Article

Complexin controls the force transfer from SNARE complexes to membranes in fusion.

Department of Neuroscience, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
Science (Impact Factor: 31.48). 02/2009; 323(5913):516-21. DOI: 10.1126/science.1166505
Source: PubMed

ABSTRACT Trans-SNAP receptor (SNARE, where SNAP is defined as soluble NSF attachment protein, and NSF is defined as N-ethylmaleimide-sensitive factor) complexes catalyze synaptic vesicle fusion and bind complexin, but the function of complexin binding to SNARE complexes remains unclear. Here we show that in neuronal synapses, complexin simultaneously suppressed spontaneous fusion and activated fast calcium ion-evoked fusion. The dual function of complexin required SNARE binding and also involved distinct amino-terminal sequences of complexin that localize to the point where trans-SNARE complexes insert into the fusing membranes, suggesting that complexin controls the force that trans-SNARE complexes apply onto the fusing membranes. Consistent with this hypothesis, a mutation in the membrane insertion sequence of the v-SNARE synaptobrevin/vesicle-associated membrane protein (VAMP) phenocopied the complexin loss-of-function state without impairing complexin binding to SNARE complexes. Thus, complexin probably activates and clamps the force transfer from assembled trans-SNARE complexes onto fusing membranes.

0 Followers
 · 
126 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: We have previously proposed that complexin cross-links multiple pre-fusion SNARE complexes via a trans interaction to function as a clamp on SNARE-mediated neurotransmitter release. A recent NMR study was unable to detect the trans clamping interaction of complexin and therefore questioned the previous interpretation of the fluorescence resonance energy transfer and isothermal titration calorimetry data on which the trans clamping model was originally based. Here we present new biochemical data that underscore the validity of our previous interpretation and the continued relevancy of the trans insertion model for complexin clamping.
    eLife Sciences 01/2015; 4. DOI:10.7554/eLife.04463 · 8.52 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: In neuroexocytosis, SNAREs and Munc18-1 may consist of the minimal membrane fusion machinery. Consistent with this notion, we observed, using single molecule fluorescence assays, that Munc18-1 stimulates SNARE zippering and SNARE-dependent lipid mixing in the absence of a major Ca2+-sensor synaptotagmin 1 (syt1), providing the structural basis for the conserved function of SM (Sec1/Munc18) proteins in exocytosis. However, when full-length syt1 is present no enhancement of SNARE zippering and no acceleration of Ca2+-triggered content mixing by Munc18-1 are observed. Thus, our results show that syt1 acts as an antagonist for Munc18-1 in SNAREs zippering and fusion pore opening. Although the SM family may serve as part of the fusion machinery in other exocytotic pathways, Munc18-1 may have evolved to play a different role such as regulating syntaxin 1a in neuroexocytosis. Copyright © 2015, The American Society for Biochemistry and Molecular Biology.
    Journal of Biological Chemistry 02/2015; 290(16). DOI:10.1074/jbc.M114.631341 · 4.60 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: SNAP-25 is a Q-SNARE protein mediating exocytosis of neurosecretory vesicles including chromaffin granules. Previous results with a SNAP-25 construct lacking the nine C terminal residues (SNAP-25Δ9) showed changed fusion pore properties (Fang et al., 2008), suggesting a model for fusion pore mechanics that couple C terminal zipping of the SNARE complex to the opening of the fusion pore. The deleted fragment contains the positively charged residues R198 and K201, adjacent to layers 7 and 8 of the SNARE complex. To determine how fusion pore conductance and dynamics depend on these residues, single exocytotic events in bovine chromaffin cells expressing R198Q, R198E, K201Q, or K201E mutants were investigated by carbon fiber amperometry and cell-attached patch capacitance measurements. Coarse grain molecular dynamics simulations revealed spontaneous transitions between a loose and tightly zippered state at the SNARE complex C terminus. The SNAP-25 K201Q mutant showed no changes compared with SNAP-25 wild-type. However, K201E, R198Q, and R198E displayed reduced release frequencies, slower release kinetics, and prolonged fusion pore duration that were correlated with reduced probability to engage in the tightly zippered state. The results show that the positively charged amino acids at the SNAP-25 C terminus promote tight SNARE complex zippering and are required for high release frequency and rapid release in individual fusion events. Copyright © 2015 the authors 0270-6474/15/353230-10$15.00/0.
    The Journal of Neuroscience : The Official Journal of the Society for Neuroscience 02/2015; 35(7):3230-9. DOI:10.1523/JNEUROSCI.2905-14.2015 · 6.75 Impact Factor

Preview

Download
1 Download
Available from