Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Review Article
  • Published:

Mechanisms of SNARE proteins in membrane fusion

Nature Reviews Molecular Cell Biology volume 25, pages 101–118 (2024)Cite this article

Subjects

Abstract

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) are a family of small conserved eukaryotic proteins that mediate membrane fusion between organelles and with the plasma membrane. SNAREs are directly or indirectly anchored to membranes. Prior to fusion, complementary SNAREs assemble between membranes with the aid of accessory proteins that provide a scaffold to initiate SNARE zippering, pulling the membranes together and mediating fusion. Recent advances have enabled the construction of detailed models describing bilayer transitions and energy barriers along the fusion pathway and have elucidated the structures of SNAREs complexed in various states with regulatory proteins. In this Review, we discuss how these advances are yielding an increasingly detailed picture of the SNARE-mediated fusion pathway, leading from first contact between the membranes via metastable non-bilayer intermediates towards the opening and expansion of a fusion pore. We describe how SNARE proteins assemble into complexes, how this assembly is regulated by accessory proteins and how SNARE complexes overcome the free energy barriers that prevent spontaneous membrane fusion.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Reaction pathway of fusion between two lipid vesicles.
Fig. 2: SNARE assembly–disassembly cycle.
Fig. 3: Pathways for trans-SNARE assembly prior to fusion.

Similar content being viewed by others

References

  1. Kalia, R. & Frost, A. Open and cut: allosteric motion and membrane fission by dynamin superfamily proteins. Mol. Biol. Cell 30, 2097–2104 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pfitzner, A. K., von Filseck, J. M. & Roux, A. Principles of membrane remodeling by dynamic ESCRT-III polymers. Trends Cell Biol. 31, 856–868 (2021).

    Article  CAS  PubMed  Google Scholar 

  3. Westermann, B. Mitochondrial fusion and fission in cell life and death. Nat. Rev. Mol. Cell Biol. 11, 872–884 (2010).

    Article  CAS  PubMed  Google Scholar 

  4. Harrison, S. C. Viral membrane fusion. Virology 479, 498–507 (2015).

    Article  PubMed  Google Scholar 

  5. White, J. M., Ward., A.E., Odongo, L. & Tamm, L.K. Viral membrane fusion: a dance between proteins and lipids. Annu. Rev. Virol. 10, https://doi.org/10.1146/annurev-virology-111821-093413 (2023).

  6. Söllner, T. et al. SNAP receptors implicated in vesicle targeting and fusion. Nature 362, 318–324 (1993).

    Article  PubMed  Google Scholar 

  7. Südhof, T. C. & Rothman, J. E. Membrane fusion: grappling with SNARE and SM proteins. Science 323, 474–477 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  8. Südhof, T. C. The synaptic vesicle cycle. Annu. Rev. Neurosci. 27, 509–547 (2004).

    Article  PubMed  Google Scholar 

  9. Schillemans, M., Karampini, E., Kat, M. & Bierings, R. Exocytosis of Weibel–Palade bodies: how to unpack a vascular emergency kit. J. Thromb. Haemost. 17, 6–18 (2019).

    Article  CAS  PubMed  Google Scholar 

  10. Jahn, R. & Scheller, R. H. SNAREs—engines for membrane fusion. Nat. Rev. Mol. Cell Biol. 7, 631–643 (2006).

    Article  CAS  PubMed  Google Scholar 

  11. Brukman, N. G., Uygur, B., Podbilewicz, B. & Chernomordik, L. V. How cells fuse. J. Cell Biol. 218, 1436–1451 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Petrany, M. J. & Millay, D. P. Cell fusion: merging membranes and making muscle. Trends Cell Biol. 29, 964–973 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Gao, S. & Hu, J. Mitochondrial fusion: the machineries in and out. Trends Cell Biol. 31, 62–74 (2021).

    Article  CAS  PubMed  Google Scholar 

  14. Wickner, W. & Rizo, J. A cascade of multiple proteins and lipids catalyzes membrane fusion. Mol. Biol. Cell 28, 707–711 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Zhang, Y. & Hughson, F. M. Chaperoning SNARE folding and assembly. Annu. Rev. Biochem. 90, 581–603 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Rizo, J. Molecular mechanisms underlying neurotransmitter release. Annu. Rev. Biophys. 51, 377–408 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Brunger, A. T., Choi, U. B., Lai, Y., Leitz, J. & Zhou, Q. Molecular mechanisms of fast neurotransmitter release. Annu. Rev. Biophys. 47, 469–497 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Bubnis, G. & Grubmüller, H. Sequential water and headgroup merger: membrane poration paths and energetics from MD simulations. Biophys. J. 119, 2418–2430 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Smirnova, Y. G., Risselada, H. J. & Müller, M. Thermodynamically reversible paths of the first fusion intermediate reveal an important role for membrane anchors of fusion proteins. Proc. Natl Acad. Sci. USA 116, 2571–2576 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Kozlov, M. M. & Markin, V. S. Possible mechanism of membrane fusion [Russian]. Biofizika 28, 242–247 (1983).

    CAS  PubMed  Google Scholar 

  21. Kuzmin, P. I., Zimmerberg, J., Chizmadzhev, Y. A. & Cohen, F. S. A quantitative model for membrane fusion based on low-energy intermediates. Proc. Natl Acad. Sci. USA 98, 7235–7240 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Kozlov, M. M. & Chernomordik, L. V. Membrane tension and membrane fusion. Curr. Opin. Struct. Biol. 33, 61–67 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Chernomordik, L. V. & Kozlov, M. M. Protein–lipid interplay in fusion and fission of biological membranes. Annu. Rev. Biochem 72, 175–207 (2003).

    Article  CAS  PubMed  Google Scholar 

  24. Cohen, F. S. & Melikyan, G. B. The energetics of membrane fusion from binding, through hemifusion, pore formation, and pore enlargement. J. Membr. Biol. 199, 1–14 (2004).

    Article  CAS  PubMed  Google Scholar 

  25. Fan, Z. A., Tsang, K. Y., Chen, S. H. & Chen, Y. F. Revisit the correlation between the elastic mechanics and fusion of lipid membranes. Sci. Rep. 6, 31470 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Chernomordik, L. V. & Kozlov, M. M. Mechanics of membrane fusion. Nat. Struct. Mol. Biol. 15, 675–683 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Marrink, S. J. & Mark, A. E. The mechanism of vesicle fusion as revealed by molecular dynamics simulations. J. Am. Chem. Soc. 125, 11144–11145 (2003).

    Article  CAS  PubMed  Google Scholar 

  28. Beaven, A. H., Sapp, K. & Sodt, A. J. Simulated dynamic cholesterol redistribution favors membrane fusion pore constriction. Biophys. J. 122, 2162–2175 (2022).

    Article  PubMed  Google Scholar 

  29. Johner, N., Harries, D. & Khelashvili, G. Implementation of a methodology for determining elastic properties of lipid assemblies from molecular dynamics simulations. BMC Bioinform. 17, 161 (2016).

