Enhancement of the Ca2+-triggering steps of native membrane fusion via thiol-reactivity

Abstract

Ca2+-triggered membrane fusion is the defining step of exocytosis. Isolated urchin cortical vesicles (CV) provide a stage-specific preparation to study the mechanisms by which Ca2+ triggers the merger of two apposed native membranes. Thiol-reactive reagents that alkylate free sulfhydryl groups on proteins have been consistently shown to inhibit triggered fusion. Here, we characterize a novel effect of the alkylating reagent iodoacetamide (IA). IA was found to enhance the kinetics and Ca2+ sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr2+, a weak Ca2+ mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca2+, suggesting that IA acts at the Ca2+-sensing step of triggered fusion. Comparison of IA to other reagents indicates that there are at least two distinct thiol sites involved in the underlying fusion mechanism: one that regulates the efficiency of fusion and one that interferes with fusion competency. © 2008 Springer-Verlag.