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Vol. 10, Issue 9, 3035-3044, September 1999
§
and¶
*Department of Neurobiology, Max-Planck-Institute for Biophysical
Chemistry, D-37077 Göttingen, Germany; and 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 Ca2+ chelator
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetra-acetic acid but not by the slow Ca2+ chelator EGTA.
Endosome fusion was restored by the addition of Ca2+ with
an optimum at a free Ca2+ concentration of 0.3 × 10Howard
Hughes Medical Institute and Departments of Pharmacology and Cell
Biology, Yale University School of Medicine, New Haven, Connecticut
06536
6 M. Other divalent cations did not substitute for
Ca2+. A membrane-permeant EGTA derivative caused inhibition
of fusion, which was reversed by addition of Ca2+. 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 Ca2+ from the endosomal interior.
These authors contributed equally to this work.
Present address: Center for Human Genetics, Group
Experimental Medicine, Gasthuisberg KU Leuven, B-3000 Leuven, Belgium.
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