    Article  Google Scholar 

  30. Rice, A., Zimmerberg, J. & Pastor, R. W. Initiation and evolution of pores formed by influenza fusion peptides probed by lysolipid inclusion. Biophys. J. 122, 1018–1032 (2023).

    Article  CAS  PubMed  Google Scholar 

  31. Grafmuller, A., Shillcock, J. & Lipowsky, R. The fusion of membranes and vesicles: pathway and energy barriers from dissipative particle dynamics. Biophys. J. 96, 2658–2675 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  32. Kawamoto, S., Klein, M. L. & Shinoda, W. Coarse-grained molecular dynamics study of membrane fusion: curvature effects on free energy barriers along the stalk mechanism. J. Chem. Phys. 143, 243112 (2015).

    Article  PubMed  Google Scholar 

  33. McLaughlin, S. The electrostatic properties of membranes. Annu. Rev. Biophys. Biophys. Chem. 18, 113–136 (1989).

    Article  CAS  PubMed  Google Scholar 

  34. Lipowsky, R. The conformation of membranes. Nature 349, 475–481 (1991).

    Article  CAS  PubMed  Google Scholar 

  35. Rand, R. P. & Parsegian, V. A. Physical force considerations in model and biological membranes. Can. J. Biochem. Cell Biol. 62, 752–759 (1984).

    Article  CAS  PubMed  Google Scholar 

  36. Israelachvili, J. N. Surface forces. In The Handbook of Surface Imaging and Visualization (ed. Hubbard, A. T.) Ch. 24, 793–817 (Taylor & Francis, 1995).

  37. Tamm, L. K. & Han, X. Viral fusion peptides: a tool set to disrupt and connect biological membranes. Biosci. Rep. 20, 501–518 (2000).

    Article  CAS  PubMed  Google Scholar 

  38. Risselada, H. J. et al. Line-tension controlled mechanism for influenza fusion. PLoS ONE 7, e38302 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Langosch, D. et al. Peptide mimics of SNARE transmembrane segments drive membrane fusion depending on their conformational plasticity. J. Mol. Biol. 311, 709–721 (2001).

    Article  CAS  PubMed  Google Scholar 

  40. Hernandez, J. M., Kreutzberger, A. J. B., Kiessling, V., Tamm, L. K. & Jahn, R. Variable cooperativity in SNARE-mediated membrane fusion. Proc. Natl Acad. Sci. USA 111, 12037–12042 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Brandt, T., Cavellini, L., Kühlbrandt, W. & Cohen, M. M. A mitofusin-dependent docking ring complex triggers mitochondrial fusion in vitro. eLife 5, e14618 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  42. Gui, L., Ebner, J. L., Mileant, A., Williams, J. A. & Lee, K. K. Visualization and sequencing of membrane remodeling leading to influenza virus fusion. J. Virol. 90, 6948–6962 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Witkowska, A., Heinz, L. P., Grubmüller, H. & Jahn, R. Tight docking of membranes before fusion represents a metastable state with unique properties. Nat. Commun. 12, 3606 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Kasson, P. M., Lindahl, E. & Pande, V. S. Atomic-resolution simulations predict a transition state for vesicle fusion defined by contact of a few lipid tails. PLoS Comput. Biol. 6, e1000829 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  45. Larsson, P. & Kasson, P. M. Lipid tail protrusion in simulations predicts fusogenic activity of influenza fusion peptide mutants and conformational models. PLoS Comput. Biol. 9, e1002950 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Scheidt, H. A. et al. Light-induced lipid mixing implies a causal role of lipid splay in membrane fusion. Biochem. Biophys. Acta Biomembr. 1862, 183483 (2020).

    Article  Google Scholar 

  47. Yang, L. & Huang, H. W. Observation of a membrane fusion intermediate structure. Science 297, 1877–1879 (2002).

    Article  CAS  PubMed  Google Scholar 

  48. Aeffner, S., Reusch, T., Weinhausen, B. & Salditt, T. Energetics of stalk intermediates in membrane fusion are controlled by lipid composition. Proc. Natl Acad. Sci. USA 109, E1609–E1618 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Salditt, T. & Aeffner, S. X-ray structural investigations of fusion intermediates: lipid model systems and beyond. Semin. Cell Dev. Biol. 60, 65–77 (2016).

    Article  CAS  PubMed  Google Scholar 

  50. Qian, S. & Rai, D. K. Grazing-angle neutron diffraction study of the water distribution in membrane hemifusion: from the lamellar to rhombohedral phase. J. Phys. Chem. Lett. 9, 5778–5784 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Tiberti, M. L., Antonny, B. & Gautier, R. The transbilayer distribution of polyunsaturated phospholipids determines their facilitating effect on membrane deformation. Soft Matter 16, 1722–1730 (2020).

    Article  CAS  PubMed  Google Scholar 

  52. Chernomordik, L. V. & Kozlov, M. M. Membrane hemifusion: crossing a chasm in two leaps. Cell 123, 375–382 (2005).

    Article  CAS  PubMed  Google Scholar 

  53. Sharma, S. & Lindau, M. The fusion pore, 60 years after the first cartoon. FEBS Lett. 592, 3542–3562 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Risselada, H. J., Bubnis, G. & Grubmüller, H. Expansion of the fusion stalk and its implication for biological membrane fusion. Proc. Natl Acad. Sci. USA 111, 11043–11048 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  55. Chanturiya, A., Chernomordik, L. V. & Zimmerberg, J. Flickering fusion pores comparable with initial exocytotic pores occur in protein-free phospholipid bilayers. Proc. Natl Acad. Sci. USA 94, 14423–14428 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kienle, N., Kloepper, T. H. & Fasshauer, D. Phylogeny of the SNARE vesicle fusion machinery yields insights into the conservation of the secretory pathway in fungi. BMC Evol. Biol. 9, 19 (2009).

    Article  PubMed  PubMed Central  Google Scholar 

  57. Fasshauer, D., Sutton, R. B., Brunger, A. T. & Jahn, R. Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. Proc. Natl Acad. Sci. USA 95, 15781–15786 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. Kloepper, T. H., Kienle, C. N. & Fasshauer, D. An elaborate classification of SNARE proteins sheds light on the conservation of the eukaryotic endomembrane system. Mol. Biol. Cell 18, 3463–3471 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Ma, D., Chen, Z. H., He, Z. P. & Huang, X. Q. A SNARE protein identification method based on iLearnPlus to efficiently solve the data imbalance problem. Front. Genet. 12, 818841 (2022).

    Article  PubMed  PubMed Central  Google Scholar 

  60. Kadkova, A., Radecke, J. & Sorensen, J. B. The SNAP-25 protein family. Neuroscience 420, 50–71 (2019).

    Article  CAS  PubMed  Google Scholar 

  61. McNew, J. A. et al. Ykt6p, a prenylated SNARE essential for endoplasmic reticulum–Golgi transport. J. Biol. Chem. 272, 17776–17783 (1997).

