Structure and Dynamics of a Two-Helix SNARE Complex in Live Cells

Abstract

SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Qa-, Qb-, Qc- and R-SNARE-motif form a complex. The core complex represents a QaQbQcR-SNARE-motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)-studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Qa), SNAP25 (Qb/Qc) and synaptobrevin 2 (R)]. We find that the Qb-SNARE-motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Qb-SNARE-motif and depends on its position within the molecule. We conclude that a zippered QaQb-SNARE complex represents a short-lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion. © 2009 John Wiley & Sons A/S.