The SNAREs syntaxin (yellow and orange in the figures), SNAP-25 (green) and synaptobrevin (red) play a crucial role in neurotransmitter release by forming a tight four helix bundle through their SNARE motifs [1, 2] (on the right in the image above). The SNARE complex brings the synaptic vesicle and plasma membranes together, which is key for membrane fusion [3, 4], and is disassembled after membrane fusion by NSF and alphaSNAP (no relation to SNAP-25) [5]. A widespread textbook model assumes that assembly of the SNARE complex involves formation of a syntaxin-SNAP-25 heterodimer on the plasma membrane that binds to synaptobrevin. Our research showed that this non-regulated assembly pathway is not productive because alphaSNAP binds to syntaxin-SNAP-25 heterodimers, preventing synaptobrevin binding, and to trans-SNARE complexes, preventing membrane fusion [6]. The resulting ‘dead-end’ alphaSNAP/SNARE complexes are disassembled by NSF [5, 7-9]. Thus, while many studies reported that the SNAREs and synaptotagmin are sufficient to induce efficient fusion between reconstituted proteoliposomes, such fusion is abolished by NSF-alphaSNAP [7]. We further demonstrated that, in the presence of NSF-alphaSNAP, liposome fusion strictly requires Munc18 and Munc13 (panel A below) because these two proteins orchestrate SNARE complex assembly via a pathway that is resistant to NSF-alphaSNAP [6-9]. These assays reconstituted synaptic vesicle fusion with the most central components of the release machinery and provided a compelling explanation for the total abrogation of neurotransmitter release observed in the absence of Munc18 or Munc13 [10-12].

Initial key findings that led us to delineate this pathway were : i) the identification of an N-terminal domain in syntaxin that forms a three-helix bundle (the Habc domain, orange) [13]; and ii) the discovery that the Habc domain binds intramolecularly to the SNARE motif to form a self-inhibited ‘closed’ conformation that binds tightly to Munc18-1 [14], as shown also by the crystal structure solved in the lab of Bill Weis [15] (on the left in the image above, with Munc18 in violet). Subsequent crystal structures of the yeast vacuolar Munc18 homologue Vps33 bound to yeast homologues of syntaxin and synaptobrevin (in the center of the image above), which were solved in the lab of Fred Hughson, showed that these interactions place the two SNARES close to each other, leading to a model whereby Munc18 and its homologues form a template for SNARE complex assembly [16]. We and others provided strong evidence that neuronal Munc18 also binds to synaptobrevin and forms such a template [16-18]. Thus, Munc18 hinders SNARE complex assembly by binding to closed syntaxin [19] but facilitates assembly in downstream events (panel B).

In parallel research, we identified a large domain in Munc13 called the MUN domain [20]  and showed that this domain facilitates opening of syntaxin, accelerating the transition from the syntaxin-Munc18 complex to the SNARE complex [19, 21, 22]. Moreover, we found that Munc13 also plays a key role in facilitating SNARE complex assembly by bridging the vesicle and plasma membranes [8, 23] through its highly elongated structure [24] (panel B, with Munc13 in cyan; see page on plasticity for further details).

The physiological relevance of this mechanism was supported my multiple evidence, including the observation that a so-called LE mutation that we designed to open syntaxin [14] leads to an increase in neurotransmitter release probability [25] and partially rescues neurotransmitter release in the absence of Unc13, the invertebrate homologue of Munc13, in C. elegans [26]. In fact, the LE mutation rescues defects in neurotransmitter release observed in a wide variety of genetic backgrounds [27], showing the critical functional importance of opening syntaxin. We also showed that the furled conformation of a Munc18-1 loop hinders binding to synaptobrevin and that a mutation that unfurls the loop leads to a strong gain of function in Munc18-1 [18]. Moreover, another mutation that results in a similar gain of function also leads to a partial rescue of release in Unc13 nulls in C. elegans [28].


