (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26), it does not promote their assembly into clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. CHem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled clathrin cages (Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It appears that the smallest functional unit that carries out all of the reported clathrin binding and assembly properties of AP-3, essentially as well as the full-length protein, is the 58-kDa C-terminal region.