Fusion of endosomes involved in synaptic vesicle recycling

Recycling of vesicles of the regulated secretory pathway presumably involve s passage through an early endosomal compartment as an intermediate step. T o learn more about the involvement of endosomes in the recycling of synapti c and secretory vesicles we studied in vitro fusion of early endosomes deri ved from pheochromocytoma (PC12) cells. Fusion was not affected by cleavage of the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins synaptobrevin and syntaxin 1 that operate at the exocyt otic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca2 + chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-acetic acid but not by t he slow Ca2+ chelator EGTA. Endosome fusion was restored by the addition of Ca2+ with an optimum at a free Ca2+ concentration of 0.3 x 10(-6) M, Other divalent cations did not substitute for Ca2+. A membrane-permeant EGTA der ivative caused inhibition of fusion, which was reversed by addition of Ca2. We conclude that the fusion of early endosomes participating in the recyc ling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca2+ from the endosomal interior.