Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion
- Huazhong University of Science and Technology, China
Figures
Figure 1
Overview of the structure features of Syt1 C2 domains and the core SNARE complex.
(A and B) Structural diagrams (A) and electrostatic surface potential (B) of Syt1 C2A (PDB entry 1BYN) and C2B (PDB entry 1TJX) domain. Residues K326 and K327 on the side, R398 and R399 on the …
Figure 2 with 2 supplements
Different Ca2+-independent interactions of Syt1 with membranes and SNARE complexes.
(A and B) Binding of Syt1 soluble fragments and their mutants to the core SNARE complex measured by GST pull-down assay (A) and quantification of the C2B binding (B). Asterisks in A show the bands …
Figure 2—figure supplement 1
The SNAP-25 3M mutation displayed less resistance to SDS and no influence on SNARE complex assembly.
(A) Assembled GST-tagged SNARE complexes were analyzed by SDS-PAGE before pull-down assays. (B) The assembly of pre-assembled syntaxin-1–SNAP-25 complex and synaptobrevin monitored by fluorescence …
Figure 2—figure supplement 2
PS-containing liposome clustering induced by C2B and its mutants in the presence of Ca2+.
https://doi.org/10.7554/eLife.14211.006
Figure 3
Persistence of the R398 R399–SNARE complex interaction in the presence of ATP and Mg2+.
(A) Schematic diagrams of bimane-labeled SNARE complex and Syt1 C2B. Tryptophan was introduced at the bottom of C2B (T285W, orange stick), which is close to residues R398 and R399; two native …
Figure 4
Binding Kd between C2B and the membrane-anchored SNARE complex.
Cis-SNARE complexes were reconstituted on liposome via the syntaxin-1 transmembrane domain. PI(4,5)P2 increased the binding affinity between Syt1 C2B and the membrane-anchored SNARE complex in the …
Figure 5 with 1 supplement
Synergistic interactions of C2B with membrane-anchored SNARE complexes, PI(4,5)P2 and PS in the presence of 0.1 mM Ca2+.
(A and B) Co-flotation of Syt1 C2B with liposomes in the absence of PI(4,5)P2 (A) and quantification of the results (B). (C and D) Co-flotation of Syt1 C2B with liposomes in the presence of 1% …
Figure 5—figure supplement 1
Binding of C2B to PS-containing liposomes in different Ca2+ concentrations.
(A) Co-flotation of C2B with liposomes (65% PC + 20% PE + 15% PS) in the presence of 250 mM KCl and different Ca2+ concentrations as indicated. (B) Quantification of the results in A. Representative …
Figure 6
Persistence of C2B–SNARE complex and C2B–PI(4,5)P2 interactions upon insertion of the Ca2+-binding loops into membranes.
(A) Schematic diagrams of C2B and membrane-embedded cis-SNARE complex. NBD was labeled on N333C or I367C (green sticks) on C2B separately; bimane was labeled on SN25 R59C (orange sphere); tryptophan …
Figure 7 with 3 supplements
Ca2+-dependent simultaneous C2B–SNARE complex–membrane interactions underlie the function of Syt1 in triggering fusion.
(A and B) Schematic diagrams of the lipid mixing (A) and content mixing (B) assay. Liposome compositions are presented below the diagram. Cpx, complexin-1. (C and I) Poly-D-lysine promoted …
Figure 7—figure supplement 1
Liposome clustering induced by Poly-D-lysine in a concentration-dependent manner.
100 μM liposomes (65% PC + 20% PE + 15% PS) mixed with different concentrations of poly-D-lysine were incubated for 40 min and particle size was monitored by DLS. Data plots are presented as the …
Figure 7—figure supplement 2
Liposome clustering and SNARE pairing monitored during liposome fusion.
(A) C2B did not cluster t-liposomes (bearing syntaxin-1–SNAP-25 complex) in the presence of Ca2+. Plain liposomes or t-liposomes (59% PC + 20% PE + 20% PS + 1% PI(4,5)P2) bearing 0.5 μM …
Figure 7—figure supplement 3
No leakiness of liposomes detected in the content-mixing experiments.
(A) Schematic diagram of the normal content mixing and the leakiness control assays. In the leakiness control, both v-liposomes and t-liposomes were loaded with 40 mM sulforhodamine. Liposome …
Figure 8
Functional analysis of the Ca2+-binding loops on full-length Syt1 in triggering liposome fusion.
(A and B) Schematic diagrams of the lipid mixing (A) and content mixing (B). (C and F) Syt1 stimulates lipid mixing (C) and content mixing (F) in the absence of Ca2+ and complexin-1 (cpx). (D and G) …
Figure 9
A working model of Syt1 in triggering membrane fusion.
(A) Binding of Syt1 C2B to PI(4,5)P2 and primed trans-SNARE complex on the plasma membrane before Ca2+ influx. (B) The simultaneous interactions of C2B with primed trans-SNARE complex and …
