BACTERIALLY EXPRESSED F1-20 AP-3 ASSEMBLES CLATHRIN INTO CAGES WITH ANARROW SIZE DISTRIBUTION - IMPLICATIONS FOR THE REGULATION OF QUANTALSIZE DURING NEUROTRANSMISSION/

F1-20/AP-3 is a synapse-specific phosphoprotein, In this study we char acterize the ability of bacterially expressed F1-20/AP-3 to bind and a ssemble clathrin cages, We find that both of two bacterially expressed alternatively spliced isoforms of F1-20/AP-3 can bind and assemble cl athrin as efficiently as preparations of F1-20/AP-3 from bovine brain, This establishes that the clathrin assembly activity found in F1-20/A P-3 preparations from brain extracts is indeed encoded by the cloned g ene for F1-20/AP-3, It also demonstrates that post-translation modific ation is not required for activation of the clathrin binding or assemb ly function of F1-20/AP-3, Ultrastructural analyses of the clathrin ca ges 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 clathri n 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 f ind that the bacterially expressed 33 kD NH2-terminus of F1-20/AP-3 bi nds to clathrin triskelia, fails to bind to preassembled clathrin cage s, and is not sufficient for clathrin assembly, The finding that the 3 3 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-term inus of F1-20/AP-3 fails to bind to clathrin cages is novel and potent ially 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 cla thrin binding; however it appears that defining the regions of the pro tein involved in clathrin binding and assembly may be more complex tha n originally anticipated. (C) 1995 Wiley-Liss, Inc.