    Article  CAS  PubMed  Google Scholar 

  62. Weimbs, T. et al. A conserved domain is present in different families of vesicular fusion proteins: a new superfamily. Proc. Natl Acad. Sci. USA 94, 3046–3051 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  63. Ungermann, C. & Wickner, W. Vam7p, a vacuolar SNAP-25 homolog, is required for SNARE complex integrity and vacuole docking and fusion. EMBO J. 17, 3269–3276 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  64. Sato, T. K., Darsow, T. & Emr, S. D. Vam7p, a SNAP-25-like molecule, and Vam3p, a syntaxin homolog, function together in yeast vacuolar protein trafficking. Mol. Cell Biol. 18, 5308–5319 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Itakura, E. & Mizushima, N. Syntaxin 17: the autophagosomal SNARE. Autophagy 9, 917–919 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Masuda, E. S., Huang, B. C., Fisher, J. M., Luo, Y. & Scheller, R. H. Tomosyn binds t-SNARE proteins via a VAMP-like coiled coil. Neuron 21, 479–480 (1998).

    Article  CAS  PubMed  Google Scholar 

  67. Pobbati, A. V., Razeto, A., Boddener, M., Becker, S. & Fasshauer, D. Structural basis for the inhibitory role of tomosyn in exocytosis. J. Biol. Chem. 279, 47192–47200 (2004).

    Article  CAS  PubMed  Google Scholar 

  68. Scales, S. J., Hesser, B. A., Masuda, E. S. & Scheller, R. H. Amisyn, a novel syntaxin-binding protein that may regulate SNARE complex assembly. J. Biol. Chem. 277, 28271–28279 (2002).

    Article  CAS  PubMed  Google Scholar 

  69. Hattendorf, D. A., Andreeva, A., Gangar, A., Brennwald, P. J. & Weis, W. I. Structure of the yeast polarity protein Sro7 reveals a SNARE regulatory mechanism. Nature 446, 567–571 (2007).

    Article  CAS  PubMed  Google Scholar 

  70. Sinha, R., Ahmed, S., Jahn, R. & Klingauf, J. Two synaptobrevin molecules are sufficient for vesicle fusion in central nervous system synapses. Proc. Natl Acad. Sci. USA 108, 14318–14323 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  71. Shi, L. et al. SNARE proteins: one to fuse and three to keep the nascent fusion pore open. Science 335, 1355–1359 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. van den Bogaart, G. et al. One SNARE complex is sufficient for membrane fusion. Nat. Struct. Mol. Biol. 17, 358–364 (2010).

    Article  PubMed  PubMed Central  Google Scholar 

  73. Hanson, P. I., Roth, R., Morisaki, H., Jahn, R. & Heuser, J. E. Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy. Cell 90, 523–535 (1997).

    Article  CAS  PubMed  Google Scholar 

  74. Weber, T. et al. SNAREpins: minimal machinery for membrane fusion. Cell 92, 759–772 (1998).

    Article  CAS  PubMed  Google Scholar 

  75. Walter, A. M., Wiederhold, K., Bruns, D., Fasshauer, D. & Sorensen, J. B. Synaptobrevin N-terminally bound to syntaxin–SNAP-25 defines the primed vesicle state in regulated exocytosis. J. Cell Biol. 188, 401–413 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  76. Stein, A., Weber, G., Wahl, M. C. & Jahn, R. Helical extension of the neuronal SNARE complex into the membrane. Nature 460, 525–528 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  77. Zhou, P., Bacaj, T., Yang, X., Pang, Z. P. & Sudhof, T. C. Lipid-anchored SNAREs lacking transmembrane regions fully support membrane fusion during neurotransmitter release. Neuron 80, 470–483 (2013).

    Article  CAS  PubMed  Google Scholar 

  78. Hong, W. SNAREs and traffic. Biochim. Biophys. Acta 1744, 120–144 (2005).

    Article  CAS  PubMed  Google Scholar 

  79. Pelham, H. R. SNAREs and the secretory pathway — lessons from yeast. Exp. Cell Res. 247, 1–8 (1999).

    Article  CAS  PubMed  Google Scholar 

  80. Zwilling, D. et al. Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes with multiple topologies. EMBO J. 26, 9–18 (2007).

    Article  CAS  PubMed  Google Scholar 

  81. Burri, L. & Lithgow, T. A complete set of SNAREs in yeast. Traffic 5, 45–52 (2004).

    Article  CAS  PubMed  Google Scholar 

  82. Koike, S. & Jahn, R. SNAREs define targeting specificity of trafficking vesicles by combinatorial interaction with tethering factors. Nat. Commun. 10, 1608 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  83. Fasshauer, D., Antonin, W., Subramaniam, V. & Jahn, R. SNARE assembly and disassembly exhibit a pronounced hysteresis. Nat. Struct. Biol. 9, 144–151 (2002).

    Article  CAS  PubMed  Google Scholar 

  84. Zhang, Y. Energetics, kinetics, and pathway of SNARE folding and assembly revealed by optical tweezers. Protein Sci. 26, 1252–1265 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  85. Wiederhold, K. & Fasshauer, D. Is assembly of the SNARE complex enough to fuel membrane fusion? J. Biol. Chem. 284, 13142–13152 (2009).

    Article  Google Scholar 

  86. Fasshauer, D., Otto, H., Eliason, W. K., Jahn, R. & Brunger, A. T. Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation. J. Biol. Chem. 272, 28036–28041 (1997).

    Article  CAS  PubMed  Google Scholar 

  87. Nicholson, K. L. et al. Regulation of SNARE complex assembly by an N-terminal domain of the t-SNARE Sso1p. Nat. Struct. Biol. 5, 793–802 (1998).

    Article  CAS  PubMed  Google Scholar 

  88. Ellena, J. F. et al. Dynamic structure of lipid-bound synaptobrevin suggests a nucleation-propagation mechanism for trans-SNARE complex formation. Proc. Natl Acad. Sci. USA 106, 20306–20311 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  89. Liang, B., Kiessling, V. & Tamm, L. K. Prefusion structure of syntaxin-1A suggests pathway for folding into neuronal trans-SNARE complex fusion intermediate. Proc. Natl Acad. Sci. USA 110, 19384–19389 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  90. Liang, B., Dawidowski, D., Ellena, J. F., Tamm, L. K. & Cafiso, D. S. The SNARE motif of synaptobrevin exhibits an aqueous-interfacial partitioning that is modulated by membrane curvature. Biochemistry 53, 1485–1494 (2014).

    Article  CAS  PubMed  Google Scholar 

  91. Lakomek, N. A., Yavuz, H., Jahn, R. & Perez-Lara, A. Structural dynamics and transient lipid binding of synaptobrevin-2 tune SNARE assembly and membrane fusion. Proc. Natl Acad. Sci. USA 116, 8699–8708 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  92. Stief, T. et al. Intrinsic disorder of the neuronal SNARE protein SNAP25a in its pre-fusion conformation. J. Mol. Biol. 435, 168069 (2023).