All these findings show that SNARE complex formation is hindered by multiple energy barriers that render neurotransmitter release highly dependent on Munc13, which acts as a master regulator of release and mediates multiple forms of presynaptic plasticity (see page on plasticity for more details). Thus, we established that assembly of the SNARE complex through the Munc18-Munc13 pathway is critical for the exquisite regulation of release, but fundamental questions remain about the molecular mechanisms underlying how Munc13 opens syntaxin to help forming the template complex of Munc18 with synaptobrevin and syntaxin, and how this template complex transits to the SNARE complex. We are addressing these questions using a combination of cryo-EM, X-ray crystallography, NMR spectroscopy, FRET and reconstitution assays. 


[1] Poirier, M. A., Xiao, W., Macosko, J. C., Chan, C., Shin, Y. K., and Bennett, M. K. (1998) The synaptic SNARE complex is a parallel four-stranded helical bundle, Nat. Struct. Biol 5, 765-769.
[2] Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 A resolution, Nature 395, 347-353.
[3] Hanson, P. I., Roth, R., Morisaki, H., Jahn, R., and Heuser, J. E. (1997) Structure and conformational changes in NSF and its membrane receptor complexes visualized by quick-freeze/deep-etch electron microscopy, Cell 90, 523-535.
[4] Weber, T., Zemelman, B. V., McNew, J. A., Westermann, B., Gmachl, M., Parlati, F., Sollner, T. H., and Rothman, J. E. (1998) SNAREpins: minimal machinery for membrane fusion, Cell 92, 759-772.
[5] Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) A protein assembly-disassembly pathway in vitro that may correspond to sequential steps of synaptic vesicle docking, activation, and fusion, Cell 75, 409-418.
[6] Stepien, K. P., Prinslow, E. A., and Rizo, J. (2019) Munc18-1 is crucial to overcome the inhibition of synaptic vesicle fusion by alphaSNAP, Nat Commun 10, 4326.
[7] Ma, C., Su, L., Seven, A. B., Xu, Y., and Rizo, J. (2013) Reconstitution of the vital functions of Munc18 and Munc13 in neurotransmitter release, Science 339, 421-425.
[8] Liu, X., Seven, A. B., Camacho, M., Esser, V., Xu, J., Trimbuch, T., Quade, B., Su, L., Ma, C., Rosenmund, C., and Rizo, J. (2016) Functional synergy between the Munc13 C-terminal C1 and C2 domains, elife 5, e13696.
[9] Prinslow, E. A., Stepien, K. P., Pan, Y. Z., Xu, J., and Rizo, J. (2019) Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and alphaSNAP, Elife 8, e38880.
[10] Verhage, M., Maia, A. S., Plomp, J. J., Brussaard, A. B., Heeroma, J. H., Vermeer, H., Toonen, R. F., Hammer, R. E., van den Berg, T. K., Missler, M., Geuze, H. J., and Sudhof, T. C. (2000) Synaptic assembly of the brain in the absence of neurotransmitter secretion, Science 287, 864-869.
[11] Richmond, J. E., Davis, W. S., and Jorgensen, E. M. (1999) UNC-13 is required for synaptic vesicle fusion in C. elegans, Nat. Neurosci 2, 959-964.
[12] Varoqueaux, F., Sigler, A., Rhee, J. S., Brose, N., Enk, C., Reim, K., and Rosenmund, C. (2002) Total arrest of spontaneous and evoked synaptic transmission but normal synaptogenesis in the absence of Munc13-mediated vesicle priming, Proc. Natl. Acad. Sci. U. S. A 99, 9037-9042.
[13] Fernandez, I., Ubach, J., Dulubova, I., Zhang, X., Sudhof, T. C., and Rizo, J. (1998) Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A, Cell 94, 841-849.
[14] Dulubova, I., Sugita, S., Hill, S., Hosaka, M., Fernandez, I., Sudhof, T. C., and Rizo, J. (1999) A conformational switch in syntaxin during exocytosis: role of munc18, EMBO J 18, 4372-4382.
