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 to PS liposomes was allowed to aggregate additional liposomes. The protein was initially bound to PS liposomes in 50-100 microM 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.(ABSTRACT TRUNCATED AT 250 WORDS)
de la Fuente
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Departamento de Fisiología y Biofísica, Facultad de Medicina, Universidad de Chile, Santiago.
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D017305:Annexin A1 / Q000378:metabolism / Q000494:pharmacology*; D001665:Binding Sites; D002118:Calcium / Q000494:pharmacology; D002462:Cell Membrane / Q000378:metabolism*; D004533:Egtazic Acid / Q000494:pharmacology; D004735:Energy Transfer; D005260:Female; D005456:Fluorescent Dyes; D006801:Humans; D008081:Liposomes / Q000378:metabolism*; D008274:Magnesium / Q000494:pharmacology; D010713:Phosphatidylcholines / Q000378:metabolism; D010714:Phosphatidylethanolamines / Q000378:metabolism; D010718:Phosphatidylserines / Q000378:metabolism; D010920:Placenta / Q000737:chemistry; D012235:Rhodamines; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry
D017305:Annexin A1; D005456:Fluorescent Dyes; D008081:Liposomes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D012235:Rhodamines; D004533:Egtazic Acid; C042077:N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)phosphatidylethanolamine; D008274:Magnesium; D002118:Calcium