    Article  CAS  PubMed  Google Scholar 

  93. Lerman, J. C., Robblee, J., Fairman, R. & Hughson, F. M. Structural analysis of the neuronal SNARE protein syntaxin-1A. Biochemistry 39, 8470–8479 (2000).

    Article  CAS  PubMed  Google Scholar 

  94. Misura, K. M., Scheller, R. H. & Weis, W. I. Self-association of the H3 region of syntaxin 1A. Implications for intermediates in SNARE complex assembly. J. Biol. Chem. 276, 13273–13282 (2001).

    Article  CAS  PubMed  Google Scholar 

  95. Sieber, J. J. et al. Anatomy and dynamics of a supramolecular membrane protein cluster. Science 317, 1072–1076 (2007).

    Article  CAS  PubMed  Google Scholar 

  96. Xiao, W., Poirier, M. A., Bennett, M. K. & Shin, Y. K. The neuronal t-SNARE complex is a parallel four-helix bundle. Nat. Struct. Biol. 8, 308–311 (2001).

    Article  CAS  PubMed  Google Scholar 

  97. Margittai, M., Fasshauer, D., Pabst, S., Jahn, R. & Langen, R. Homo- and heterooligomeric SNARE complexes studied by site-directed spin labeling. J. Biol. Chem. 276, 13169–13177 (2001).

    Article  CAS  PubMed  Google Scholar 

  98. Misura, K. M., Gonzalez, L. C. Jr., May, A. P., Scheller, R. H. & Weis, W. I. Crystal structure and biophysical properties of a complex between the N-terminal SNARE region of SNAP25 and syntaxin 1a. J. Biol. Chem. 276, 41301–41309 (2001).

    Article  CAS  PubMed  Google Scholar 

  99. Hesselbarth, J. & Schmidt, C. Mass spectrometry uncovers intermediates and off-pathway complexes for SNARE complex assembly. Commun. Biol. 6, 198 (2023).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  100. Dulubova, I. et al. A conformational switch in syntaxin during exocytosis: role of munc18. EMBO J. 18, 4372–4382 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  101. Margittai, M. et al. Single-molecule fluorescence resonance energy transfer reveals a dynamic equilibrium between closed and open conformations of syntaxin 1. Proc. Natl Acad. Sci. USA 100, 15516–15521 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  102. Fasshauer, D. & Margittai, M. A transient N-terminal interaction of SNAP-25 and syntaxin nucleates SNARE assembly. J. Biol. Chem. 279, 7613–7621 (2004).

    Article  CAS  PubMed  Google Scholar 

  103. Furukawa, N. & Mima, J. Multiple and distinct strategies of yeast SNAREs to confer the specificity of membrane fusion. Sci. Rep. 4, 4277 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  104. Antonin, W. et al. A SNARE complex mediating fusion of late endosomes defines conserved properties of SNARE structure and function. EMBO J. 19, 6453–6464 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  105. Wilhelm, B. G. et al. Composition of isolated synaptic boutons reveals the amounts of vesicle trafficking proteins. Science 344, 1023–1028 (2014).

    Article  CAS  PubMed  Google Scholar 

  106. Bethani, I. et al. The specificity of SNARE pairing in biological membranes is mediated by both proof-reading and spatial segregation. EMBO J. 26, 3981–3992 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  107. Behnia, R. & Munro, S. Organelle identity and the signposts for membrane traffic. Nature 438, 597–604 (2005).

    Article  CAS  PubMed  Google Scholar 

  108. Ungermann, C. & Kummel, D. Structure of membrane tethers and their role in fusion. Traffic 20, 479–490 (2019).

    Article  CAS  PubMed  Google Scholar 

  109. Archbold, J. K., Whitten, A. E., Hu, S. H., Collins, B. M. & Martin, J. L. SNARE-ing the structures of Sec1/Munc18 proteins. Curr. Opin. Struct. Biol. 29, 44–51 (2014).

    Article  CAS  PubMed  Google Scholar 

  110. Toonen, R. F. & Verhage, M. Vesicle trafficking: pleasure and pain from SM genes. Trends Cell Biol. 13, 177–186 (2003).

    Article  CAS  PubMed  Google Scholar 

  111. Yu, I. M. & Hughson, F. M. Tethering factors as organizers of intracellular vesicular traffic. Annu. Rev. Cell Dev. Biol. 26, 137–156 (2010).

    Article  CAS  PubMed  Google Scholar 

  112. Santana-Molina, C., Gutierrez, F. & Devos, D. P. Homology and modular evolution of CATCHR at the origin of the eukaryotic endomembrane system. Genome Biol. Evol. 13, evab125 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  113. Pobbati, A. V., Stein, A. & Fasshauer, D. N- to C-terminal SNARE complex assembly promotes rapid membrane fusion. Science 313, 673–676 (2006).

    Article  CAS  PubMed  Google Scholar 

  114. Liu, Y. et al. SNARE zippering is suppressed by a conformational constraint that Is removed by v-SNARE splitting. Cell Rep. 34, 108611 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  115. Gerber, S. H. et al. Conformational switch of syntaxin-1 controls synaptic vesicle fusion. Science 321, 1507–1510 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  116. Andre, T. et al. The interaction of Munc18-1 helix 11 and 12 with the central region of the VAMP2 SNARE motif is essential for SNARE templating and synaptic transmission. eNeuro 7, https://doi.org/10.1523/ENEURO.0278-20.2020 (2020).

  117. Stepien, K. P., Xu, J., Zhang, X., Bai, X. C. & Rizo, J. SNARE assembly enlightened by cryo-EM structures of a synaptobrevin–Munc18-1–syntaxin-1 complex. Sci. Adv. 8, eabo5272 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  118. Dawidowski, D. & Cafiso, D. S. Munc18-1 and the syntaxin-1 N terminus regulate open-closed states in a t-SNARE complex. Structure 24, 392–400 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  119. Jakhanwal, S., Lee, C. T., Urlaub, H. & Jahn, R. An activated Q-SNARE/SM protein complex as a possible intermediate in SNARE assembly. EMBO J. 36, 1788–1802 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  120. Kraynack, B. A. et al. Dsl1p, Tip20p, and the novel Dsl3(Sec39) protein are required for the stability of the Q/t-SNARE complex at the endoplasmic reticulum in yeast. Mol. Biol. Cell 16, 3963–3977 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  121. Tripathi, A., Ren, Y., Jeffrey, P. D. & Hughson, F. M. Structural characterization of Tip20p and Dsl1p, subunits of the Dsl1p vesicle tethering complex. Nat. Struct. Mol. Biol. 16, 114–123 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  122. Magdziarek, M. et al. Re-examining how Munc13-1 facilitates opening of syntaxin-1. Protein Sci. 29, 1440–1458 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  123. Rizo, J., David, G., Fealey, M. E. & Jaczynska, K. On the difficulties of characterizing weak protein interactions that are critical for neurotransmitter release. FEBS Open Bio 12, 1912–1938 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  124. Kummel, D. et al. Complexin cross-links prefusion SNAREs into a zigzag array. Nat. Struct. Mol. Biol. 18, 927–933 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  125. Zhou, Q. J. et al. Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis. Nature 525, 62–67 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  126. Zhou, Q. J. et al. The primed SNARE–complexin–synaptotagmin complex for neuronal exocytosis. Nature 548, 420–425 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  127. Brunger, A. T. & Leitz, J. The core complex of the Ca2+-triggered presynaptic fusion machinery. J. Mol. Biol. 435, 167853 (2023).