[15] Misura, K. M., Scheller, R. H., and Weis, W. I. (2000) Three-dimensional structure of the neuronal-Sec1-syntaxin 1a complex, Nature 404, 355-362.
[16] Baker, R. W., Jeffrey, P. D., Zick, M., Phillips, B. P., Wickner, W. T., and Hughson, F. M. (2015) A direct role for the Sec1/Munc18-family protein Vps33 as a template for SNARE assembly, Science 349, 1111-1114.
[17] Parisotto, D., Pfau, M., Scheutzow, A., Wild, K., Mayer, M. P., Malsam, J., Sinning, I., and Sollner, T. H. (2014) An extended helical conformation in domain 3a of Munc18-1 provides a template for SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complex assembly, J. Biol. Chem 289, 9639-9650.
[18] Sitarska, E., Xu, J., Park, S., Liu, X., Quade, B., Stepien, K., Sugita, K., Brautigam, C. A., Sugita, S., and Rizo, J. (2017) Autoinhibition of Munc18-1 modulates synaptobrevin binding and helps to enable Munc13-dependent regulation of membrane fusion, Elife 6, e24278.
[19] Ma, C., Li, W., Xu, Y., and Rizo, J. (2011) Munc13 mediates the transition from the closed syntaxin-Munc18 complex to the SNARE complex, Nat. Struct. Mol. Biol 18, 542-549.
[20] Basu, J., Shen, N., Dulubova, I., Lu, J., Guan, R., Guryev, O., Grishin, N. V., Rosenmund, C., and Rizo, J. (2005) A minimal domain responsible for Munc13 activity, Nat. Struct. Mol. Biol 12, 1017-1018.
[21] Yang, X., Wang, S., Sheng, Y., Zhang, M., Zou, W., Wu, L., Kang, L., Rizo, J., Zhang, R., Xu, T., and Ma, C. (2015) Syntaxin opening by the MUN domain underlies the function of Munc13 in synaptic-vesicle priming, Nat. Struct. Mol. Biol 22, 547-554.
[22] Magdziarek, M., Bolembach, A. A., Stepien, K. P., Quade, B., Liu, X., and Rizo, J. (2020) Re-examining how Munc13-1 facilitates opening of syntaxin-1, Protein Sci 29, 1440-1458.
[23] Quade, B., Camacho, M., Zhao, X., Orlando, M., Trimbuch, T., Xu, J., Li, W., Nicastro, D., Rosenmund, C., and Rizo, J. (2019) Membrane bridging by Munc13-1 is crucial for neurotransmitter release, Elife 8, e42806.
[24] Xu, J., Camacho, M., Xu, Y., Esser, V., Liu, X., Trimbuch, T., Pan, Y. Z., Ma, C., Tomchick, D. R., Rosenmund, C., and Rizo, J. (2017) Mechanistic insights into neurotransmitter release and presynaptic plasticity from the crystal structure of Munc13-1 C1C2BMUN, Elife 6, e22567.
[25] Gerber, S. H., Rah, J. C., Min, S. W., Liu, X., de, W. H., Dulubova, I., Meyer, A. C., Rizo, J., Arancillo, M., Hammer, R. E., Verhage, M., Rosenmund, C., and Sudhof, T. C. (2008) Conformational switch of syntaxin-1 controls synaptic vesicle fusion, Science 321, 1507-1510.
[26] Richmond, J. E., Weimer, R. M., and Jorgensen, E. M. (2001) An open form of syntaxin bypasses the requirement for UNC-13 in vesicle priming, Nature 412, 338-341.
[27] Stradley, S. J., Rizo, J., Bruch, M. D., Stroup, A. N., and Gierasch, L. M. (1990) Cyclic pentapeptides as models for reverse turns: determination of the equilibrium distribution between type I and type II conformations of Pro-Asn and Pro-Ala beta-turns, Biopolymers 29, 263-287.
[28] Park, S., Bin, N. R., Yu, B., Wong, R., Sitarska, E., Sugita, K., Ma, K., Xu, J., Tien, C. W., Algouneh, A., Turlova, E., Wang, S., Siriya, P., Shahid, W., Kalia, L., Feng, Z. P., Monnier, P. P., Sun, H. S., Zhen, M., Gao, S., Rizo, J., and Sugita, S. (2017) UNC-18 and Tomosyn Antagonistically Control Synaptic Vesicle Priming Downstream of UNC-13 in Caenorhabditis elegans, J Neurosci 37, 8797-8815.