Cell-cycle-dependent binding kinetics for the early endosomal tethering factor EEA1.
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.
Bergeland
Trygve
T
Department of Molecular Biosciences, University of Oslo, PO Box 1041, Blindernveien 31, Oslo 0316, Norway.
Haugen
Linda
L
Landsverk
Ole J B
OJ
Stenmark
Harald
H
Bakke
Oddmund
O
D000818:Animals; D000944:Antigens, Differentiation, B-Lymphocyte / Q000378:metabolism; D002453:Cell Cycle / Q000502:physiology*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D004285:Dogs; D011992:Endosomes / Q000378:metabolism*; D036681:Fluorescence Recovery After Photobleaching; D000949:Histocompatibility Antigens Class II / Q000378:metabolism; D007399:Interphase; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008677:Metaphase; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000378:metabolism*; D020696:rab5 GTP-Binding Proteins / Q000378:metabolism
D000944:Antigens, Differentiation, B-Lymphocyte; D000949:Histocompatibility Antigens Class II; D008565:Membrane Proteins; D018129:Phosphatidylinositol Phosphates; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; C093846:early endosome antigen 1; C045084:invariant chain; C055525:phosphatidylinositol 3-phosphate; D020696:rab5 GTP-Binding Proteins