Dynamic Ca2+-Dependent Activity of Membrane-Anchored Synaptotagmin1 Observed at the Content Mixing Level

Abstract

SNARE proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here we used optical tweezers to observe in real time a long-sought SNARE assembly intermediate in which only the membranedistal N-terminal half of the bundle is assembled. Our finding supports the zippering hypothesis, but suggests that zippering proceeds through three sequential binary switches, not continuously, in the N-and C-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force which mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 kBT to mediate fusion. 464-Pos Board B233Mechanical Unzipping and Rezipping of a Single SNARE Complex Reveals Large Hysteresis as the Force Generating Mechanism