scholarly journals Synaptotagmin-1 C2B domain interacts simultaneously with SNAREs and membranes to promote membrane fusion

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Shen Wang ◽  
Yun Li ◽  
Cong Ma
Keyword(s):  
Membrane Fusion ◽  
Neurotransmitter Release ◽  
Functional Significance ◽  
Underlying Mechanism ◽  
C2 Domains ◽  
Membrane Deformation ◽  
Synaptotagmin 1 ◽  
Bottom Face ◽  
Liposome Fusion

Synaptotagmin-1 (Syt1) acts as a Ca2+ sensor for neurotransmitter release through its C2 domains. It has been proposed that Syt1 promotes SNARE-dependent fusion mainly through its C2B domain, but the underlying mechanism is poorly understood. In this study, we show that the C2B domain interacts simultaneously with acidic membranes and SNARE complexes via the top Ca2+-binding loops, the side polybasic patch, and the bottom face in response to Ca2+. Disruption of the simultaneous interactions completely abrogates the triggering activity of the C2B domain in liposome fusion. We hypothesize that the simultaneous interactions endow the C2B domain with an ability to deform local membranes, and this membrane-deformation activity might underlie the functional significance of the Syt1 C2B domain in vivo.

scholarly journals Functional analysis of the interface between the tandem C2 domains of synaptotagmin-1

2016 ◽  
Vol 27 (6) ◽  
pp. 979-989 ◽  
Author(s):  
Chantell S. Evans ◽  
Zixuan He ◽  
Hua Bai ◽  
Xiaochu Lou ◽  
Pia Jeggle ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Membrane Fusion ◽  
Functional Significance ◽  
Wild Type ◽  
C2 Domains ◽  
Parent Protein ◽  
Synaptotagmin 1 ◽  
Open Issue ◽  
Physical Interactions

C2 domains are widespread motifs that often serve as Ca2+-binding modules; some proteins have more than one copy. An open issue is whether these domains, when duplicated within the same parent protein, interact with one another to regulate function. In the present study, we address the functional significance of interfacial residues between the tandem C2 domains of synaptotagmin (syt)-1, a Ca2+ sensor for neuronal exocytosis. Substitution of four residues, YHRD, at the domain interface, disrupted the interaction between the tandem C2 domains, altered the intrinsic affinity of syt-1 for Ca2+, and shifted the Ca2+ dependency for binding to membranes and driving membrane fusion in vitro. When expressed in syt-1 knockout neurons, the YHRD mutant yielded reductions in synaptic transmission, as compared with the wild-type protein. These results indicate that physical interactions between the tandem C2 domains of syt-1 contribute to excitation–secretion coupling.

scholarly journals SNAREs support atlastin-mediated homotypic ER fusion in Saccharomyces cerevisiae

2015 ◽  
Vol 210 (3) ◽  
pp. 451-470 ◽  
Author(s):  
Miriam Lee ◽  
Young-Joon Ko ◽  
Yeojin Moon ◽  
Minsoo Han ◽  
Hyung-Wook Kim ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Underlying Mechanism ◽  
In Vitro Assay ◽  
Physical Interaction ◽  
Physiological Concentration ◽  
Vitro Assay ◽  
Lack Of Fusion ◽  
Fold Excess

Dynamin-like GTPases of the atlastin family are thought to mediate homotypic endoplasmic reticulum (ER) membrane fusion; however, the underlying mechanism remains largely unclear. Here, we developed a simple and quantitative in vitro assay using isolated yeast microsomes for measuring yeast atlastin Sey1p-dependent ER fusion. Using this assay, we found that the ER SNAREs Sec22p and Sec20p were required for Sey1p-mediated ER fusion. Consistently, ER fusion was significantly reduced by inhibition of Sec18p and Sec17p, which regulate SNARE-mediated membrane fusion. The involvement of SNAREs in Sey1p-dependent ER fusion was further supported by the physical interaction of Sey1p with Sec22p and Ufe1p, another ER SNARE. Furthermore, our estimation of the concentration of Sey1p on isolated microsomes, together with the lack of fusion between Sey1p proteoliposomes even with a 25-fold excess of the physiological concentration of Sey1p, suggests that Sey1p requires additional factors to support ER fusion in vivo. Collectively, our data strongly suggest that SNARE-mediated membrane fusion is involved in atlastin-initiated homotypic ER fusion.

