Identification and characterization of a novel type of annexin-membrane interaction: Ca²⁺ is not required for the association of annexin II with early endosomes

Annexin II, a member of a family of Ca²⁺ and membrane binding proteins, has been implicated in regulating membrane organization and membrane transport during endocytosis and Ca²⁺ regulated secretion. To characterize the mechanistic aspects of the annexin II action we studied parameters which determine the endosomal association of annexin II. Immunoblot analysis of subcellular membrane fractions prepared from BHK cells in the presence of a Ca²⁺ chelating agent reveals that annexin II remains associated with endosomal membranes under such conditions. This annexin II behaviour is atypical for the Ca²⁺ regulated annexins and is corroborated by the finding that ectopically expressed annexin II mutants with inactivated Ca²⁺ binding sites continue to co-fractionate with endosomal membranes. The Ca²⁺-independent membrane association of annexin II is also not affected by introducing mutations interfering with the complex formation of annexin II with its intracellular protein ligand p11. However, a deletion of the unique N-terminal domain of annexin II, in particular the sequence spanning residues 15 to 24, abolishes the Ca²⁺-independent association of the protein with endosomes. These results describe a novel, Ca²⁺-independent type of annexin-membrane interaction and provide a first explanation for the observed preference of different annexins for different cellular membranes. In the case of annexin II this specificity could be mediated through specific membrane receptors interacting with a unique sequence in the annexin II molecule.