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Originally published as MBC in Press, 10.1091/mbc.E08-04-0344 on July 2, 2008

Vol. 19, Issue 9, 3944-3955, September 2008

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The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Li Wang*,{dagger}, Mary A. Bittner*,{dagger}, Daniel Axelrod{ddagger}, and Ronald W. Holz*

Departments of *Pharmacology and {ddagger}Physics and LSA Biophysics, University of Michigan, Ann Arbor, MI 48109-5632

Submitted April 3, 2008; Revised May 29, 2008; Accepted June 19, 2008
Monitoring Editor: Thomas F. J. Martin

InCytes from MBC

We investigated the functional and structural implications of SNAP25 having two SNARE motifs (SN1 and SN2). A membrane-bound, intramolecular FRET probe was constructed to report on the folding of N-terminal SN1 and C-terminal SN2 in living cells. Membrane-bound constructs containing either or both SNARE motifs were also singly labeled with donor or acceptor fluorophores. Interaction of probes with other SNAREs was monitored by the formation of SDS-resistant complexes and by changes in FRET measured in vitro using spectroscopy and in the plasma membrane of living cells using TIRF microscopy. The probes formed the predicted SDS-resistant SNARE complexes. FRET measurements revealed that syntaxin induced a close association of the N-termini of SN1 and SN2. This association required that the SNARE motifs reside in the same molecule. Unexpectedly, the syntaxin-induced FRET was prevented by VAMP. Both full-length SNAP25 constructs and the combination of its separated, membrane-bound constituent chains supported secretion in permeabilized chromaffin cells that had been allowed to rundown. However, only full-length SNAP25 constructs enabled robust secretion from intact cells or permeabilized cells before rundown. The experiments suggest that the bidentate structure permits specific conformations in complexes with syntaxin and VAMP and facilitates the function of SN1 and SN2 in exocytosis.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E08-04-0344) on July 2, 2008.

{dagger} These authors contributed equally to this work.

Address correspondence to: Ronald W. Holz (holz{at}umich.edu)


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