scholarly journals Polybasic patches in both C2 domains of Synaptotagmin-1 are required for evoked neurotransmitter release

2021 ◽  
Author(s):  
Zhenyong Wu ◽  
Lu Ma ◽  
Nicholas A Courtney ◽  
Jie Zhu ◽  
Yongli Zhang ◽  
...  
Keyword(s):  
Optical Tweezers ◽  
Single Molecule ◽  
Calcium Binding ◽  
Neurotransmitter Release ◽  
Transmembrane Domain ◽  
Snare Complex ◽  
Critical Feature ◽  
C2 Domains ◽  
Electrophysiological Recordings ◽  
Synaptotagmin 1

Synaptotagmin-1 (Syt1) is a vesicular calcium sensor required for synchronous neurotransmitter release. It is composed of a single-pass transmembrane domain linked to two tandem C2 domains (C2A and C2B) that bind calcium, acidic lipids, and SNARE proteins that drive fusion of the synaptic vesicle with the plasma membrane. Despite its essential role, how Syt1 couples calcium entry to synchronous release is not well understood. Calcium binding to C2B, but not to C2A, is critical for synchronous release and C2B additionally binds the SNARE complex. The C2A domain is also required for Syt1 function, but it is not clear why. Here we asked what critical feature of C2A may be responsible for its functional role, and compared this to the analogous feature in C2B. We focused on highly conserved poly-lysine patches located on the sides of C2A (K189-192) and C2B (K324-327). We tested effects of charge-neutralization mutations in either region (Syt1K189-192A and Syt1K326-327A) side-by-side to determine their relative contributions to Syt1 function in cultured cortical mouse neurons and in single-molecule experiments. Combining electrophysiological recordings and optical tweezers measurements to probe dynamic single C2 domain-membrane interactions, we show that both C2A and C2B polybasic patches contribute to membrane binding, and both are required for evoked release. The readily releasable vesicle pool or spontaneous release were not affected, so both patches are specifically required for synchronization of release. We suggest these patches contribute to cooperative binding to membranes, increasing the overall affinity of Syt1 for negatively charged membranes and facilitating evoked release.

scholarly journals Phosphatidylinositol 4,5 bisphosphate controls the cis and trans interactions of synaptotagmin 1

2019 ◽  
Author(s):  
S.B. Nyenhuis ◽  
A. Thapa ◽  
D. S. Cafiso
Keyword(s):  
Neurotransmitter Release ◽  
Plasma Membranes ◽  
Full Length ◽  
Membrane Insertion ◽  
Membrane Interface ◽  
Vesicle Membrane ◽  
C2 Domains ◽  
Synaptotagmin 1 ◽  
Site Directed Spin Labeling ◽  
Cis And Trans

AbstractSynaptotagmin 1 acts as the Ca2+-sensor for synchronous neurotransmitter release; however, the mechanism by which it functions is not understood and is presently a topic of considerable interest. Here we describe measurements on full-length membrane reconstituted synaptotagmin 1 using site-directed spin labeling where we characterize the linker region as well as the cis (vesicle membrane) and trans (cytoplasmic membrane) binding of its two C2 domains. In the full-length protein, the C2A domain does not undergo membrane insertion in the absence of Ca2+; however, the C2B domain will bind to and penetrate in trans to a membrane containing phosphatidylinositol 4,5 bisphosphate (PIP2), even if phosphatidylserine (PS) is present in the cis membrane. In the presence of Ca2+, the Ca2+-binding loops of C2A and C2B both insert into the membrane interface; moreover, C2A preferentially inserts into PS containing bilayers and will bind in a cis configuration to membranes containing PS even if a PIP2 membrane is presented in trans. The data are consistent with a bridging activity for Syt1 where the two domains bind to opposing vesicle and plasma membranes. The failure of C2A to bind membranes in the absence of Ca2+ and the long unstructured segment linking C2A to the vesicle membrane indicates that synaptotagmin 1 could act to significantly shorten the vesicle-plasma membrane distance with increasing levels of Ca2+.

