The ability of neurons to communicate with each other is fundamental for neural function. Information transfer occurs via neurotransmitters that are released from presynaptic terminals by Ca²⁺-evoked synaptic vesicle exocytosis and act on receptors in postsynaptic membranes. In addition to triggering exocytosis, Ca²⁺ regulates the efficiency of release in diverse processes of presynaptic plasticity that mediate some forms of information processing in the brain. The laboratory of Josep Rizo (also known as Jose Rizo-Rey) is interested in elucidating the mechanisms of neurotransmitter release and its regulation through analysis of the three-dimensional structure and interactions of proteins involved in this process. This research is not only relevant to neurotransmission but also to protein sorting and secretion in general, since most types of intracellular membrane traffic share a common membrane fusion machinery.

Our studies use a wide range of biophysical techniques that include multidimensional NMR spectroscopy, X-ray crystallography, cryo-electron microscopy and fluorescence spectroscopy among others. Our philosophy is to use whichever technique is most suitable to address specific, critical questions that arise as we and others keep making progress in shedding light onto the mechanisms of neurotransmitter release and intracellular membrane fusion in general. This work is performed in close collaboration with several laboratories, in particular those of Thomas Sudhof and Christian Rosenmund, where a variety of neurobiological approaches including mouse genetics and electrophysiology are being applied to study neurotransmitter release. The combined expertise from our laboratories allows us to study the mechanism of release using a wide range of techniques, in an endeavor where the structural studies are driven by specific biological questions and the results of these studies in turn drive the biology.

The primary targets of our research can be divided in two groups: i) components of the universal membrane fusion machinery such as the Sec1/Munc18 (SM) homologue munc18-1, the small Rab GTPase Rab3, and the SNARE proteins syntaxin 1, SNAP-25 and synaptobrevin; and ii) proteins with specialized roles in release such as synaptotagmin 1, complexin, munc13 and RIM. Our research has provided key insights into the structures, interactions and functions of these proteins, but there are still critical unanswered questions about the mechanisms underlying the different steps that lead to neurotransmitter release, i.e. synaptic vesicle docking, priming of the fusion machinery to a release-ready state, and Ca²⁺-dependent membrane fusion. After having analyzed the structures and interactions of many of these proteins using soluble fragments that lacked transmembrane regions, we are now focusing on the reconstitution into membranes of the macromolecular assemblies that mediate the different steps of release. A key aspect of these current efforts is the development of methodologies to analyze in detail protein-protein interactions on lipid bilayers.

Selected publications

X. Shao, B. A. Davletov, R. B. Sutton, T. C. Südhof and J. Rizo, “A Bipartite Ca²⁺-Binding Motif in C₂ Domains of Synaptotagmin and Protein Kinase C”, Science 273, 248-251 (1996).

X. Shao, C. Li, I. Fernandez, X. Zhang, T. C. Südhof and J. Rizo, “Synaptotagmin-Syntaxin Interaction: the C₂-Domain as a Ca²⁺-Dependent Electrostatic Switch”, Neuron 18,133-142 (1997).

J. Ubach, X. Zhang, X. Shao, T. C. Südhof and J. Rizo, “Ca²⁺-binding to synaptotagmin: how many Ca²⁺ ions bind at the tip of a C₂-domain?”, EMBO J. 17, 3921-3930 (1998).

I. Fernandez, J. Ubach, X. Zhang, T. C. Südhof and J. Rizo, “Three-dimensional structure of an evolutionarily conserved N-terminal domain of syntaxin 1A”, Cell 18, 841-849 (1998).

I. Dulubova, S. Sugita, S. Hill, M. Hosaka, I. Fernandez, T. C. Südhof and J. Rizo, “A conformational switch in syntaxin during exocytosis”, EMBO J. 18, 4372-4382 (1999).

R. Fernandez-Chacon, A. Königstorfer, S. H. Gerber, J. Garcia, M. F. Matos, C. F. Stevens, N. Brose, J. Rizo, C. Rosenmund and T. C. Südhof, “Synaptotagmin I functions as a Ca²⁺-regulator of release probability”, Nature 410, 41-49 (2001).

I. Fernandez, D. Arac, J. Ubach, S. H. Gerber, O. Shin, Y. Gao, R. G. W. Anderson, T. C. Südhof and J. Rizo, “Three-dimensional structure of the synaptotagmin 1 C₂B-domain: synaptotagmin 1 as a phospholipid binding machine”, Neuron 32, 1057-1069 (2001).

X. Chen, D. Tomchick, E. Kovrigin, D. Arac, M. Machius, T. C. Südhof and J. Rizo, “Three-dimensional structure of the complexin/SNARE complex”, Neuron 33, 397-409 (2002).

I. Dulubova, T. Yamaguchi, Y. Gao, S. W. Min, I. Huryeva, T. C. Südhof and J.Rizo, “How Tlg2p/syntaxin 16 ‘snares’ Vps45p”, EMBO J. 21, 3620-3631 (2002).

J. Garcia, S. H. Gerber, S. Sugita, T. C. Südhof and J. Rizo, “A conformational switch in the piccolo C₂A-domain regulated by alternative splicing”, Nat. Struct. Molec. Biol. 11, 45-53 (2004).

I. Dulubova, X. Lou, J. Lu, I. Huryeva, A. Alam, R. Schneggenburger, T. C. Südhof and J. Rizo, “A Munc13/RIM/Rab3 tripartite complex: from priming to plasticity?”, EMBO J. 24, 2839-2850 (2005).

