Abstract
Complexins facilitate and inhibit neurotransmitter release through distinct domains, and their function was proposed to be coupled to the Ca2+ sensor synaptotagmin-1 (Syt1). However, the mechanisms underlying complexin function remain unclear. We now uncover an interaction between the complexin N terminus and the SNARE complex C terminus, and we show that disrupting this interaction abolishes the facilitatory function of complexins in mouse neurons. Analyses of hypertonically induced exocytosis show that complexins enhance synaptic-vesicle fusogenicity. Genetic experiments crossing complexin- and Syt1-null mice indicate a functional interaction between these proteins but also show that complexins can promote Ca2+-triggered release in the absence of Syt1. We propose that the complexin N terminus stabilizes the SNARE complex C terminus and/or helps release the inhibitory function of complexins, thereby activating the fusion machinery in a manner that may cooperate with Syt1 but does not require Syt1.
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References
Wickner, W. & Schekman, R. Membrane fusion. Nat. Struct. Mol. Biol. 15, 658–664 (2008).
Jahn, R. & Scheller, R.H. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).
Wojcik, S.M. & Brose, N. Regulation of membrane fusion in synaptic excitation-secretion coupling: speed and accuracy matter. Neuron 55, 11–24 (2007).
Rizo, J. & Rosenmund, C. Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 15, 665–674 (2008).
McMahon, H.T., Missler, M., Li, C. & Sudhof, T.C. Complexins: cytosolic proteins that regulate SNAP receptor function. Cell 83, 111–119 (1995).
Takahashi, S. et al. Identification of two highly homologous presynaptic proteins distinctly localized at the dendritic and somatic synapses. FEBS Lett. 368, 455–460 (1995).
Ishizuka, T., Saisu, H., Odani, S. & Abe, T. Synaphin: a protein associated with the docking/fusion complex in presynaptic terminals. Biochem. Biophys. Res. Commun. 213, 1107–1114 (1995).
Reim, K. et al. Structurally and functionally unique complexins at retinal ribbon synapses. J. Cell Biol. 169, 669–680 (2005).
Pabst, S. et al. Selective interaction of complexin with the neuronal SNARE complex. Determination of the binding regions. J. Biol. Chem. 275, 19808–19818 (2000).
Pabst, S. et al. Rapid and selective binding to the synaptic SNARE complex suggests a modulatory role of complexins in neuroexocytosis. J. Biol. Chem. 277, 7838–7848 (2002).
Li, Y., Augustine, G.J. & Weninger, K. Kinetics of complexin binding to the SNARE complex: correcting single molecule FRET measurements for hidden events. Biophys. J. 93, 2178–2187 (2007).
Bowen, M.E., Weninger, K., Ernst, J., Chu, S. & Brunger, A.T. Single-molecule studies of synaptotagmin and complexin binding to the SNARE complex. Biophys. J. 89, 690–702 (2005).
Chen, X. et al. Three-dimensional structure of the complexin/SNARE complex. Neuron 33, 397–409 (2002).
Bracher, A., Kadlec, J., Betz, H. & Weissenhorn, W. X-ray structure of a neuronal complexin–SNARE complex from squid. J. Biol. Chem. 277, 26517–26523 (2002).
Brose, N. For better or for worse: complexins regulate SNARE function and vesicle fusion. Traffic 9, 1403–1413 (2008).
Huntwork, S. & Littleton, J.T. A complexin fusion clamp regulates spontaneous neurotransmitter release and synaptic growth. Nat. Neurosci. 10, 1235–1237 (2007).
Maximov, A., Tang, J., Yang, X., Pang, Z.P. & Sudhof, T.C. Complexin controls the force transfer from SNARE complexes to membranes in fusion. Science 323, 516–521 (2009).
Reim, K. et al. Complexins regulate a late step in Ca2+-dependent neurotransmitter release. Cell 104, 71–81 (2001).
Xue, M. et al. Complexins facilitate neurotransmitter release at excitatory and inhibitory synapses in mammalian central nervous system. Proc. Natl. Acad. Sci. USA 105, 7875–7880 (2008).
Strenzke, N. et al. Complexin-I is required for high-fidelity transmission at the endbulb of held auditory synapse. J. Neurosci. 29, 7991–8004 (2009).
Cai, H. et al. Complexin II plays a positive role in Ca2+-triggered exocytosis by facilitating vesicle priming. Proc. Natl. Acad. Sci. USA 105, 19538–19543 (2008).
Giraudo, C.G., Eng, W.S., Melia, T.J. & Rothman, J.E. A clamping mechanism involved in SNARE-dependent exocytosis. Science 313, 676–680 (2006).
Schaub, J.R., Lu, X., Doneske, B., Shin, Y.K. & McNew, J.A. Hemifusion arrest by complexin is relieved by Ca2+-synaptotagmin I. Nat. Struct. Mol. Biol. 13, 748–750 (2006).
Yoon, T.Y. et al. Complexin and Ca2+ stimulate SNARE-mediated membrane fusion. Nat. Struct. Mol. Biol. 15, 707–713 (2008).