scholarly journals Ca2+-dependent release of synaptotagmin-1 from the SNARE complex on phosphatidylinositol 4,5-bisphosphate-containing membranes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Rashmi Voleti ◽  
Klaudia Jaczynska ◽  
Josep Rizo
Keyword(s):  
Fluorescence Spectroscopy ◽  
Membrane Fusion ◽  
Neurotransmitter Release ◽  
Tight Binding ◽  
Snare Complex ◽  
Structural Studies ◽  
Binding Modes ◽  
Specific Effects ◽  
Synaptotagmin 1 ◽  
Polybasic Region

The Ca2+ sensor synaptotagmin-1 and the SNARE complex cooperate to trigger neurotransmitter release. Structural studies elucidated three distinct synaptotagmin-1-SNARE complex binding modes involving ‘polybasic’, ‘primary’ and ‘tripartite’ interfaces of synaptotagmin-1. We investigated these interactions using NMR and fluorescence spectroscopy. Synaptotagmin-1 binds to the SNARE complex through the polybasic and primary interfaces in solution. Ca2+-free synaptotagmin-1 binds to SNARE complexes anchored on PIP2-containing nanodiscs. R398Q/R399Q and E295A/Y338W mutations at the primary interface, which strongly impair neurotransmitter release, disrupt and enhance synaptotagmin-1-SNARE complex binding, respectively. Ca2+ induces tight binding of synaptotagmin-1 to PIP2-containing nanodiscs, disrupting synaptotagmin-1-SNARE interactions. Specific effects of mutations in the polybasic region on Ca2+-dependent synaptotagmin-1-PIP2-membrane interactions correlate with their effects on release. Our data suggest that synaptotagmin-1 binds to the SNARE complex through the primary interface and that Ca2+ releases this interaction, inducing PIP2/membrane binding and allowing cooperation between synaptotagmin-1 and the SNAREs in membrane fusion to trigger release.

scholarly journals A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

2021 ◽  
Vol 220 (4) ◽  
Author(s):  
Lei Li ◽  
Haowen Liu ◽  
Mia Krout ◽  
Janet E. Richmond ◽  
Yu Wang ◽  
...  
Keyword(s):  
Neurotransmitter Release ◽  
Release Kinetics ◽  
Sensor System ◽  
C2 Domains ◽  
Loosely Coupled ◽  
C Elegans ◽  
Membrane Tethering ◽  
Synaptotagmin 1 ◽  
Tm Domain ◽  
Fast Release

Ca2+-dependent neurotransmitter release requires synaptotagmins as Ca2+ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca2+. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca2+ sensor in Caenorhabditis elegans. Here, we report a new Ca2+ sensor, SNT-3, which triggers delayed Ca2+-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca2+ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca2+ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca2+ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca2+ sensor system in C. elegans and provide significant insights into Ca2+-regulated exocytosis.

scholarly journals Synaptotagmin C2A Loop 2 Mediates Ca2+-dependent SNARE Interactions Essential for Ca2+-triggered Vesicle Exocytosis

2007 ◽  
Vol 18 (12) ◽  
pp. 4957-4968 ◽  
Author(s):  
K. L. Lynch ◽  
R.R.L. Gerona ◽  
E. C. Larsen ◽  
R. F. Marcia ◽  
J. C. Mitchell ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding ◽  
Binding Surface ◽  
Essential Components ◽  
Vesicle Exocytosis ◽  
Synaptotagmin 1 ◽  
Liposome Fusion ◽  
And Function ◽  
Loop 2

