Multiple factors maintain assembled trans-SNARE complexes in the presence of NSF and αSNAP
- University of Texas Southwestern Medical Center, United States
Figures
Figure 1
Design of a SNAP-25 mutation that abrogates its ability to support membrane fusion.
(A) Ribbon diagram of the crystal structure of the SNARE complex (PDB accession code 1SFC) (Sutton et al., 1998). Synaptobrevin is red, syntaxin-1 yellow and SNAP-25 green, with the side chains of …
Figure 2 with 4 supplements
An assay to measure assembly of trans-SNARE complexes and disassembly by NSF-αSNAP.
(A) Diagram illustrating the assay used to monitor trans-SNARE complex assembly and disassembly. V-liposomes containing synaptobrevin labeled with a FRET donor (Alexa488, green star) at residue 26 …
Figure 2—figure supplement 1
Control experiments acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes containing Alexa488-synaptobrevin.
Spectra were acquired before (V) (black traces) or after (red traces) addition of Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13), complexin-1 (Cpx), Ca2+-bound synaptotagmin-1 C2AB, NSF, αSNAP or …
Figure 2—figure supplement 2
Syb49-93 strongly accelerates trans-SNARE complex assembly.
(A) Kinetic assays where the donor fluorescence emission intensity was measured to monitor trans-SNARE complex assembly as a function of time upon mixing V- and T-liposomes (1:4 ratio) in the …
Figure 2—figure supplement 3
Representative cryo-EM image of a mixture of V- and T-liposomes (1:4 ratio) that had been incubated with Syb49-93 for 5 hours before rapid freezing (scale bar, 100 nm).
The yellow arrows point to somewhat extended interfaces between liposomes.
Figure 2—figure supplement 4
Additional supporting fluorescence emission spectra.
(A) Fluorescence emission spectra (excitation at 468 nm) of separate samples of V-liposomes containing Alexa488-synaptobrevin (green trace) and T-liposomes containing TMR-syntaxin-1-SNAP-25m (1:4 V- …
Figure 3 with 2 supplements
Influence of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 on trans-SNARE complex assembly-disassembly in the presence of NSF-αSNAP.
(A,B) Kinetic assays monitoring trans-SNARE complex assembly between V- and T-liposomes (1:4 ratio) in the presence of NSF-αSNAP from the decrease in the donor fluorescence emission intensity. The …
Figure 3—figure supplement 1
Complexin-1 increases the efficiency of Ca2+-independent trans-SNARE complex assembly between VSyt1- and T-liposomes in the presence of NSF-αSNAP.
The traces show kinetic assays monitoring trans-SNARE complex assembly between VSyt1- and T-liposomes (1:4 ratio) in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C, NSF-αSNAP and 100 μM EGTA, …
Figure 3—figure supplement 2
Ca2+-dependent fusion between VSyt1- and T-liposomes.
(A,B) Lipid mixing (A) between VSyt1-liposomes (synaptobrevin-to-lipid ratio 1:10,000; synaptotagmin-1-to-lipid ratio 1:1,000) and T-liposomes was monitored from the fluorescence de-quenching of …
Figure 4 with 1 supplement
Influence of Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 on trans-SNARE complex assembly in the absence of NSF-αSNAP and on protection against disassembly upon addition of NSF-αSNAP.
(A) Kinetic assays monitoring trans-SNARE complex assembly upon mixing V- and T-liposomes (1:4 ratio) in the presence of Syb49-93 and disassembly upon addition of NSF-αSNAP (indicated by the …
Figure 4—figure supplement 1
The SNARE motif of synaptobrevin does not affect the results observed upon addition of NSF-αSNAP to trans-SNARE complexes formed in the presence of Munc18-1, Munc13-1 C1C2BMUNC2C and Ca2+.
The plots show kinetic assays monitoring trans-SNARE complex assembly upon mixing V- and T-liposomes (1:4 ratio) in the presence of Munc18-1 (M18), Munc13-1 C1C2BMUNC2C (M13) or both (blue, green …
Figure 5 with 5 supplements
Quantitative analysis of how Munc18-1, Munc13-1 C1C2BMUNC2C, complexin-1 and synaptotagmin-1 protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP.
(A) Fluorescence emission spectra of mixtures of V-liposomes containing Alexa488-synaptobrevin and T-liposomes containing TMR-syntaxin-1-SNAP-25m (1:4 V- to T-liposome ratio) that were incubated for …
Figure 5—figure supplement 1
Representative fluorescence emission spectra used in the experiments of Figure 5B to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1, synaptotagmin-1 and Ca2+ in different combinations protect pre-formed trans-SNARE complexes against disassembly by NSF-αSNAP.
V- and T-liposomes were incubated for five hours with Syb49-93 to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1 (M18), Munc13-1 …
Figure 5—figure supplement 2
Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of V-liposomes incorporated into trans-SNARE complexes in the absence of FRET.
V-liposomes containing Alexa488-synaptobrevin (V*) were incubated for five hours with Syb49-93 and T-liposomes containing syntaxin-1-SNAP-25m (T) (1:4 V- to T-liposome ratio). The mixture was then …
Figure 5—figure supplement 3
Control spectra acquired to assess the effects of various factors on the fluorescence emission spectra of T-liposomes incorporated into trans-SNARE complexes in the absence of FRET.
V-liposomes containing synaptobrevin (V) were incubated for five hours with Syb49-93 and T-liposomes containing TMR-syntaxin-1-SNAP-25m (T*) (1:4 V- to T-liposome ratio). The mixture was then …
Figure 5—figure supplement 4
Representative fluorescence emission spectra used in the experiments of Figure 5C to obtain a quantitative measurement of how Munc18-1, Munc13-1, complexin-1 and Ca2+ in different combinations protect pre-formed trans-SNARE complexes between VSyt1- and T-liposomes against disassembly by NSF-αSNAP.
VSyt1- and T-liposomes were incubated with Syb49-93 (but for 24 hr at 4°C) to preform trans-SNARE complexes and then they were incubated for five minutes with different combinations of Munc18-1, …
Figure 5—figure supplement 5
Complexin-1 concentration dependence of protection of trans-SNARE complexes against disassembly by NSF-αSNAP.
Protection experiments with pre-formed trans-SNARE complexes between VSyt1- and T-liposomes analogous to those of Figure 5C were performed by adding different complexin-1 concentrations, incubating …
Figure 6 with 1 supplement
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 …
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 …
Figure 7
Models illustrating the different geometric constraints of cis- and trans-SNARE complex disassembly.
(A,B) Models showing ribbon diagrams of the cryo-electron microscopy structure of the 20S complex (PDB accession code 3J96) (Zhao et al., 2015) assembled on a cis-SNARE complex on one membrane (A) …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.38880.022
