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EMBO reports 9, 2, 171–178 (2008)
doi:10.1038/sj.embor.7401152
AOP Published online: 11 January 2008
Cell-cycle-dependent binding kinetics for the early endosomal tethering factor EEA1
Trygve Bergeland1, Linda Haugen1, Ole J B Landsverk1, Harald Stenmark2 & Oddmund Bakke1, 3
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1 Centre for Immune Regulation, Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindernveien 31, Oslo 0316, Norway
2 Department of Biochemistry, Institute for Cancer Research, The Norwegian Radium Hospital, Oslo 0310, Norway
3 The Gade Institute, University of Bergen, N-5021 Bergen, Norway
To whom correspondence should be addressed
Oddmund Bakke Tel: +47 22 851 479; E-mail: oddmund.bakke@imbv.uio.no
Received 22 June 2007; Accepted 16 November 2007; Published online 11 January 2008.
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Abstract
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.
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