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 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 Phosphatidylinositol 4,5-bisphosphate regulates SNARE-dependent membrane fusion

2008 ◽  
Vol 182 (2) ◽  
pp. 355-366 ◽  
Author(s):  
Declan J. James ◽  
Chuenchanok Khodthong ◽  
Judith A. Kowalchyk ◽  
Thomas F.J. Martin
Keyword(s):  
Plasma Membrane ◽  
Membrane Fusion ◽  
Negative Curvature ◽  
Positive Curvature ◽  
Membrane Microdomains ◽  
Attachment Protein ◽  
Protein Receptor ◽  
Vesicle Exocytosis ◽  
Liposome Fusion ◽  
Plasma Membrane Microdomains

Phosphatidylinositol 4,5-bisphosphate (PI 4,5-P2) on the plasma membrane is essential for vesicle exocytosis but its role in membrane fusion has not been determined. Here, we quantify the concentration of PI 4,5-P2 as ∼6 mol% in the cytoplasmic leaflet of plasma membrane microdomains at sites of docked vesicles. At this concentration of PI 4,5-P2 soluble NSF attachment protein receptor (SNARE)–dependent liposome fusion is inhibited. Inhibition by PI 4,5-P2 likely results from its intrinsic positive curvature–promoting properties that inhibit formation of high negative curvature membrane fusion intermediates. Mutation of juxtamembrane basic residues in the plasma membrane SNARE syntaxin-1 increase inhibition by PI 4,5-P2, suggesting that syntaxin sequesters PI 4,5-P2 to alleviate inhibition. To define an essential rather than inhibitory role for PI 4,5-P2, we test a PI 4,5-P2–binding priming factor required for vesicle exocytosis. Ca2+-dependent activator protein for secretion promotes increased rates of SNARE-dependent fusion that are PI 4,5-P2 dependent. These results indicate that PI 4,5-P2 regulates fusion both as a fusion restraint that syntaxin-1 alleviates and as an essential cofactor that recruits protein priming factors to facilitate SNARE-dependent fusion.

Role of electrostatics on membrane binding, aggregation and destabilization induced by NAD(P)H dehydrogenases. Implication in membrane fusion

2008 ◽  
Vol 137 (2-3) ◽  
pp. 126-132 ◽  
Author(s):  
César L. Avila ◽  
Beatriz F. de Arcuri ◽  
Fernando Gonzalez-Nilo ◽  
Javier De Las Rivas ◽  
Rosana Chehín ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Binding

scholarly journals Role of synaptotagmin in Ca2+-triggered exocytosis

2002 ◽  
Vol 366 (1) ◽  
pp. 1-13 ◽  
Author(s):  
Ward C. TUCKER ◽  
Edwin R. CHAPMAN
Keyword(s):  
Nervous System ◽  
Gene Family ◽  
Membrane Fusion ◽  
Cell Types ◽  
Neuroendocrine Cells ◽  
Biophysical Properties ◽  
Synaptotagmin I ◽  
And Function ◽  
Fusion Pores

The Ca2+-binding synaptic-vesicle protein synaptotagmin I has attracted considerable interest as a potential Ca2+ sensor that regulates exocytosis from neurons and neuroendocrine cells. Recent studies have shed new light on the structure, biochemical/biophysical properties and function of synaptotagmin, and the emerging view is that it plays an important role in both exocytosis and endocytosis. At least a dozen additional isoforms exist, some of which are expressed outside of the nervous system, suggesting that synaptotagmins might regulate membrane traffic in a variety of cell types. Here we provide an overview of the members of this gene family, with particular emphasis on the question of whether and how synaptotagmin I functions during the final stages of membrane fusion: does it regulate the Ca2+-triggered opening and dilation of fusion pores?

scholarly journals Accessory proteins of the zDHHC family of S-acylation enzymes

2020 ◽  
Vol 133 (22) ◽  
pp. jcs251819
Author(s):  
Christine Salaun ◽  
Carolina Locatelli ◽  
Filip Zmuda ◽  
Juan Cabrera González ◽  
Luke H. Chamberlain
Keyword(s):  
Fatty Acyl ◽  
Accessory Proteins ◽  
Enzyme Substrate ◽  
Essential Components ◽  
Selenoprotein K ◽  
Acyl Chains ◽  
The Stability ◽  
And Function ◽  
Encoding Genes

ABSTRACTAlmost two decades have passed since seminal work in Saccharomyces cerevisiae identified zinc finger DHHC domain-containing (zDHHC) enzymes as S-acyltransferases. These enzymes are ubiquitous in the eukarya domain, with 23 distinct zDHHC-encoding genes in the human genome. zDHHC enzymes mediate the bulk of S-acylation (also known as palmitoylation) reactions in cells, transferring acyl chains to cysteine thiolates, and in so-doing affecting the stability, localisation and function of several thousand proteins. Studies using purified components have shown that the minimal requirements for S-acylation are an appropriate zDHHC enzyme–substrate pair and fatty acyl-CoA. However, additional proteins including GCP16 (also known as Golga7), Golga7b, huntingtin and selenoprotein K, have been suggested to regulate the activity, stability and trafficking of certain zDHHC enzymes. In this Review, we discuss the role of these accessory proteins as essential components of the cellular S-acylation system.

