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© The Rockefeller University Press, 0021-9525/2002/8/751 $5.00
The Journal of Cell Biology, Volume 158, Number 4, August 19, 2002 751-760

SNAREs in native plasma membranes are active and readily form core complexes with endogenous and exogenous SNAREs

¹ Department of Neurobiology, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany
² High Resolution Optical Microscopy Group, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany

Address correspondence to Reinhard Jahn, Dept. of Neurobiology, Max Planck Institute for Biophysical Chemistry, Am Fassberg, 37077 Göttin-gen, Germany. Tel.: 49-551-201-1635. Fax: 49-551-201-1639. E-mail: rjahn{at}gwdg.de

During neuronal exocytosis, the vesicle-bound soluble NSF attachment protein (SNAP) receptor (SNARE) synaptobrevin 2 forms complexes with the plasma membrane–bound SNAREs syntaxin 1A and SNAP25 to initiate the fusion reaction. However, it is not known whether in the native membrane SNAREs are constitutively active or whether they are unable to enter SNARE complexes unless activated before membrane fusion. Here we used binding of labeled recombinant SNAREs to inside-out carrier supported plasma membrane sheets of PC12 cells to probe for the activity of endogenous SNAREs. Binding was specific, saturable, and depended on the presence of membrane-resident SNARE partners. Our data show that virtually all of the endogenous syntaxin 1 and SNAP-25 are highly reactive and readily form SNARE complexes with exogenously added SNAREs. Furthermore, complexes between endogenous SNAREs were not detectable when the membranes are freshly prepared, but they slowly form upon prolonged incubation in vitro. We conclude that the activity of membrane-resident SNAREs is not downregulated by control proteins but is constitutively active even if not engaged in fusion events.

Key Words: exocytosis; membrane fusion; PC12 cells; in vitro; SNARE reactivity

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