Fusion of rat erythrocytes by membrane-mobility agent A2C depends on membrane proteolysis by a cytoplasmic calpain.

The membrane-mobility agent 2-(2-methoxyethoxy)ethyl-cis-8-(2-octylcyclopropyl)octanoate (A2C) promotes fusion of rat, but not of human, erythrocytes. The difference in fusibility was shown to be correlated with membrane proteolysis, a process induced by Ca2+ in the rat erythrocytes or hemolysate-loaded ghosts, but not in the human cell. Membrane proteolysis is necessary but not sufficient for fusion. Fusion requires both Ca2+ and A2C [Kosower, N. S., Glaser, T. and Kosower, E. M. (1983) Proc. Natl Acad. Sci USA 80, 7542-7546]. Membrane proteolysis (Ca2+-dependent) and fusion (Ca2+ and A2C-dependent) requires a Ca2+-activated cytoplasmic thiol protease, as shown by the following observations. In intact rat erythrocytes, proteolysis and fusion are prevented by thiol alkylation and by inhibitors of Ca2+-dependent thiol proteases. Inhibitors to other proteases have no effect. Erythrocyte ghosts undergo proteolysis and fusion only when loaded with non-inhibited hemolysate, irrespective of membrane status (native or alkylated membrane). A partially purified cytosolic enzyme, identified as calpain I, promotes proteolysis in rat erythrocyte ghosts. A2C induces fusion only in such calpain-treated ghosts.