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Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10536-41. doi: 10.1073/pnas.1604000113. Epub 2016 Sep 6.

Kinetic barriers to SNAREpin assembly in the regulation of membrane docking/priming and fusion.

Author information

1
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520;
2
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; pincet@lps.ens.fr james.rothman@yale.edu.
3
Department of Cell Biology, School of Medicine, Yale University, New Haven, CT 06520; Nanobiology Institute, School of Medicine, Yale University, New Haven, CT 06520; Laboratoire de Physique Statistique, Ecole Normale Supérieure, Paris Sciences et Lettres Research University, 75005 Paris, France; Laboratoire de Physique Statistique, Université Paris Diderot Sorbonne Paris Cité, 75005 Paris, France; Laboratoire de Physique Statistique, Sorbonne Universités, Université Pierre et Marie Curie, Univ Paris 06, CNRS, 75005 Paris, France pincet@lps.ens.fr james.rothman@yale.edu.

Abstract

Neurotransmission is achieved by soluble NSF attachment protein receptor (SNARE)-driven fusion of readily releasable vesicles that are docked and primed at the presynaptic plasma membrane. After neurotransmission, the readily releasable pool of vesicles must be refilled in less than 100 ms for subsequent release. Here we show that the initial association of SNARE complexes, SNAREpins, is far too slow to support this rapid refilling owing to an inherently high activation energy barrier. Our data suggest that acceleration of this process, i.e., lowering of the barrier, is physiologically necessary and can be achieved by molecular factors. Furthermore, under zero force, a low second energy barrier transiently traps SNAREpins in a half-zippered state similar to the partial assembly that engages calcium-sensitive regulatory machinery. This result suggests that the barrier must be actively raised in vivo to generate a sufficient pause in the zippering process for the regulators to set in place. We show that the heights of the activation energy barriers can be selectively changed by molecular factors. Thus, it is possible to modify, both in vitro and in vivo, the lifespan of each metastable state. This controllability provides a simple model in which vesicle docking/priming, an intrinsically slow process, can be substantially accelerated. It also explains how the machinery that regulates vesicle fusion can be set in place while SNAREpins are trapped in a half-zippered state.

KEYWORDS:

RRP refilling; SNAREpin assembly; Tomosyn; fluorescence anisotropy; fusion regulation

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