Association of annexin 2 with recycling endosomes requires either calcium- or cholesterol-stabilized membrane domains

D Zeuschner, W Stoorvogel, V Gerke

    Research output: Contribution to journalArticleAcademicpeer-review

    Abstract

    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.

    Original languageEnglish
    Pages (from-to)499-507
    Number of pages9
    JournalEuropean Journal of Cell Biology
    Volume80
    Issue number8
    DOIs
    Publication statusPublished - Aug 2001

    Keywords

    • Animals
    • Annexin A2
    • Binding Sites
    • Calcium
    • Cell Line
    • Cholesterol
    • Cricetinae
    • Endosomes
    • Intracellular Membranes
    • Microscopy, Fluorescence
    • Microscopy, Immunoelectron

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