Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network.
We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.
Wu
Xufeng
X
Laboratory of Cell Biology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Zhao
Xiaohong
X
Puertollano
Rosa
R
Bonifacino
Juan S
JS
Eisenberg
Evan
E
Greene
Lois E
LE
D033961:Adaptor Protein Complex 1 / Q000378:metabolism; D033962:Adaptor Protein Complex 2 / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002966:Clathrin / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D011188:Potassium / Q000378:metabolism; D013696:Temperature; D013997:Time Factors; D014162:Transfection; D021601:trans-Golgi Network / Q000378:metabolism*
D033961:Adaptor Protein Complex 1; D033962:Adaptor Protein Complex 2; D002966:Clathrin; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D004247:DNA; D011188:Potassium