Fusion of endosomes involved in synaptic vesicle recycling

Recycling of vesicles of the regulated secretory pathway presumably involves passage through an early endosomal compartment as an intermediate step. To learn more about the involvement of endosomes in the recycling of synaptic and secretory vesicles we studied in vitro fusion of early endosomes derived 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 exocytotic limb of the pathway. Furthermore, fusion was inhibited by the fast Ca(2+) chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the slow Ca(2+) chelator EGTA. Endosome fusion was restored by the addition of Ca(2+) with an optimum at a free Ca(2+) concentration of 0.3 x 10(-6) M. Other divalent cations did not substitute for Ca(2+). A membrane-permeant EGTA derivative caused inhibition of fusion, which was reversed by addition of Ca(2+). We conclude that the fusion of early endosomes participating in the recycling of synaptic and neurosecretory vesicles is mediated by a set of SNAREs distinct from those involved in exocytosis and requires the local release of Ca(2+) from the endosomal interior.