    Article  CAS  PubMed  Google Scholar 

  128. Snyder, D. A., Kelly, M. L. & Woodbury, D. J. SNARE complex regulation by phosphorylation. Cell Biochem. Biophys. 45, 111–123 (2006).

    Article  CAS  PubMed  Google Scholar 

  129. Warner, H., Mahajan, S. & van den Bogaart, G. Rerouting trafficking circuits through posttranslational SNARE modifications. J. Cell Sci. 135, jcs.260112 (2022).

    Article  Google Scholar 

  130. Sabatini, B. L. & Regehr, W. G. Timing of synaptic transmission. Annu. Rev. Physiol. 61, 521–542 (1999).

    Article  CAS  PubMed  Google Scholar 

  131. Imig, C. et al. The morphological and molecular nature of synaptic vesicle priming at presynaptic active zones. Neuron 84, 416–431 (2014).

    Article  CAS  PubMed  Google Scholar 

  132. Pang, Z. P. & Sudhof, T. C. Cell biology of Ca2+-triggered exocytosis. Curr. Opin. Cell Biol. 22, 496–505 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  133. Wolfes, A. C. & Dean, C. The diversity of synaptotagmin isoforms. Curr. Opin. Neurobiol. 63, 198–209 (2020).

    Article  CAS  PubMed  Google Scholar 

  134. Park, Y. & Ryu, J. K. Models of synaptotagmin-1 to trigger Ca2+-dependent vesicle fusion. FEBS Lett. 592, 3480–3492 (2018).

    Article  CAS  PubMed  Google Scholar 

  135. Mohrmann, R., Dhara, M. & Bruns, D. Complexins: small but capable. Cell Mol. Life Sci. 72, 4221–4235 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  136. Lottermoser, J. A. & Dittman, J. S. Complexin membrane interactions: implications for synapse evolution and function. J. Mol. Biol. 435, 167774 (2023).

    Article  CAS  PubMed  Google Scholar 

  137. van den Bogaart, G. et al. Membrane protein sequestering by ionic protein–lipid interactions. Nature 479, 552–555 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  138. Bai, J., Tucker, W. C. & Chapman, E. R. PIP2 increases the speed of response of synaptotagmin and steers its membrane-penetration activity toward the plasma membrane. Nat. Struct. Mol. Biol. 11, 36–44 (2004).

    Article  CAS  PubMed  Google Scholar 

  139. Li, L. Y. et al. Phosphatidylinositol phosphates as co-activators of Ca2+ binding to C-2 domains of synaptotagmin 1. J. Biol. Chem. 281, 15845–15852 (2006).

    Article  CAS  PubMed  Google Scholar 

  140. Söllner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H. & Rothman, J. E. A protein assembly–disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion. Cell 75, 409–418 (1993).

    Article  PubMed  Google Scholar 

  141. Puchades, C., Sandate, C. R. & Lander, G. C. The molecular principles governing the activity and functional diversity of AAA plus proteins. Nat. Rev. Mol. Cell Biol. 21, 43–58 (2020).

    Article  CAS  PubMed  Google Scholar 

  142. Khan, Y. A., White, K. I. & Brunger, A. T. The AAA plus superfamily: a review of the structural and mechanistic principles of these molecular machines. Crit. Rev. Biochem. Mol. 57, 156–187 (2021).

    Article  Google Scholar 

  143. Wickner, W. & Schekman, R. Membrane fusion. Nat. Struct. Mol. Biol. 15, 658–664 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  144. Clary, D. O., Griff, I. C. & Rothman, J. E. SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast. Cell 61, 709–721 (1990).

    Article  CAS  PubMed  Google Scholar 

  145. Zhao, M. L. et al. Mechanistic insights into the recycling machine of the SNARE complex. Nature 518, 61 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  146. Vivona, S. et al. Disassembly of all SNARE complexes by N-ethylmaleimide-sensitive factor (NSF) is initiated by a conserved 1:1 interaction between α-soluble NSF attachment protein (SNAP) and SNARE complex. J. Biol. Chem. 288, 24984–24991 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  147. Winter, U., Chen, X. & Fasshauer, D. A conserved membrane attachment site in α-SNAP facilitates N-ethylmaleimide-sensitive factor (NSF)-driven SNARE complex disassembly. J. Biol. Chem. 284, 31817–31826 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  148. Cipriano, D. J. et al. Processive ATP-driven substrate disassembly by the N-ethylmaleimide-sensitive factor (NSF) molecular machine. J. Biol. Chem. 288, 23436–23445 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  149. White, K. I., Zhao, M., Choi, U. B., Pfuetzner, R. A. & Brunger, A. T. Structural principles of SNARE complex recognition by the AAA plus protein NSF. eLife 7, e38888 (2018).

    Article  PubMed  PubMed Central  Google Scholar 

  150. Ryu, J. K. et al. Spring-loaded unraveling of a single SNARE complex by NSF in one round of ATP turnover. Science 347, 1485–1489 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  151. Yavuz, H. et al. Arrest of trans-SNARE zippering uncovers loosely and tightly docked intermediates in membrane fusion. J. Biol. Chem. 293, 8645–8655 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  152. Xu, H., Jun, Y., Thompson, J., Yates, J. & Wickner, W. HOPS prevents the disassembly of trans-SNARE complexes by Sec17p/Sec18p during membrane fusion. EMBO J. 29, 1948–1960 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  153. Prinslow, E. A., Stepien, K. P., Pan, Y. Z., Xu, J. J. & Rizo, J. Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP. eLife 8, e38880 (2019).

