Trafficking of yellow-fluorescent-protein-tagged mu1 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 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 revealed that YFP-mu1/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-mu1 determined that the structural requirements for its binding to tyrosine-containing sequence motifs are similar to those previously defined in mu2 subunit of AP-2. Moreover, the carboxyl-terminal half of mu2 could replace the corresponding fragment 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. 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 mu1 protein can serve as a useful tool for visualizing the dynamics of AP-1 in living cells and for the structure-function analysis of mu1-cargo interactions.
Huang
F
F
Department of Pharmacology, University of Colorado Health Science Center, Denver, CO 80262, USA.
Nesterov
A
A
Carter
R E
RE
Sorkin
A
A
D001426:Bacterial Proteins / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D005456:Fluorescent Dyes; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007202:Indicators and Reagents; D008164:Luminescent Proteins / Q000378:metabolism*; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D017354:Point Mutation; D011233:Precipitin Tests; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D014443:Tyrosine