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Cited by 75Cellular transport and secretionNeuroscience and Neuropharmacology ResearchExtracellular vesicles in disease

Synaptotagmin C2B Domain Regulates Ca2+-triggered Fusion in Vitro

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Gaffaney, Jon D., Dunning, F. Mark, Wang, Zhao, Hui, Enfu, Chapman, Edwin R.·2008·Journal of Biological Chemistry
DOI 10.1074/jbc.m803355200PMC PMC2581593
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Abstract

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

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  • The effect of the Syt1 R322E, K325E mutant and reported observations suggest a functional role for the polybasic region.

    “The effect of the Syt1 R322E, K325E mutant (Fig. 4) as well as reported observations29,30,35,44,45 suggests a functional role for the polybasic region.”

    From Architecture of the synaptotagmin–SNARE machinery for neuronal exocytosis (2015) · cites this paper as [35]

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Cited by

Sorted by citations

Authors
Gaffaney, J. D., Dunning, F. M., Wang, Z., Hui, E., & Chapman, E. R.
Affiliations
  • University of Wisconsin–Madison (US)
  • Howard Hughes Medical Institute (US)
Corresponding author
Chapman, Edwin R.
ORCID
Venue
Journal of Biological Chemistry
Publisher
Elsevier BV
Published
2008-11
Volume
283
Issue
46
Pages
31763-31775
ISSN
0021-9258 (ISSN-L)
DOI
10.1074/jbc.m803355200
PMC
PMC2581593
OpenAlex
W2051707340
Cited by
75
References
1
Citations by year
2025: 2 · 2024: 4 · 2023: 5 · 2022: 2 · 2021: 1 · 2020: 2 · 2019: 2 · 2018: 9 · 2017: 2 · 2016: 5 · 2015: 7 · 2014: 5 · 2013: 5 · 2012: 3
FWCI
2.92
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Biological Chemistry (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by-nc)
  • PubMed (publishedVersion)
Funders
  • Howard Hughes Medical Institute
  • American Heart Association (0440168N)
  • National Institutes of Health (MH61876)
  • National Institute of Mental Health (MH61876, R01MH061876)
  • National Institute of General Medical Sciences (GM56827, R01GM056827)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Cellular transport and secretion
  • Life Sciences › Neuroscience › Cellular and Molecular Neuroscience › Neuroscience and Neuropharmacology Research
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
Keywords
synaptotagmin-1, C2B domain, C2A domain, synaptic vesicle exocytosis, phosphatidylethanolamine, alanine scanning, calcium binding, vesicle fusion
MeSH terms
Alanine, Animals, Calcium, Cell Membrane, Cytosol, Liposomes, Models, Molecular, Protein Binding, Mutagenesis, Protein Structure, Tertiary, Protein Structure, Quaternary, Qa-SNARE Proteins, Synaptotagmins, Mice

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