    Article  PubMed  PubMed Central  Google Scholar 

  154. Lobingier, B. T., Nickerson, D. P., Lo, S. Y. & Merz, A. J. SM proteins Sly1 and Vps33 co-assemble with Sec17 and SNARE complexes to oppose SNARE disassembly by Sec18. eLife 3, e02272 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  155. Zick, M., Orr, A., Schwartz, M. L., Merz, A. J. & Wickner, W. T. Sec17 can trigger fusion of trans-SNARE paired membranes without Sec18. Proc. Natl Acad. Sci. USA 112, E2290–E2297 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  156. Finkelstein, A. V., Bogatyreva, N. S., Ivankov, D. N. & Garbuzynskiy, S. O. Protein folding problem: enigma, paradox, solution. Biophys. Rev. 14, 1255–1272 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  157. Manca, F. et al. SNARE machinery is optimized for ultrafast fusion. Proc. Natl Acad. Sci. USA 116, 2435–2442 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  158. Dubuke, M. L. & Munson, M. The secret life of tethers: the role of tethering factors in SNARE complex regulation. Front. Cell Dev. Biol. 4, 42 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  159. Baker, R. W. & Hughson, F. M. Chaperoning SNARE assembly and disassembly. Nat. Rev. Mol. Cell Biol. 17, 465–479 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  160. Chamberlain, L. H., Burgoyne, R. D. & Gould, G. W. SNARE proteins are highly enriched in lipid rafts in PC12 cells: implications for the spatial control of exocytosis. Proc. Natl Acad. Sci. USA 98, 5619–5624 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  161. Lang, T. et al. SNAREs are concentrated in cholesterol-dependent clusters that define docking and fusion sites for exocytosis. EMBO J. 20, 2202–2213 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  162. Barg, S., Knowles, M. K., Chen, X., Midorikawa, M. & Almers, W. Syntaxin clusters assemble reversibly at sites of secretory granules in live cells. Proc. Natl Acad. Sci. USA 107, 20804–20809 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  163. Li, M. L., Oh, T. J., Fan, H. X., Diao, J. J. & Zhang, K. Syntaxin clustering and optogenetic control for synaptic membrane fusion. J. Mol. Biol. 432, 4773–4782 (2020).

    Article  CAS  PubMed  Google Scholar 

  164. Gandasi, N. R. & Barg, S. Contact-induced clustering of syntaxin and munc18 docks secretory granules at the exocytosis site. Nat. Commun. 5, 3914 (2014).

    Article  CAS  PubMed  Google Scholar 

  165. Koike, S. & Jahn, R. SNARE proteins: zip codes in vesicle targeting. Biochem. J. 479, 273–288 (2022).

    Article  CAS  PubMed  Google Scholar 

  166. Hernandez, J. M. et al. Membrane fusion intermediates via directional and full assembly of the SNARE complex. Science 336, 1581–1584 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  167. Ginger, L. et al. Arrangements of proteins at reconstituted synaptic vesicle fusion sites depend on membrane separation. FEBS Lett. 594, 3450–3463 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  168. Kiessling, V. et al. A molecular mechanism for calcium-mediated synaptotagmin-triggered exocytosis. Nat. Struct. Mol. Biol. 25, 911 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  169. Risselada, H. J. & Mayer, A. SNAREs, tethers and SM proteins: how to overcome the final barriers to membrane fusion? Biochem. J. 477, 243–258 (2020).

    Article  CAS  PubMed  Google Scholar 

  170. Domanska, M. K., Kiessling, V. & Tamm, L. K. Docking and fast fusion of synaptobrevin vesicles depends on the lipid compositions of the vesicle and the acceptor SNARE complex-containing target membrane. Biophys. J. 99, 2936–2946 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  171. Wang, L., Seeley, E. S., Wickner, W. & Merz, A. J. Vacuole fusion at a ring of vertex docking sites leaves membrane fragments within the organelle. Cell 108, 357–369 (2002).

    Article  CAS  PubMed  Google Scholar 

  172. Rizzoli, S. O. & Betz, W. J. The structural organization of the readily releasable pool of synaptic vesicles. Science 303, 2037–2039 (2004).

    Article  CAS  PubMed  Google Scholar 

  173. Pieren, M., Desfougeres, Y., Michaillat, L., Schmidt, A. & Mayer, A. Vacuolar SNARE protein transmembrane domains serve as nonspecific membrane anchors with unequal roles in lipid mixing. J. Biol. Chem. 290, 12821–12832 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  174. Dhara, M. et al. Synergistic actions of v-SNARE transmembrane domains and membrane-curvature modifying lipids in neurotransmitter release. eLife 9, e55152 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  175. Hu, Y. R., Zhu, L. & Ma, C. Structural roles for the juxtamembrane linker region and transmembrane region of synaptobrevin 2 in membrane fusion. Front. Cell Dev. Biol. 8, 609708 (2021).

    Article  PubMed  PubMed Central  Google Scholar 

  176. Grote, E., Baba, M., Ohsumi, Y. & Novick, P. J. Geranylgeranylated SNAREs are dominant inhibitors of membrane fusion. J. Cell Biol. 151, 453–466 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  177. Chang, C. W., Chiang, C. W., Gaffaney, J. D., Chapman, E. R. & Jackson, M. B. Lipid-anchored synaptobrevin provides little or no support for exocytosis or liposome fusion. J. Biol. Chem. 291, 2848–2857 (2016).

    Article  CAS  PubMed  Google Scholar 

  178. Ucar, H. et al. Mechanical actions of dendritic-spine enlargement on presynaptic exocytosis. Nature 600, 686–689 (2021).

    Article  CAS  PubMed  Google Scholar 

  179. Rosenmund, C. & Stevens, C. F. Definition of the readily releasable pool of vesicles at hippocampal synapses. Neuron 16, 1197–1207 (1996).

    Article  CAS  PubMed  Google Scholar 

  180. Chakraborty, H., Tarafdar, P. K., Bruno, M. J., Sengupta, T. & Lentz, B. R. Activation thermodynamics of poly(ethylene glycol)-mediated model membrane fusion support mechanistic models of stalk and pore formation. Biophys. J. 102, 2751–2760 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  181. D’Agostino, M., Risselada, H. J., Lurick, A., Ungermann, C. & Mayer, A. A tethering complex drives the terminal stage of SNARE-dependent membrane fusion. Nature 551, 634–638 (2017).

    Article  PubMed  Google Scholar 

  182. Orr, A., Song, H. & Wickner, W. Fusion with wild-type SNARE domains is controlled by juxtamembrane domains, transmembrane anchors, and Sec17. Mol. Biol. Cell 33, ar38 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  183. Lindau, M. & Almers, W. Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion. Curr. Opin. Cell Biol. 7, 509–517 (1995).

    Article  CAS  PubMed  Google Scholar 

  184. Chang, C. W., Chiang, C. W. & Jackson, M. B. Fusion pores and their control of neurotransmitter and hormone release. J. Gen. Physiol. 149, 301–322 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  185. Spruce, A. E., Breckenridge, L. J., Lee, A. K. & Almers, W. Properties of the fusion pore that forms during exocytosis of a mast cell secretory vesicle. Neuron 4, 643–654 (1990).

    Article  CAS  PubMed  Google Scholar 

  186. Chow, R. H., von Ruden, L. & Neher, E. Delay in vesicle fusion revealed by electrochemical monitoring of single secretory events in adrenal chromaffin cells. Nature 356, 60–63 (1992).

    Article  CAS  PubMed  Google Scholar 

  187. Takahashi, N., Kishimoto, T., Nemoto, T., Kadowaki, T. & Kasai, H. Fusion pore dynamics and insulin granule exocytosis in the pancreatic islet. Science 297, 1349–1352 (2002).