Synaptotagmins contain tandem C2 domains and function as Ca2+ sensors for vesicle exocytosis but the mechanism for coupling Ca2+ rises to membrane fusion remains undefined. Synaptotagmins bind SNAREs, essential components of the membrane fusion machinery, but the role of these interactions in Ca2+-triggered vesicle exocytosis has not been directly assessed. We identified sites on synaptotagmin−1 that mediate Ca2+-dependent SNAP25 binding by zero-length cross-linking. Mutation of these sites in C2A and C2B eliminated Ca2+-dependent synaptotagmin−1 binding to SNAREs without affecting Ca2+-dependent membrane binding. The mutants failed to confer Ca2+ regulation on SNARE-dependent liposome fusion and failed to restore Ca2+-triggered vesicle exocytosis in synaptotagmin-deficient PC12 cells. The results provide direct evidence that Ca2+-dependent SNARE binding by synaptotagmin is essential for Ca2+-triggered vesicle exocytosis and that Ca2+-dependent membrane binding by itself is insufficient to trigger fusion. A structure-based model of the SNARE-binding surface of C2A provided a new view of how Ca2+-dependent SNARE and membrane binding occur simultaneously.

scholarly journals Synaptotagmin-1 C2B domains cooperatively stabilize the fusion stalk via a master-servant mechanism

2021 ◽  
Author(s):  
Ary Lautaro Di Bartolo ◽  
Diego Masone
Keyword(s):  
Free Energy ◽  
Membrane Fusion ◽  
Membrane Binding ◽  
Vesicle Fusion ◽  
Fusion Process ◽  
Total Work ◽  
C2 Domains ◽  
Domain Interactions ◽  
Synaptotagmin 1 ◽  
Data Point

Synaptotagmin-1 is a low-affinity Ca2+ sensor that triggers synchronous vesicle fusion. It contains two similar C2 domains (C2A and C2B) that cooperate in membrane binding, being the C2B domain the main responsible for the membrane fusion process due to its polybasic patch KRLKKKKTTIKK (321-332). In this work, a master-servant mechanism between two identical C2B domains is shown to control the formation of the fusion stalk. Two regions in C2B are essential for the process, the well-known polybasic patch and a recently described pair of arginines (398,399). The master domain shows strong PIP2 interactions with its polybasic patch and its pair of arginines. At the same time, the servant analogously cooperates with the master to reduce the total work to form the fusion stalk. The strategic mutation (T328E,T329E) in both master and servant domains disrupts the cooperative mechanism, drastically increasing the free energy needed to induce the fusion stalk, however with negligible effects on the master domain interactions with PIP2. These data point to a difference in the behavior of the servant domain, which is unable to sustain its PIP2 interactions neither through its polybasic patch nor through its pair of arginines, in the end losing its ability to assist the master in the formation of the fusion stalk.

scholarly journals Structural Roles for the Juxtamembrane Linker Region and Transmembrane Region of Synaptobrevin 2 in Membrane Fusion

2021 ◽  
Vol 8 ◽  
Author(s):  
Yaru Hu ◽  
Le Zhu ◽  
Cong Ma
Keyword(s):  
Membrane Fusion ◽  
Underlying Mechanism ◽  
Snare Complex ◽  
Transmembrane Region ◽  
Tight Coupling ◽  
Linker Region ◽  
Force Transfer ◽  
Tryptophan Residues ◽  
Snare Motif ◽  
Liposome Fusion

Formation of the trans-SNARE complex is believed to generate a force transfer to the membranes to promote membrane fusion, but the underlying mechanism remains elusive. In this study, we show that helix-breaking and/or length-increasing insertions in the juxtamembrane linker region of synaptobrevin-2 exert diverse effects on liposome fusion, in a manner dependent on the insertion position relative to the two conserved tryptophan residues (W89/W90). Helical extension of synaptobrevin-2 to W89/W90 is a prerequisite for initiating membrane merger. The transmembrane region of synaptobrevin-2 enables proper localization of W89/W90 at the membrane interface to gate force transfer. Besides, our data indicate that the SNARE regulatory components Munc18-1 and Munc13-1 impose liposome fusion strong demand on tight coupling between the SNARE motif and the transmembrane region of synaptobrevin-2.

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