Analysis of annexin I-mediated membrane aggregation

The main objective of this thesis is to elucidate the mechanisms of annexin I-mediated membrane aggregation. Annexin I belongs to a large family of proteins that reversibly bind the membrane containing anionic phospholipids in a Ca2+-dependent manner. Annexin I and a few other annexins can promote membrane aggregation in vitro, which have led to a hypothesis that annexins are involved in endocytosis and exocytosis. This thesis has three parts.;In the first part, a comprehensive mutagenesis of all six Ca2+ -binding sites of annexin I was performed to elucidate the relationship between membrane binding and membrane aggregation activity of the protein. Results indicate that a type II site in domain II is essential for Ca 2+-dependent vesicle binding of annexin I.;In the second part, the role of amino-terminal region in membrane-aggregation activity of annexin I was investigated. The series of truncated mutants of human annexin I lacking various parts of the amino-terminal region were generated and their membrane binding and aggregation activities were evaluated. Results indicate that both the amino-terminal region of annexin I spanning residues 26--29 and the carboxy-terminal core are involved in membrane aggregation. Lys-26 and Lys-29 were found to be essential for annexin I membrane aggregation activity. Their primary role is to induce and stabilize a secondary site for aggregation for membrane-bound annexin I.;In the third part, detailed molecular mechanism of annexin I-mediated membrane aggregation was determined by various biophysical techniques. First, surface plasmon resonance measurements allowed us to characterize membrane aggregation activity of annexin I independently of its membrane binding. Results suggest that the secondary interaction site can bind secondary membrane directly via hydrophobic interaction and this activity can be modulated by specific mutations in the amino-terminal region of the protein. Also, annexin I was found to bind membranes as a protein monolayer with thickness of (31 +/- 2)A and laterally aggregate on the membranes by x-ray reflectivity and crosslinking, respectively. Overall, the results of these experiments are consistent with a model of annexin I-mediated membrane aggregation in which laterally associated annexin I monolayer directly interacts with a secondary membrane via hydrophobic interaction.