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Science 339 (6118): 421-425

Copyright © 2013 by the American Association for the Advancement of Science

Reconstitution of the Vital Functions of Munc18 and Munc13 in Neurotransmitter Release

Cong Ma1,2,3,4,*,{dagger}, Lijing Su2,3,4,*, Alpay B. Seven2,3,4, Yibin Xu2,3,4, and Josep Rizo2,3,4,{dagger}

1 Key Laboratory of Molecular Biophysics, Ministry of Education, and Institute of Biophysics and Biochemistry, Huazhong University of Science and Technology, Wuhan 430074, China.
2 Department of Biophysics, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
3 Department of Biochemistry, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.
4 Department of Pharmacology, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA.


Figure 1
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Fig. 1. Munc18-1 displaces SNAP-25 from syntaxin-1. (A to C) 1H-13C HMQC spectra of 2H-Ile-13CH3–syntaxin-1 (A) bound to Munc18-1 (black) or to SNAP-25 (red); (B) initially bound to Munc18-1 and then incubated with the SNAP-25 SNARE motifs (SNN and SNC) for 11 hours; and (C) initially bound to SNAP-25 and then incubated with Munc18-1 for 11 hours. In (A), the available cross-peak assignments for the syntaxin-1–Munc18-1 complex are indicated in black and those for the syntaxin-1-SNAP-25 complex in red [(13) for assignments; H3 identifies the SNARE motif; asterisk indicates tentative assignments]. Because of the 2:1 stoichiometry of the syntaxin-1–SNAP-25 complex, the cross-peak of I203 is double. (D) Proteoliposomes containing coexpressed syntaxin-1–Munc18-1 complex were incubated with SNAP-25 or buffer, cofloatation assays were performed, and the top fraction was analyzed by means of SDS–polyacrylamide gel electrophoresis (SDS-PAGE) and Coomassie Blue staining (left two lanes). (E) Proteoliposomes containing syntaxin-1 were incubated with Munc18-1, SNAP-25, NSF, α-SNAP, ATP, Mg2+, and/or EDTA as indicated. Cofloatation assays were then performed, and the results analyzed by means of SDS-PAGE and Coomassie Blue staining. The five lanes on the right of (D) show loading controls with soluble proteins.

 

Figure 2
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Fig. 2. Requirement of Munc13 for lipid mixing of syntaxin-1–Munc18-1 liposomes with synaptobrevin liposomes. (A) Diagram summarizing the lipid mixing experiments, which were performed with acceptor liposomes containing syntaxin-1–Munc18-1 and donor liposomes containing synaptobrevin and NBD-lipids quenched by rhodamine-lipids. (B and C) Traces showing the lipid mixing observed in the presence of SNAP-25, Munc13-1 C1C2BMUN (M13), synaptotagmin-1 C2AB fragment (C2AB), synaptobrevin cytoplasmic domain (Sybcd), and/or 0.5 mM Ca2+ in various combinations. The y axis represents NBD fluorescence normalized to the maximum fluorescence observed upon detergent addition. (D) Quantification of the results obtained in the experiments of (B) and (C). (E) Lipid mixing observed in the presence of SNAP-25, C1C2BMUN, and C2AB fragment as a function of Ca2+ concentration. (F) Plot of the normalized NBD fluorescence intensity at 1000 s as a function of Ca2+ observed in (E). (G and H) Dependence of lipid mixing on the presence of DAG and/or PIP2 in the acceptor syntaxin-1–Munc18-1 liposomes (G), and quantification of the results (H). (I and J) Lipid mixing obtained with acceptor liposomes that contained syntaxin-1 bound to wild-type (WT) or E66A mutant Munc18-1 (I) and quantification of the results (J). In (D), (H), and (J), bars represent averages of the normalized NBD fluorescence observed after 1000 s in repeated experiments performed under the same conditions. Error bars represent SDs.

 

Figure 3
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Fig. 3. Content mixing assays with syntaxin-1–Munc18-1 liposomes and synaptobrevin-liposomes. (A) Traces showing the content mixing between syntaxin-1–Munc18-1 acceptor liposomes and synaptobrevin donor liposomes in the presence of SNAP-25, Munc13-1 C1C2BMUN (M13), and/or synaptotagmin-1 C2AB fragment (C2AB) (all in 0.5 mM Ca2+). The donor liposomes contained encapsulated self-quenched sulforhodamine and self-quenched DiD lipids. The y axis represents sulforhodamine fluorescence normalized to the maximum fluorescence observed upon detergent addition. (B) Quantification of the results obtained in the experiments of (A). (C and D) Traces showing the lipid mixing observed from DiD fluorescence dequenching in the same experiments (C) and quantification of the results (D). Error bars represent SDs.

 

Figure 4
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Fig. 4. NSF–α-SNAPs inhibit lipid mixing between syntaxin-1–SNAP-25 liposomes and synaptobrevin-liposomes, and Munc18-1–Munc13-1 activate lipid mixing. (A) Traces showing the lipid mixing observed between syntaxin-1–SNAP-25 acceptor liposomes and synaptobrevin donor liposomes containing NBD-lipids quenched by rhodamine-lipids in the presence of Munc18-1 (M18), Munc13-1 C1C2BMUN (M13), synaptotagmin-1 C2AB fragment (C2AB), NSF, α-SNAP, and/or Mg2+-ATP in various combinations (all in 0.5 mM Ca2+). The y axis represents NBD fluorescence normalized to the maximum fluorescence observed upon detergent addition. (B) Quantification of the results obtained in the experiments of (A). (C) Lipid mixing experiments performed as in (A) with various additions, all in the presence of 2 μM SNAP-25 excess. (D) Quantification of the results of (C). Error bars represent SDs.

 

Figure 5
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Fig. 5. Model of synaptic vesicle fusion integrating the function of eight major components of the release machinery. In the top right, syntaxin-1 (yellow) is shown in a closed conformation bound to Munc18-1 and in an open conformation bound to SNAP-25. To illustrate the likely heterogeneity of syntaxin-1–SNAP-25 heterodimers, two complexes with 2:1 or 4:2 stoichiometries are shown, but larger complexes bridged by SNAP-25 (not shown) are also likely to exist. The model postulates that the syntaxin-1–SNAP-25 heterodimers are converted to syntaxin-1–Munc18-1 complexes (top left) and that Munc13 helps to open syntaxin-1 and to orchestrate trans-SNARE complex assembly together with Munc18-1, leading to a partially assembled SNARE complex that remains bound to Munc18-1 and Munc13 (bottom left). This state, which may correspond to that of primed synaptic vesicles and cannot be disassembled by NSF-SNAPs, serves as the substrate for synaptotagmin-1-Ca2+ to trigger fast synaptic vesicle fusion (bottom right). The arrangement of Munc18-1, Munc13, and synaptotagmin-1 with respect to the SNARE complex is unknown but is drawn to suggest the possibility that the three proteins may bind simultaneously to the SNARE complex and may also help to bridge the two membranes to help induce fusion. The interaction of synaptotagmin-1 with the SNARE complex may occur before Ca2+ influx (not shown in bottom left for simplicity).

 


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