The Structural and Functional Implications of Linked SNARE Motifs in SNAP25

Abstract

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 SDSresistant 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.

Rapid structural change in synaptosomal-associated protein 25 (SNAP25) precedes the fusion of single vesicles with the plasma membrane in live chromaffin cells

“…2 C and D, faint traces and Table S1). The transient FRET increase is therefore specifically associated with individual fusion events, indicating that SCORE can function in fusion, consistent with a previous report (8), although endogenous unlabeled SNAP25 may also participate.…”

“…The specific correlation of the FRET change with fusion events suggests a trans interaction that also involves synaptobrevin 2 in the vesicle membrane. In a cis complex, synaptobrevin seems to reverse the FRET increase of SCORE produced by the syntaxin interaction (8). It is possible that the reversal of the FRET increase that occurs ∼2 s after fusion is associated with the transition to the cis conformation.…”

“…What molecular interactions does the FRET signal report? It has been shown that SCORE and similar constructs show an increase in FRET ratio upon syntaxin binding (5,7,8). The specific correlation of the FRET change with fusion events suggests a trans interaction that also involves synaptobrevin 2 in the vesicle membrane.…”

“…SCORE and constructs like it (5, 7-9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8). In PC12 cells expressing SCORE, a FRET change was evoked by high [K + ] stimulation, but with this method the time scale was tens of seconds (7).…”

“…To determine whether a conformational change in SNAREs is linked to fusion, the synaptosomal-associated protein 25 (SNAP25) mutant SCORE (SNARE COmplex REporter) has been developed (7), which contains two fluorescent proteins, CFP as a fluorescence resonance energy transfer (FRET) donor and Venus as a FRET acceptor. SCORE and constructs like it (5,(7)(8)(9) have the advantage that donor and acceptor exist at fixed stoichiometry, facilitating the analysis and interpretation of the measurements. Like SNAP25, SCORE forms SNARE complexes with syntaxin and vesicle-associated membrane protein (VAMP)/synaptobrevin (7,8), and when endogenous SNAP25 in chromaffin cells is cleaved by botulinum toxin E, a toxin-resistant SCORE rescues exocytosis (8).…”