Malsam, J. et al. The carboxy-terminal domain of complexin I stimulates liposome fusion. Proc. Natl. Acad. Sci. USA 106, 2001–2006 (2009).
Xue, M. et al. Distinct domains of complexin I differentially regulate neurotransmitter release. Nat. Struct. Mol. Biol. 14, 949–958 (2007).
Xue, M. et al. Tilting the balance between facilitatory and inhibitory functions of mammalian and Drosophila complexins orchestrates synaptic vesicle exocytosis. Neuron 64, 367–380 (2009).
Tang, J. et al. A complexin/synaptotagmin 1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175–1187 (2006).
Roggero, C.M. et al. Complexin/synaptotagmin interplay controls acrosomal exocytosis. J. Biol. Chem. 282, 26335–26343 (2007).
Dai, H., Shen, N., Arac, D. & Rizo, J. A quaternary SNARE-synaptotagmin-Ca2+-phospholipid complex in neurotransmitter release. J. Mol. Biol. 367, 848–863 (2007).
Rosenmund, C. & Stevens, C.F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).
Seiler, F., Malsam, J., Krause, J.M. & Sollner, T.H. A role of complexin-lipid interactions in membrane fusion. FEBS Lett. 583, 2343–2348 (2009).
Arac, D. et al. Close membrane-membrane proximity induced by Ca2+-dependent multivalent binding of synaptotagmin-1 to phospholipids. Nat. Struct. Mol. Biol. 13, 209–217 (2006).
Battiste, J.L. & Wagner, G. Utilization of site-directed spin labeling and high-resolution heteronuclear nuclear magnetic resonance for global fold determination of large proteins with limited nuclear overhauser effect data. Biochemistry 39, 5355–5365 (2000).
Stevens, C.F. & Wesseling, J.F. Augmentation is a potentiation of the exocytotic process. Neuron 22, 139–146 (1999).
Basu, J., Betz, A., Brose, N. & Rosenmund, C. Munc13–1 C1 domain activation lowers the energy barrier for synaptic vesicle fusion. J. Neurosci. 27, 1200–1210 (2007).
Gerber, S.H. et al. Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507–1510 (2008).
Geppert, M. et al. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse. Cell 79, 717–727 (1994).
Guan, R., Dai, H. & Rizo, J. Binding of the Munc13–1 MUN domain to membrane-anchored SNARE complexes. Biochemistry 47, 1474–1481 (2008).
Weninger, K., Bowen, M.E., Choi, U.B., Chu, S. & Brunger, A.T. Accessory proteins stabilize the scceptor complex for synaptobrevin, the 1:1 syntaxin/SNAP-25 complex. Structure 16, 308–320 (2008).
Giraudo, C.G. et al. Alternative zippering as an on-off switch for SNARE-mediated fusion. Science 323, 512–516 (2009).
Lu, B., Song, S. & Shin, Y.K. Accessory α-helix of complexin I can displace VAMP2 locally in the complexin–SNARE quaternary complex. J. Mol. Biol. 396, 602–609 (2010).
Xue, M., Ma, C., Craig, T.K., Rosenmund, C. & Rizo, J. The Janus-faced nature of the C(2)B domain is fundamental for synaptotagmin-1 function. Nat. Struct. Mol. Biol. 15, 1160–1168 (2008).
Ashery, U., Betz, A., Xu, T., Brose, N. & Rettig, J. An efficient method for infection of adrenal chromaffin cells using the Semliki Forest virus gene expression system. Eur. J. Cell Biol. 78, 525–532 (1999).
Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).
Chen, X. et al. SNARE-mediated lipid mixing depends on the physical state of the vesicles. Biophys. J. 90, 2062–2074 (2006).
Acknowledgements
We thank H. Deng, H. Chen and X. Zhu for technical assistance, K. Reim and N. Brose (Max Planck Institute of Experimental Medicine, Germany) for providing CplxI KO, CplxII KO and CplxIII KO mice and T. Sudhof (Stanford University) for providing Syt1 KO mice. This work was supported by the US National Institutes of Health (F31MH078678 to H.T.C., NS037200 to J.R. and NS050655 to C.R.), the Welch Foundation (grant I-1304 to J.R.), Baylor Research Advocates for Student Scientists and a McNair Fellowship (both to H.T.C.) and Baylor College of Medicine Mental Retardation and Developmental Disabilities Research Center.
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M.X. performed the physiological studies of Cplx and the Cplx-Syt1 interaction; T.K.C. and J.X. performed the biophysical studies of CplxI–SNARE complex interaction; H.-T.C. contributed to the immunocytochemistry and molecular cloning; M.X., J.R. and C.R. wrote the paper.
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Xue, M., Craig, T., Xu, J. et al. Binding of the complexin N terminus to the SNARE complex potentiates synaptic-vesicle fusogenicity. Nat Struct Mol Biol 17, 568–575 (2010). https://doi.org/10.1038/nsmb.1791
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DOI: https://doi.org/10.1038/nsmb.1791
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