    Article  CAS  PubMed  Google Scholar 

  188. Lindau, M. & Alvarez, D. T. The fusion pore. Biochim. Biophys. Acta 1641, 167–173 (2003).

    Article  CAS  PubMed  Google Scholar 

  189. Fesce, R., Grohovaz, F., Valtorta, F. & Meldolesi, J. Neurotransmitter release: fusion or ‘kiss-and-run’? Trends Cell Biol. 4, 1–4 (1994).

    Article  CAS  PubMed  Google Scholar 

  190. Alabi, A. A. & Tsien, R. W. Perspectives on kiss-and-run: role in exocytosis, endocytosis, and neurotransmission. Annu. Rev. Physiol. 75, 393–422 (2013).

    Article  CAS  PubMed  Google Scholar 

  191. Watanabe, S. et al. Ultrafast endocytosis at mouse hippocampal synapses. Nature 504, 242–247 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  192. Holroyd, P., Lang, T., Wenzel, D., De Camilli, P. & Jahn, R. Imaging direct, dynamin-dependent recapture of fusing secretory granules on plasma membrane lawns from PC12 cells. Proc. Natl Acad. Sci. USA 99, 16806–16811 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  193. Taraska, J. W., Perrais, D., Ohara-Imaizumi, M., Nagamatsu, S. & Almers, W. Secretory granules are recaptured largely intact after stimulated exocytosis in cultured endocrine cells. Proc. Natl Acad. Sci. USA 100, 2070–2075 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  194. Taraska, J. W. & Almers, W. Bilayers merge even when exocytosis is transient. Proc. Natl Acad. Sci. USA 101, 8780–8785 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  195. Jackson, M. B. & Chapman, E. R. Fusion pores and fusion machines in Ca2+-triggered exocytosis. Annu. Rev. Biophys. Biomol. Struct. 35, 135–160 (2006).

    Article  CAS  PubMed  Google Scholar 

  196. Wu, Z., Thiyagarajan, S., O’Shaughnessy, B. & Karatekin, E. Regulation of exocytotic fusion pores by SNARE protein transmembrane domains. Front. Mol. Neurosci. 10, 315 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  197. Hastoy, B. et al. A central small amino acid in the VAMP2 transmembrane domain regulates the fusion pore in exocytosis. Sci. Rep. 7, 2835 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  198. Dhara, M., Mohrmann, R. & Bruns, D. v-SNARE function in chromaffin cells. Pflug. Arch. 470, 169–180 (2018).

    Article  CAS  Google Scholar 

  199. Zhang, Y., Ma, L. & Bao, H. Energetics, kinetics, and pathways of SNARE assembly in membrane fusion. Crit. Rev. Biochem. Mol. Biol. 57, 443–460 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  200. Bretou, M., Anne, C. & Darchen, F. A fast mode of membrane fusion dependent on tight SNARE zippering. J. Neurosci. 28, 8470–8476 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  201. Stratton, B. S. et al. Cholesterol increases the openness of SNARE-mediated flickering fusion pores. Biophys. J. 110, 1538–1550 (2016).

    Article  PubMed  PubMed Central  Google Scholar 

  202. Wu, L., Courtney, K. C. & Chapman, E. R. Cholesterol stabilizes recombinant exocytic fusion pores by altering membrane bending rigidity. Biophys. J. 120, 1367–1377 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  203. Kreutzberger, A. J., Kiessling, V. & Tamm, L. K. High cholesterol obviates a prolonged hemifusion intermediate in fast SNARE-mediated membrane fusion. Biophys. J. 109, 319–329 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  204. Kreutzberger, A. J. B. et al. Asymmetric phosphatidylethanolamine distribution controls fusion pore lifetime and probability. Biophys. J. 113, 1912–1915 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  205. Wu, Z. Y. et al. Nanodisc-cell fusion: control of fusion pore nucleation and lifetimes by SNARE protein transmembrane domains. Sci. Rep. 6, 27287 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  206. Bao, H. et al. Exocytotic fusion pores are composed of both lipids and proteins. Nat. Struct. Mol. Biol. 23, 67–73 (2016).

    Article  CAS  PubMed  Google Scholar 

  207. Bao, H. et al. Dynamics and number of trans-SNARE complexes determine nascent fusion pore properties. Nature 554, 260–263 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  208. Das, D., Bao, H., Courtney, K. C., Wu, L. & Chapman, E. R. Resolving kinetic intermediates during the regulated assembly and disassembly of fusion pores. Nat. Commun. 11, 231 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  209. Karatekin, E. Toward a unified picture of the exocytotic fusion pore. FEBS Lett. 592, 3563–3585 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  210. Sharma, S. & Lindau, M. Molecular mechanism of fusion pore formation driven by the neuronal SNARE complex. Proc. Natl Acad. Sci. USA 115, 12751–12756 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  211. Grushin, K., Kalyana Sundaram, R. V., Sindelar, C. V. & Rothman, J. E. Munc13 structural transitions and oligomers that may choreograph successive stages in vesicle priming for neurotransmitter release. Proc. Natl Acad. Sci. USA 119, e2121259119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  212. Holt, M., Riedel, D., Stein, A., Schuette, C. & Jahn, R. Synaptic vesicles are constitutively active fusion machines that function independently of Ca2+. Curr. Biol. 18, 715–722 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  213. Park, Y. et al. Controlling synaptotagmin activity by electrostatic screening. Nat. Struct. Mol. Biol. 19, 991–997 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  214. Kreutzberger, A. J. B. et al. Reconstitution of calcium-mediated exocytosis of dense-core vesicles. Sci. Adv. 3, e1603208 (2017).

    Article  PubMed  PubMed Central  Google Scholar 

  215. Kiessling, V. et al. Rapid fusion of synaptic vesicles with reconstituted target SNARE membranes. Biophys. J. 104, 1950–1958 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  216. Bello, O. D., Auclair, S. M., Rothman, J. E. & Krishnakumar, S. S. Using ApoE nanolipoprotein particles to analyze SNARE-induced fusion pores. Langmuir 32, 3015–3023 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  217. Malsam, J. et al. Complexin arrests a pool of docked vesicles for fast Ca2+-dependent release. EMBO J. 31, 3270–3281 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  218. Schuette, C. G. et al. Determinants of liposome fusion mediated by synaptic SNARE proteins. Proc. Natl Acad. Sci. USA 101, 2858–2863 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  219. Nickel, W. et al. Content mixing and membrane integrity during membrane fusion driven by pairing of isolated v-SNAREs and t-SNAREs. Proc. Natl Acad. Sci. USA 96, 12571–12576 (1999).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  220. Diao, J. et al. Single-vesicle fusion assay reveals Munc18-1 binding to the SNARE core is sufficient for stimulating membrane fusion. ACS Chem. Neurosci. 1, 168–174 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  221. Kyoung, M. et al. In vitro system capable of differentiating fast Ca2+-triggered content mixing from lipid exchange for mechanistic studies of neurotransmitter release. Proc. Natl Acad. Sci. USA 108, E304–E313 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  222. Yoon, T. Y., Okumus, B., Zhang, F., Shin, Y. K. & Ha, T. Multiple intermediates in SNARE-induced membrane fusion. Proc. Natl Acad. Sci. USA 103, 19731–19736 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  223. Cypionka, A. et al. Discrimination between docking and fusion of liposomes reconstituted with neuronal SNARE-proteins using FCS. Proc. Natl Acad. Sci. USA 106, 18575–18580 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  224. Bhalla, A., Chicka, M. C., Tucker, W. C. & Chapman, E. R. Ca2+-synaptotagmin directly regulates t-SNARE function during reconstituted membrane fusion. Nat. Struct. Mol. Biol. 13, 323–330 (2006).