scholarly journals Glycosylation Is Dispensable for Sorting of Synaptotagmin 1 but Is Critical for Targeting of SV2 and Synaptophysin to Recycling Synaptic Vesicles

2012 ◽  
Vol 287 (42) ◽  
pp. 35658-35668 ◽  
Author(s):  
Sung E. Kwon ◽  
Edwin R. Chapman
Keyword(s):  
Synaptic Vesicle ◽  
Post Translational Modification ◽  
Vesicle Membrane ◽  
Knock Out ◽  
Synaptic Localization ◽  
Glycosylation Sites ◽  
Synaptotagmin 1 ◽  
Knock Out Mice ◽  
And Function

Glycosylation is a major form of post-translational modification of synaptic vesicle membrane proteins. For example, the three major synaptic vesicle glycoproteins, synaptotagmin 1, synaptophysin, and SV2, represent ∼30% of the total copy number of vesicle proteins. Previous studies suggested that glycosylation is required for the vesicular targeting of synaptotagmin 1, but the role of glycosylation of synaptophysin and SV2 has not been explored in detail. In this study, we analyzed all glycosylation sites on synaptotagmin 1, synaptophysin, and SV2A via mutagenesis and optical imaging of pHluorin-tagged proteins in cultured neurons from knock-out mice lacking each protein. Surprisingly, these experiments revealed that glycosylation is completely dispensable for the sorting of synaptotagmin 1 to SVs whereas the N-glycans on SV2A are only partially dispensable. In contrast, N-glycan addition is essential for the synaptic localization and function of synaptophysin. Thus, glycosylation plays distinct roles in the trafficking of each of the three major synaptic vesicle glycoproteins.

scholarly journals The role of PI(4,5)P2 and synaptotagmin in membrane fusion - an in vitro study

2021 ◽  
Author(s):  
Joern Dietz ◽  
Marieelen Oelkers ◽  
Raphael Hubrich ◽  
Angel Perez-Lara ◽  
Reinhard Jahn ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Membrane Lipids ◽  
In Vitro Study ◽  
Snare Proteins ◽  
Large Unilamellar Vesicles ◽  
Intermediate States ◽  
Synaptotagmin 1 ◽  
Fusion Efficiency

Synaptotagmin-1 (syt-1) is known to trigger fusion of neuronal synaptic vesicles with the presynaptic membrane by recognizing acidic membrane lipids. In particular, binding to PI(4,5)P2 is believed to be crucial for its function as a calcium sensor. We propose a mechanism for syt-1 to interact with anionic bilayers and promote fusion in the presence of SNARE proteins. We found that in the absence of Ca2+ the binding of syt-1 to membranes depends on the PI(4,5)P2 content. Addition of Ca2+ switches the interaction forces from weak to strong eventually exceeding the cohesion of the C2A domain, while the interaction between PI(4,5)P2 and the C2B domain was preserved even in the absence of Ca2+ or phosphatidylserine. Fusion of large unilamellar vesicles equipped with syt-1 and synaptobrevin with freestanding target membranes composed of PS/PI(4,5)P2 show an increased fusion speed, and by effective suppression of stalled intermediate states, a larger number of full fusion events. Fusion efficiency could be maximized when irreversible docking is additionally prevented by addition of multivalent anions. The picture that emerges is that syt-1 remodels the membrane in the presence of calcium and PIP2, thereby substantially increasing the efficiency of membrane fusion by avoiding stalled intermediate states.

scholarly journals N-terminal phosphorylation of Huntingtin: A molecular switch for regulating Htt aggregation, helical conformation, internalization and nuclear targeting

2018 ◽  
Author(s):  
Sean M. DeGuire ◽  
Francesco. S. Ruggeri ◽  
Mohamed-Bilal Fares ◽  
Anass Chiki ◽  
Urszula Cendrowska ◽  
...  
Keyword(s):  
Membrane Binding ◽  
Serine Phosphorylation ◽  
Helical Conformation ◽  
Nuclear Targeting ◽  
Huntingtin Protein ◽  
Health And Disease ◽  
Bona Fide ◽  
Α Helix ◽  
And Function

AbstractPhosphorylation of exon1 of the Huntingtin protein (Httex1) has been shown to play important roles in regulating the structure, toxicity and cellular properties of N-terminal fragments and the full-length Huntingtin protein. Here, we investigated and compared the effect of bona fide phosphorylation at S13 and/or S16 on the structure, aggregation, membrane binding, and subcellular properties of mutant Httex1-Q18A. We show that serine phosphorylation at either S13 or S16 strongly disrupts the amphipathic α-helix of the N-terminus, inhibits the aggregation of mutant Httex1 and prompts the internalization and nuclear targeting of Httex1 preformed aggregates. In synthetic peptides phosphorylation at S13 and/or S16 strongly disrupted the amphipathic α-helix of the N-terminal 17 residues (Nt17) of Httex1 and Nt17 membrane binding. Our studies on peptides bearing a different combination of phosphorylation sites within Nt17 revealed a novel phosphorylation-dependent switch for regulating the structure of Httex1 involving crosstalk between phosphorylation at T3 and S13 or S16. Together, our results provide novel insights into the role of phosphorylation in regulating Httex1 structure and function in health and disease and underscore the critical importance of identifying enzymes responsible for regulating Htt phosphorylation and their potential as therapeutic targets for the treatment of Huntington’s disease.

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.

Sign in / Sign up

Export Citation Format

Share Document