VESICLE AGGREGATION BY ANNEXIN-I - ROLE OF A SECONDARY MEMBRANE-BINDING SITE

Proteins of the annexin family bind to and aggregate secretion granules or liposomes in the presence of Ca2+. We investigated the mechanism of vesicle aggregation performing experiments in which annexin I bound tb PS liposomes was allowed to aggregate additional liposomes. The protein was initially bound to PS liposomes in 50-100 mu M Ca2+ under nonaggregating conditions; then further liposomes were added, and aggregation was started by increasing Ca2+ to 0.5-1 mM. Coaggregation between both liposome populations was followed using resonance energy transfer (RET) and turbidimetric techniques. In RET experiments, annexin I was bound to liposomes containing N-(7-nitro-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine (NBD-PE), and the second liposome population contained lissamine-Rhodamine-PS. Coaggregation was estimated from NBD fluorescence quenching. Rapid fluorescence and turbidimetric changes were observed, demonstrating coaggregation between both populations of liposomes. Therefore, annexin I molecules may bind two membranes in a bivalent fashion. Rates of coaggregation were similar to the rates of aggregation observed when all vesicles contained protein, indicating that aggregation is mediated only by bivalent annexin I molecules. Thus, membrane aggregation is due to a secondary membrane binding site in annexin I. PS liposomes containing annexin I coaggregated with phosphatidylcholine (PC) liposomes, demonstrating that membrane-bound annexin I binds PC, in contrast with soluble annexin I. Secondary binding to PC was significantly slower than secondary binding to PS, pointing to the importance of negative charge in the secondary membrane. Coaggregation with PS liposomes was stimulated by Ca2+ or by Mg2+ in the millimolar range concentration, whereas the rate of coaggregation with PC was not stimulated by Ca2+ above 50 mu M. Annexin I binding to secondary vesicles was reversible upon addition of EGTA. Hence, secondary binding probably requires Ca2+ in micromolar concentrations, similarly to primary binding.