Complexin and Ca2+ stimulate SNARE-mediated membrane fusion

Abstract

Ca2+-triggered, synchronized synaptic vesicle fusion underlies interneuronal communication. Complexin is a major binding partner of the SNARE complex, the core fusion machinery at the presynapse. The physiological data on complexin, however, have been at odds with each other, making delineation of its molecular function difficult. Here we report direct observation of two-faceted functions of complexin using the single-vesicle fluorescence fusion assay and EPR. We show that complexin I has two opposing effects on trans-SNARE assembly: inhibition of SNARE complex formation and stabilization of assembled SNARE complexes. Of note, SNARE-mediated fusion is markedly stimulated by complexin, and it is further accelerated by two orders of magnitude in response to an externally applied Ca2+ wave. We suggest that SNARE complexes, complexins and phospholipids collectively form a complex substrate for Ca2+ and Ca2+-sensing fusion effectors in neurotransmitter release.

C-terminal domain of mammalian complexin-1 localizes to highly curved membranes

“…To study the molecular mechanisms of Cpx, several reconstituted systems have been developed (19,(39)(40)(41)(42). As we reported previously, the C-terminal domain is important for suppression of Ca 2+ -independent fusion in a single-vesicle content mixing assay with reconstituted full-length neuronal SNAREs, and synaptotagmin-1 (19,43,44).…”

Minimal membrane docking requirements revealed by reconstitution of Rab GTPase-dependent membrane fusion from purified components

“…Regulation of SNARE-dependent membrane fusion by the SNARE-interacting protein Sec1/Munc18 (SM) (14,15) and the SNARE-binding proteins synaptotagmin and complexin (16)(17)(18)(19)(20)(21)(22)(23)(24)(25) has also been reconstituted. We have recently reported reconstituted membrane fusion that requires yeast vacuolar SNAREs, regulatory lipids, and the homotypic vacuole fusion and protein sorting (HOPS)/class C vacuole protein sorting (class C Vps) complex, a six-subunit effector and GEF for the yeast vacuolar Rab GTPase Ypt7p (26)(27)(28).…”

Complexin Controls the Force Transfer from SNARE Complexes to Membranes in Fusion

“…Complexin binds to SNARE complexes via its central α-helix, which inserts in an anti-parallel orientation into a groove formed by synaptobrevin/VAMP and syntaxin-1 [8,9]. Although multiple approaches have revealed an essential role of complexin in synaptic fusion [7,[10][11][12][13][14][15], the nature of this role remains unclear. In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10].…”

“…In vertebrate autapses, deletion of complexin selectively impairs fast synchronous neurotransmitter release without changing asynchronous or spontaneous release [7,10]. In in vitro fusion assays, conversely, addition of complexin causes a general block of SNARE-dependent fusion, indicating that complexin is a SNARE clamp [11][12][13][14]. In Drosophila neuromuscular synapses, deletion of complexin produces a >20-fold increase in spontaneous release but only a small decrease in evoked release [15].…”

“…S4). Thus, the complexin knock-down greatly impaired fast synchronous but not asynchronous synaptic vesicle fusion, and increased spontaneous fusion, thereby reconciling the divergent phenotypes observed in vertebrate autapses, Drosophila neuromuscular junctions, and in vitro fusion assays [7,[10][11][12][13][14][15].We next examined which complexin sequences mediate spontaneous and evoked fusion (Fig. 1C).…”