Trafficking of yellow-fluorescent-protein-tagged mu 1 subunit of clathrin adaptor AP-1 complex in living cells

Clathrin adaptor protein AP-1 complex is thought to function in forming cla thrin-coated vesicles at the trans-Golgi network (TGN) and mediating transp ort of cargo between the TGN and endosomes. To study trafficking of AP-1 in living cells, yellow fluorescent protein (YFP) was inserted in the middle of mu1 A subunit of AP-1. When expressed in a tetracycline-dependent manner in HeLa cells, YFP-mu1 was efficiently incorporated into the AP-1 complex, replacing endogenous mu1 in most of cellular AP-1. Time-lapse imaging reve aled that YFP-mu1/AP-1 departs from TGN as isolated vesicles and spherical structures, or varicosities, associated with fine tubular processes. Typica lly, several vesicles or varicosities were seen moving sequentially along t he same 'tracks' from TGN to cell periphery. These data suggest that AP-1 m ay function after formation of Golgi transport intermediates in facilitatin g their intracellular movement. Mutagenesis of YFP-mu1 determined that the structural requirements for its binding to tyrosine-containing sequence mot ifs are similar to those previously defined in mu2 subunit of AP-2. Moreove r, the carboxyl-terminal half of mu2 could replace the corresponding fragme nt of mu1 without loss of the ability of the resulting mu1-YFP-mu2 chimeric protein to incorporate into AP-1 and bind tyrosine-containing motifs. Muta tions that abolish binding capacity for tyrosine motifs did not mistarget A P-1 in the cell, suggesting that AP-1 interactions with this type of sortin g signals are not essential for membrane docking of AP-1 at the TGN. Altoge ther, this study demonstrates that YFP-tagged mu1 protein can serve as a us eful tool for visualizing the dynamics of AP-1 in living cells and for the structure-function analysis of mu1-cargo interactions.