Enhancement of the Ca(2+)-triggering steps of native membrane fusion via thiol-reactivity.
Ca(2+)-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 Ca(2+) 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 Ca(2+) sensitivity of both CV-plasma membrane and CV-CV fusion. If Sr(2+), a weak Ca(2+) mimetic, was used to trigger fusion, the potentiation was even greater than that observed for Ca(2+), suggesting that IA acts at the Ca(2+)-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.
Furber
Kendra L
KL
Department of Physiology and Biophysics, University of Calgary Faculty of Medicine, Calgary, AB, T2N 4N1, Canada.
Brandman
David M
DM
Coorssen
Jens R
JR