Trafficking of Yellow‐Fluorescent‐Protein‐Tagged µ1 Subunit of Clathrin Adaptor AP‐1 Complex in Living Cells

Clathrin adaptor protein AP‐1 complex is thought to function in forming clathrin‐coated vesicles at the trans ‐Golgi network (TGN) and mediating transport 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 µ1 A subunit of AP‐1. When expressed in a tetracycline‐dependent manner in HeLa cells, YFP‐µ1 was efficiently incorporated into the AP‐1 complex, replacing endogenous µ1 in most of cellular AP‐1. Time‐lapse imaging revealed that YFP‐µ1/AP‐1 departs from TGN as isolated vesicles and spherical structures, or varicosities, associated with fine tubular processes. Typically, several vesicles or varicosities were seen moving sequentially along the same ‘tracks’ from TGN to cell periphery. These data suggest that AP‐1 may function after formation of Golgi transport intermediates in facilitating their intracellular movement. Mutagenesis of YFP‐µ1 determined that the structural requirements for its binding to tyrosine‐containing sequence motifs are similar to those previously defined in µ2 subunit of AP‐2. Moreover, the carboxyl‐terminal half of µ2 could replace the corresponding fragment of µ1 without loss of the ability of the resulting µ1‐YFP‐µ2 chimeric protein to incorporate into AP‐1 and bind tyrosine‐containing motifs. Mutations that abolish binding capacity for tyrosine motifs did not mistarget AP‐1 in the cell, suggesting that AP‐1 interactions with this type of sorting signals are not essential for membrane docking of AP‐1 at the TGN. Altogether, this study demonstrates that YFP‐tagged µ1 protein can serve as a useful tool for visualizing the dynamics of AP‐1 in living cells and for the structure‐function analysis of µ1–cargo interactions.