J. Basu, N. Shen, I. Dulubova, J. Lu, R. Guan, O. Guryev, N. V. Grishin, C. Rosenmund and J. Rizo, “Identification of a minimal domain responsible for Munc13 activity”, Nat. Struct. Molec. Biol. 12, 1017-1018 (2005).

X. Chen, D. Araç, T.-M. Wang, C. J. Gilpin, J. Zimmerberg and J. Rizo, “SNARE-mediated lipid mixing depends on the physical state of the vesicles”, Biophys. J. 90, 2062-2074 (2006).

D. Araç, X. Chen, H. A. Khant, J. Ubach, S. J. Ludtke, M. Kikkawa, A. E. Johnson, W. Chiu, T. C. Südhof and J. Rizo, “Close membrane-membrane proximity induced by Ca²⁺-dependent multivalent binding of synaptotagmin 1 to phospholipids”, Nat. Struct. Molec. Biol. 13, 209-217 (2006).

Lu,J., Machius,M., Dulubova,I., Dai,H., Sudhof,T.C., Tomchick,D.R., and Rizo,J. Structural Basis for a Munc13-1 Homodimer to Munc13-1/RIM Heterodimer Switch. PLoS. Biol. 4, 1159-1172 (e192) (2006).

Rizo,J., Chen,X., and Arac,D. Unraveling the mechanisms of synaptotagmin and SNARE function in neurotransmitter release. Trends Cell Biol. 16, 339-350 (2006).

Tang, J., Maximov, A., Shin, O.-H., Dai, H., Rizo, J. and Sudhof, T. C. A complexin/synaptotagmin-1 switch controls fast synaptic vesicle exocytosis. Cell 126, 1175-1187 (2006).

Dulubova, I., Khvotchev, M., Liu, S., Huryeva, I., Südhof, T. C. and Rizo, J. Munc18-1 binds directly to the neuronal SNARE complex. Proc. Natl. Acad. Sci. USA 104, 2697-2702 (2007).

Dai, H., Shen, N., Araç, D. and Rizo, J. A quaternary SNARE-synaptotagmin-Ca²⁺-phospholipid complex in neurotransmitter release. J. Mol. Biol. 367, 848-863 (2007).

Yang, M., Li, B., Tomchick, D. R., Machius, M., Rizo, J., Yu, H. and Luo, X. p31(comet) blocks Mad2 activation through structural mimicry. Cell 131, 744-755 (2007).

Rizo, J. and Rosenmund, C. Synaptic vesicle fusion. Nat. Struct. Mol. Biol. 15, 665-674 (2008).

Gerber, S. H., Rah, J.-C., Min, S.W., Liu, X., de Wit, H., Dulubova, I., Meyer, A. C., Rizo, J., Arancillo, M., Hammer, R. E., Verhage, M., Rosenmund, C. and Südhof, T. C. Conformational switch of Syntaxin-1 controls synaptic vesicle fusion, Science 321, 1507-1510 (2008).

Xue, M., Ma, C., Craig, T. K., Rosenmund, C. and Rizo, J. The Janus-faced nature of the C₂B domain is fundamental for synaptotagmin-1 function. Nat. Struct. Mol. Biol. 15, 1160-1168 (2008).

Deák. F., Xu, Y., Chang, W.-P., Dulubova, I., Khvotchev, M., Liu, X., Sudhof, T. C. and Rizo, J. Munc18-1 binding to the neuronal SNARE complex controls synaptic vesicle priming. J. Cell Biol. 184, 751-764 (2009).

Shin, O.-H., Lu, J., Rhee, J.-S., Tomchick, D. R., Pang, Z. P., Wojcik, S. M., Camacho-Perez, M., Brose, N., Machius, M., Rizo J.^*, Rosenmund, C.^* and Sudhof, T. C.^* Munc13 C₂B domain –an activity-dependent Ca²⁺-regulator of synaptic exocytosis. Nat. Struct. Mol. Biol. 17, 280-288 (2010).

Xue, M., Craig, T.K., Xu, J., Chao, H.-T., Rizo, J.^* and Rosenmund, C.^* Binding of the Complexin N terminus to the SNARE complex C terminus potentiates synaptic vesicle fusogenicity. Nat. Struct. Mol. Biol. 17, 568-575 (2010).

Sheard, L.B., Tan, X., Mao, H., Withers, J., Nissan, G.B., Hinds, T.R., Hsu, F.-F., Sharon, M., Browse, J., He, S.Y., Rizo, J., Howe, G.A. and Zheng, N. Jasmonate perception by inositol-phosphate-potentiated COI1-JAZ co-receptor. Nature 468, 400-405 (2010).

Kaeser, P.S., Deng, L., Wang, Y., Dulubova, I., Liu, X., Rizo J. and Sudhof, T.C. RIM proteins tether Ca²⁺-channels to presynaptic active zones via a direct PDZ-domain interaction. Cell 144, 282-295 (2011).

Ma, C., Li, W., Xu, Y. and Rizo, J. Munc13 mediates the transition from the closed syntaxin/Munc18 complex to the SNARE complex, Nat. Struct. Mol. Biol. 18, 542-549 (2011).

Brewer, K.D., Li, W., Horne, E.B. and Rizo J. Reluctance to membrane binding enables accessibility of the synaptobrevin SNARE motif for SNARE complex formation. Proc. Natl. Acad. Sci. USA 108, 12723-12728 (2011).

Li, W., Ma, C., Guan, R., Xu, Y., Tomchick, D.R.^* and Rizo, J.^* The crystal structure of a Munc13 C-terminal module exhibits a remarkable similarity to vesicle tethering factors. Structure 19, 1443-1455 (2011).