Figure 6. Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1 and synaptotagmin-1 C₂AB do not decrease the overall amount of cis-SNARE complex disassembly caused by NSF-αSNAP.

(A) Kinetic assays monitoring changes in the donor fluorescence emission intensity due to cis-SNARE complex formation upon mixing V-liposomes containing Alexa488-synaptobrevin with an excess of TMR-labeled syntaxin-1 (2–253) and SNAP-25m in the presence of NSF-αSNAP with no additions (ctrl) (light gray trace) or with different combinations of Munc18-1 (M18), Munc13-1 C₁C₂BMUNC₂C (M13), complexin-1 (Cpx) and synaptotagmin-1 C₂AB as indicated. Ca²⁺ was added at 550 s. For comparison purposes, the dark gray trace shows a cis-SNARE complex assembly reaction performed in the presence of Syb49-93 and absence of NSF-αSNAP. (B) Kinetic assays of cis-SNARE complex assembly analogous to those of (A), but performed in the absence of NSF-αSNAP and the presence of Syb49-93 alone (black trace) or together with complexin-1 (Cpx) (red trace) or synaptotagmin-1 C₂AB plus Ca²⁺ (blue trace). NSF-αSNAP were added when the reactions reached a plateau (black arrow) to monitor cis-SNARE complex disassembly. (C) Kinetic assays analogous to those in (B), but in the presence of Munc18-1 (M18) and Munc13-1 C₁C₂BMUNC₂C (M13) without (red trace) or with (dark gray trace) Syb49-93. Ca²⁺ was added after 950 s. (D) Kinetic assays where cis-SNARE complex formation was initially catalyzed by Syb49-93 and, after reaching a plateau, Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1, synaptotagmin-1 C₂AB and Ca²⁺ were added (red arrow); after five minutes, NSF-αSNAP were added to test for disassembly (red trace). The black trace shows a control experiment where the four proteins were not included before adding NSF-αSNAP. In the experiments shown in (C–D), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of (A–D), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents.

Figure 6—figure supplement 1. Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1 and synaptotagmin-1 C₂AB do not protect cis-SNARE complexes against disassembly by NSF-αSNAP.

(A) Kinetic assays where cis-SNARE complex formation was catalyzed by Syb49-93, as in Figure 6D, and different concentrations of complexin-1 (Cpx) were added five minutes before disassembly with NSF-αSNAP. (B) Kinetic assays analogous to those of Figure 6D, but using WT SNAP-25 instead of SNAP-25m to ensure that the mutation in SNAP-25m did not affect the disassembly of cis-SNARE complexes by NSF-αSNAP in the presence of Munc18-1, Munc13-1 C₁C₂BMUNC₂C, complexin-1, synaptotagmin-1 C₂AB and Ca²⁺. (C) Kinetic assays analogous to those of panels (A), but adding 1 μM complexin-1 five minutes before disassembly with NSF-αSNAP (red and orange traces). In these experiments, the concentrations of NSF and αSNAP were 0.1 μM and 0.5 μM, respectively, which were lower than those of our standard conditions (0.5 μM and 2 μM, respectively) to test whether complexin-1 might hinder disassembly at a higher molar ratio with respect to αSNAP. The experiments were performed with SNAP-25m (black and red traces) or WT SNAP-25 (gray and orange traces). The black and gray traces are controls where complexin-1 was not added. In the experiments shown in (A–C), we stopped monitoring the donor fluorescence intensity to add the reagents for disassembly, and a few minutes elapsed until we started to monitor the reaction again (indicated by the double slanted bars on the traces and on the x axis). For all traces of (A–C), fluorescence emission intensities were normalized with the intensity observed in the first point and corrected for the dilution caused by the addition of reagents to make the data comparable.