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Cell‐cycle‐dependent binding kinetics for the early endosomal tethering factor EEA1
Author(s) -
Bergeland Trygve,
Haugen Linda,
Landsverk Ole J B,
Stenmark Harald,
Bakke Oddmund
Publication year - 2008
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.1038/sj.embor.7401152
Subject(s) - endosome , microbiology and biotechnology , fluorescence recovery after photobleaching , mitosis , cytosol , tethering , lipid bilayer fusion , chemistry , biophysics , biology , biochemistry , membrane , intracellular , enzyme
Early endosomal antigen 1 (EEA1) is a cytosolic protein that specifically binds to early endosomal membranes where it has a crucial role in the tethering process leading to homotypic endosome fusion. Green fluorescent protein‐tagged EEA1 (EEA1‐GFP) was bound to the endosomal membrane throughout the cell cycle, and measurements using fluorescent recovery after photobleaching showed two fractions: one rapidly exchanging with the cytosolic pool, and the other with a long half‐life. The exchange consists of a release and binding process, and we have separated these two by using GFP and photoactivable GFP. The release rate was identical to the exchange rate, showing that the dissociation characteristics determine the cycling of this molecule. During mitosis, we found that the dissociation rate was markedly accelerated and, in addition, the long‐lived fraction was markedly reduced. This indicates that a fusion arrest in mitosis is not the result of EEA1 not binding to early endosomes, but rather due to the marked shift in membrane‐binding characteristics. This might be a general mechanism to fine‐tune and control tethering and fusion of early endosomes.