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 locatio n and its stimulatory effect on membrane transport annexin 2 has been propo sed to play a role in the structural organization and dynamics of endosomal membranes. Validation of this view requires a detailed analysis of the dis tribution of annexin 2 over the endosomal compartment and a characterizatio n of the parameters governing this distribution. Towards this end we have d evised an immunoisolation protocol to purify annexin 2-positive membrane ve sicles from subcellular fractions of BHK cells containing early endosomes. We show that this approach leads to the isolation of intact endosomal vesic les containing internalized fluid-phase marker and that the immunoisolated membranes are positive for the transferrin receptor and Rab4 but not for th e early endosomal antigen EEA1. A distinct and non-uniform distribution of annexin 2 over the early endosomal compartment is also observed in immunoel ectron microscopy analyses of whole-mount specimens of BHK cells. Annexin 2 antibodies labeled transferrin receptor-containing tubular early endosomal structures, but not EEA1-positive endosomal vacuoles. We also observed tha t the Ca2+-independent association of annexin 2 with endosomal membranes wa s disrupted by the cholesterol-binding glycerid saponin, while Ca2+ could t rigger annexin 2 binding to saponin-treated endosomal membranes. Thus, eith er Ca2+- or cholesterol-stabilized membrane domains are required for the bi nding of annexin 2 to endosomes suggesting that both factors may regulate t his interaction.