Single reconstituted neuronal SNARE complexes zipper in three distinct stages.
Soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins drive membrane fusion by assembling into a four-helix bundle in a zippering process. Here, we used optical tweezers to observe in a cell-free reconstitution experiment in real time a long-sought SNARE assembly intermediate in which only the membrane-distal amino-terminal half of the bundle is assembled. Our findings support the zippering hypothesis, but suggest that zippering proceeds through three sequential binary switches, not continuously, in the amino- and carboxyl-terminal halves of the bundle and the linker domain. The half-zippered intermediate was stabilized by externally applied force that mimicked the repulsion between apposed membranes being forced to fuse. This intermediate then rapidly and forcefully zippered, delivering free energy of 36 k(B)T (where k(B) is Boltzmann's constant and T is temperature) to mediate fusion.
Department of Cell Biology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520, USA.
Ying
Y
Gao
Sylvain
S
Zorman
Gregory
G
Gundersen
Zhiqun
Z
Xi
Lu
L
Ma
George
G
Sirinakis
James E
JE
Rothman
Yongli
Y
Zhang
D002474:Cell-Free System; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D019277:Entropy; D009474:Neurons / Q000378:metabolism; D052898:Optical Tweezers; D050765:Qa-SNARE Proteins / Q000737:chemistry; D050600:SNARE Proteins / Q000737:chemistry*; D050988:Vesicle-Associated Membrane Protein 2 / Q000737:chemistry
D050765:Qa-SNARE Proteins; D050600:SNARE Proteins; D050988:Vesicle-Associated Membrane Protein 2; D004247:DNA