Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains.
Annexin 2 is a Ca2+- and phospholipid-binding protein previously identified on endosomal membranes and the plasma membrane. Inferred from this location and its stimulatory effect on membrane transport annexin 2 has been proposed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the distribution of annexin 2 over the endosomal compartment and a characterization of the parameters governing this distribution. Towards this end we have devised an immunoisolation protocol to purify annexin 2-positive membrane vesicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesicles containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for the early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoelectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEAl-positive endosomal vacuoles. We also observed that the Ca2+-independent association of annexin 2 with endosomal membranes was disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could trigger annexin 2 binding to saponin-treated endosomal membranes. Thus, either Ca2+- or cholesterol-stabilized membrane domains are required for the binding of annexin 2 to endosomes suggesting that both factors may regulate this interaction.
Zeuschner
D
D
Institute for Medical Biochemistry, ZMBE, University of Münster, Germany.
Stoorvogel
W
W
Gerke
V
V
D000818:Animals; D017306:Annexin A2 / Q000378:metabolism*; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D002784:Cholesterol / Q000378:metabolism*; D006224:Cricetinae; D011992:Endosomes / Q000378:metabolism*; D007425:Intracellular Membranes / Q000378:metabolism; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron
D017306:Annexin A2; D002784:Cholesterol; D002118:Calcium