    Article  CAS  PubMed  Google Scholar 

  225. Ma, C., Su, L., Seven, A. B., Xu, Y. & Rizo, J. Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release. Science 339, 421–425 (2013).

    Article  CAS  PubMed  Google Scholar 

  226. Tareste, D., Shen, J., Melia, T. J. & Rothman, J. E. SNAREpin/Munc18 promotes adhesion and fusion of large vesicles to giant membranes. Proc. Natl Acad. Sci. USA 105, 2380–2385 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  227. Fix, M. et al. Imaging single membrane fusion events mediated by SNARE proteins. Proc. Natl Acad. Sci. USA 101, 7311–7316 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  228. Bowen, M. E., Weninger, K., Brunger, A. T. & Chu, S. Single molecule observation of liposome-bilayer fusion thermally induced by soluble N-ethyl maleimide sensitive-factor attachment protein receptors (SNAREs). Biophys. J. 87, 3569–3584 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  229. Karatekin, E. et al. A fast, single-vesicle fusion assay mimics physiological SNARE requirements. Proc. Natl Acad. Sci. USA 107, 3517–3521 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  230. Domanska, M. K., Kiessling, V., Stein, A., Fasshauer, D. & Tamm, L. K. Single vesicle millisecond fusion kinetics reveals number of SNARE complexes optimal for fast SNARE-mediated membrane fusion. J. Biol. Chem. 284, 32158–32166 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  231. Kiessling, V., Domanska, M. K. & Tamm, L. K. Single SNARE-mediated vesicle fusion observed in vitro by polarized TIRFM. Biophys. J. 99, 4047–4055 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  232. Schwenen, L. L. et al. Resolving single membrane fusion events on planar pore-spanning membranes. Sci. Rep. 5, 12006 (2015).

    Article  PubMed  PubMed Central  Google Scholar 

  233. Sutton, R. B., Fasshauer, D., Jahn, R. & Brunger, A. T. Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution. Nature 395, 347–353 (1998).

    Article  CAS  PubMed  Google Scholar 

  234. Tucker, W. C., Weber, T. & Chapman, E. R. Reconstitution of Ca2+-regulated membrane fusion by synaptotagmin and SNAREs. Science 304, 435–438 (2004).

    Article  CAS  PubMed  Google Scholar 

  235. van den Bogaart, G. et al. Synaptotagmin-1 may be a distance regulator acting upstream of SNARE nucleation. Nat. Struct. Mol. Biol. 18, 805–812 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  236. Zdanowicz, R. et al. Complexin binding to membranes and acceptor t-SNAREs explains its clamping effect on fusion. Biophys. J. 113, 1235–1250 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  237. Li, F. et al. A half-zippered SNARE complex represents a functional intermediate in membrane fusion. J. Am. Chem. Soc. 136, 3456–3464 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  238. Brunger, A. T. et al. The pre-synaptic fusion machinery. Curr. Opin. Struct. Biol. 54, 179–188 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  239. Li, X. et al. Symmetrical organization of proteins under docked synaptic vesicles. FEBS Lett. 593, 144–153 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  240. Zhu, J. et al. Synaptotagmin rings as high-sensitivity regulators of synaptic vesicle docking and fusion. Proc. Natl Acad. Sci. USA 119, e2208337119 (2022).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  241. Gao, Y. et al. Single reconstituted neuronal SNARE complexes zipper in three distinct stages. Science 337, 1340–1343 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  242. Park, Y. et al. Synaptotagmin-1 binds to PIP2-containing membrane but not to SNAREs at physiological ionic strength. Nat. Struct. Mol. Biol. 22, 815–823 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

The authors thank H. Grubmüller, M. Müller, S. Pribićević and A. Stein (all Max-Planck Institute for Multidisciplinary Sciences, Göttingen) for critical comments, and A. Chizhik (Third Institute of Physics, University of Göttingen) for help in preparing Fig. 1. Work in the authors’ laboratory was supported by Program Project Grant 2 P01 GM072694 from the National Institutes of Health (NIH) awarded to L.K.T., R.J. and D.S.C.

Author information

Authors and Affiliations

  1. Laboratory of Neurobiology, Max-Planck Institute for Multidisciplinary Sciences, Göttingen, Germany

    Reinhard Jahn

  2. Department of Chemistry, University of Virginia, Charlottesville, VA, USA

    David C. Cafiso

  3. Department of Molecular Physiology and Biological Physics, University of Virginia, Charlottesville, VA, USA

    Lukas K. Tamm

Authors
  1. Reinhard Jahn
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David C. Cafiso
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lukas K. Tamm
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

R.J. researched data for the article. All authors contributed substantially to discussion of the content. R.J. and L.K.T. wrote the article. All authors reviewed and/or edited the manuscript before submission.

Corresponding author

Correspondence to Reinhard Jahn.

Ethics declarations

Competing interests

The authors declare no competing interests.

Peer review

Peer review information

Nature Reviews Molecular Cell Biology thanks Axel Brunger and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Glossary

Black lipid membranes

An experimental model in which single bilayers span a hole 10–100 µm wide created in a Teflon sheet immersed in aqueous media. They appear black because light reflected off the back of the membrane interferes with light reflected from the front.

Content mixing

Mixing of the aqueous interior of two vesicles undergoing fusion. Only occurs after opening of a fusion pore.

Content release

Release of the enclosed content of a vesicle into the environment upon fusion of a vesicle with a planar membrane.

Hysteresis

The state of a system depends on the history (or pathway) by which the state has been reached. Hysteretic systems therefore cannot be modelled using equilibrium thermodynamics where a system can only assume a single state under a defined set of conditions, independent of how it got there.

kT

The product of the Boltzmann constant k with the absolute temperature T, a measure of the thermal energy per molecule. Can be converted into other energy units such as calories, joules or electronvolts.

Lipid mixing

Mixing of membrane lipids during fusion, often describing the stage when the contacting lipid leaflets have mixed and the two distal leaflets remain separated.

Secretory pathway

A system that connects most intracellular membranes, including the plasma membrane of eukaryotic cells, by vesicles that dissociate from the ‘donor’ membrane and then fuse with the target membrane, including the plasma membrane, resulting in secretion of the vesicle content.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jahn, R., Cafiso, D.C. & Tamm, L.K. Mechanisms of SNARE proteins in membrane fusion. Nat Rev Mol Cell Biol 25, 101–118 (2024). https://doi.org/10.1038/s41580-023-00668-x

Download citation

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41580-023-00668-x

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing