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PMID: 10974000 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

The C2B domain of synaptotagmin is a Ca(2+)-sensing module essential for exocytosis.

The Journal of cell biology ·Vol. 150 ·No. 5 ·2000-09-04 ·Pages 1125-36

Desai RC, Vyas B, Earles CA, Littleton JT, Kowalchyck JA, Martin TF, Chapman ER

Abstract

The synaptic vesicle protein synaptotagmin I has been proposed to serve as a Ca(2+) sensor for rapid exocytosis. Synaptotagmin spans the vesicle membrane once and possesses a large cytoplasmic domain that contains two C2 domains, C2A and C2B. Multiple Ca(2+) ions bind to the membrane proximal C2A domain. However, it is not known whether the C2B domain also functions as a Ca(2+)-sensing module. Here, we report that Ca(2+) drives conformational changes in the C2B domain of synaptotagmin and triggers the homo- and hetero-oligomerization of multiple isoforms of the protein. These effects of Ca(2)+ are mediated by a set of conserved acidic Ca(2)+ ligands within C2B; neutralization of these residues results in constitutive clustering activity. We addressed the function of oligomerization using a dominant negative approach. Two distinct reagents that block synaptotagmin clustering potently inhibited secretion from semi-intact PC12 cells. Together, these data indicate that the Ca(2)+-driven clustering of the C2B domain of synaptotagmin is an essential step in excitation-secretion coupling. We propose that clustering may regulate the opening or dilation of the exocytotic fusion pore.

MeSH Terms
Amino Acid Sequence Animals Binding Sites Calcium/physiology Calcium Signaling/physiology Calcium-Binding Proteins/chemistry,physiology Cytoplasmic Granules/physiology Endocytosis Exocytosis/physiology Intracellular Membranes/physiology Macromolecular Substances Membrane Fusion Membrane Glycoproteins/chemistry,physiology Molecular Sequence Data Nerve Tissue Proteins/chemistry,physiology PC12 Cells Protein Isoforms/chemistry,physiology Rats Recombinant Proteins/chemistry,metabolism Sequence Alignment Sequence Homology, Amino Acid Synaptotagmin I Synaptotagmins
Chemicals
Calcium-Binding Proteins Macromolecular Substances Membrane Glycoproteins Nerve Tissue Proteins Protein Isoforms Recombinant Proteins Synaptotagmin I Syt1 protein, rat Synaptotagmins Calcium
Authors & Affiliations
7 authors, click to expand affiliations / ORCID
Desai R C
Department of Physiology, University of Wisconsin, Madison, Wisconsin 53706, USA.
Vyas B
Earles C A
Littleton J T
Kowalchyck J A
Martin T F
Chapman E R
References (65)
65 references, click to expand
  1. Oligomeric complexes link Rab5 effectors with NSF and drive membrane fusion via interactions between EEA1 and syntaxin 13.
    Cell. 1999 Aug 6;98(3):377-86 PMID: 10458612
  2. Synaptic function modulated by changes in the ratio of synaptotagmin I and IV.
    Nature. 1999 Aug 19;400(6746):757-60 PMID: 10466723
  3. A novel alternatively spliced variant of synaptotagmin VI lacking a transmembrane domain. Implications for distinct functions of the two isoforms.
    J Biol Chem. 1999 Oct 29;274(44):31428-34 PMID: 10531344
  4. Crystal structure of the cytosolic C2A-C2B domains of synaptotagmin III. Implications for Ca(+2)-independent snare complex interaction.
    J Cell Biol. 1999 Nov 1;147(3):589-98 PMID: 10545502
  5. Structure of the Janus-faced C2B domain of rabphilin.
    Nat Cell Biol. 1999 Jun;1(2):106-12 PMID: 10559882
  6. Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes.
    Neuron. 1999 Oct;24(2):363-76 PMID: 10571230
  7. Crystal structures of the membrane-binding C2 domain of human coagulation factor V.
    Nature. 1999 Nov 25;402(6760):434-9 PMID: 10586886
  8. Structure of the C2 domain of human factor VIII at 1.5 A resolution.
    Nature. 1999 Nov 25;402(6760):439-42 PMID: 10586887
  9. The C terminus of SNAP25 is essential for Ca(2+)-dependent binding of synaptotagmin to SNARE complexes.
    J Biol Chem. 2000 Mar 3;275(9):6328-36 PMID: 10692432
  10. Membrane-embedded synaptotagmin penetrates cis or trans target membranes and clusters via a novel mechanism.
    J Biol Chem. 2000 Aug 18;275(33):25427-35 PMID: 10840045
  11. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue.
    J Cell Biol. 1981 Oct;91(1):257-69 PMID: 7298720
  12. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C.
    Nature. 1990 May 17;345(6272):260-3 PMID: 2333096
  13. Synaptotagmin: a calcium sensor on the synaptic vesicle surface.
    Science. 1992 May 15;256(5059):1021-5 PMID: 1589771
  14. Neurotransmitter release from synaptotagmin-deficient clonal variants of PC12 cells.
    Science. 1992 Jun 26;256(5065):1821-3 PMID: 1352065
  15. Resolution of regulated secretion into sequential MgATP-dependent and calcium-dependent stages mediated by distinct cytosolic proteins.
    J Cell Biol. 1992 Oct;119(1):139-51 PMID: 1527165
  16. A role for synaptotagmin (p65) in regulated exocytosis.
    Cell. 1993 Jan 15;72(1):153-9 PMID: 8422678
  17. SNAP receptors implicated in vesicle targeting and fusion.
    Nature. 1993 Mar 25;362(6418):318-24 PMID: 8455717
  18. Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin.
    Cell. 1993 Jul 2;73(7):1291-305 PMID: 8391930
  19. Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release.
    Cell. 1993 Sep 24;74(6):1125-34 PMID: 8104705
  20. A single C2 domain from synaptotagmin I is sufficient for high affinity Ca2+/phospholipid binding.
    J Biol Chem. 1993 Dec 15;268(35):26386-90 PMID: 8253763
  21. Calcium-dependent interaction of the cytoplasmic region of synaptotagmin with membranes. Autonomous function of a single C2-homologous domain.
    J Biol Chem. 1994 Feb 25;269(8):5735-41 PMID: 8119912
  22. The effect on synaptic physiology of synaptotagmin mutations in Drosophila.
    Neuron. 1994 Apr;12(4):909-20 PMID: 7909234
  23. Synaptotagmin III is a novel isoform of rat synaptotagmin expressed in endocrine and neuronal cells.
    J Biol Chem. 1994 Apr 22;269(16):11675-8 PMID: 8163462
  24. Synaptotagmin I is a high affinity receptor for clathrin AP-2: implications for membrane recycling.
    Cell. 1994 Sep 9;78(5):751-60 PMID: 8087843
  25. Calcium dependence of the rate of exocytosis in a synaptic terminal.
    Nature. 1994 Oct 6;371(6497):513-5 PMID: 7935764
  26. Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse.
    Cell. 1994 Nov 18;79(4):717-27 PMID: 7954835
  27. Ca(2+)-dependent conformational change in synaptotagmin I.
    J Biol Chem. 1994 Nov 18;269(46):28547-50 PMID: 7961798
  28. Calcium dependence of neurotransmitter release and rate of spontaneous vesicle fusions are altered in Drosophila synaptotagmin mutants.
    Proc Natl Acad Sci U S A. 1994 Nov 8;91(23):10888-92 PMID: 7971978
  29. ATP-dependent inositide phosphorylation required for Ca(2+)-activated secretion.
    Nature. 1995 Mar 9;374(6518):173-7 PMID: 7877690
  30. Synaptotagmin IV is an immediate early gene induced by depolarization in PC12 cells and in brain.
    Proc Natl Acad Sci U S A. 1995 Mar 14;92(6):2164-8 PMID: 7892240
  31. Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold.
    Cell. 1995 Mar 24;80(6):929-38 PMID: 7697723
  32. Cytosolic calcium facilitates release of secretory products after exocytotic vesicle fusion.
    FEBS Lett. 1995 Apr 24;363(3):221-5 PMID: 7737406
  33. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins.
    Nature. 1995 Jun 15;375(6532):594-9 PMID: 7791877
  34. Identification of a nonneuronal isoform of synaptotagmin.
    Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5895-9 PMID: 7597049
  35. Structure and function of fusion pores in exocytosis and ectoplasmic membrane fusion.
    Curr Opin Cell Biol. 1995 Aug;7(4):509-17 PMID: 7495570
  36. Ca2+ regulates the interaction between synaptotagmin and syntaxin 1.
    J Biol Chem. 1995 Oct 6;270(40):23667-71 PMID: 7559535
  37. Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain.
    J Biol Chem. 1995 Nov 3;270(44):26523-7 PMID: 7592870
  38. Defective recycling of synaptic vesicles in synaptotagmin mutants of Caenorhabditis elegans.
    Nature. 1995 Nov 9;378(6553):196-9 PMID: 7477324
  39. A possible docking and fusion particle for synaptic transmission.
    Nature. 1995 Dec 14;378(6558):733-6 PMID: 7501022
  40. Distinct Ca(2+)-dependent properties of the first and second C2-domains of synaptotagmin I.
    J Biol Chem. 1996 Jan 19;271(3):1262-5 PMID: 8576108
  41. A novel function for the second C2 domain of synaptotagmin. Ca2+-triggered dimerization.
    J Biol Chem. 1996 Mar 8;271(10):5844-9 PMID: 8621455
  42. Membrane fusion mediated by the influenza virus hemagglutinin requires the concerted action of at least three hemagglutinin trimers.
    J Cell Biol. 1996 May;133(3):559-69 PMID: 8636231
  43. C2 domain conformational changes in phospholipase C-delta 1.
    Nat Struct Biol. 1996 Sep;3(9):788-95 PMID: 8784353
  44. Dilation of the influenza hemagglutinin fusion pore revealed by the kinetics of individual cell-cell fusion events.
    J Cell Biol. 1996 Oct;135(1):63-71 PMID: 8858163
  45. Inhibition of neurotransmission by peptides containing the synaptic protein interaction site of N-type Ca2+ channels.
    Neuron. 1996 Oct;17(4):781-8 PMID: 8893034
  46. Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin.
    J Biol Chem. 1996 Nov 1;271(44):27770-5 PMID: 8910372
  47. The C2 domain calcium-binding motif: structural and functional diversity.
    Protein Sci. 1996 Dec;5(12):2375-90 PMID: 8976547
  48. Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses.
    Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):997-1001 PMID: 9023371
  49. Interaction of the synprint site of N-type Ca2+ channels with the C2B domain of synaptotagmin I.
    Proc Natl Acad Sci U S A. 1997 May 13;94(10):5405-10 PMID: 9144250
  50. Alteration of Ca2+ dependence of neurotransmitter release by disruption of Ca2+ channel/syntaxin interaction.
    J Neurosci. 1997 Sep 1;17(17):6647-56 PMID: 9254677
  51. Structural changes are associated with soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor complex formation.
    J Biol Chem. 1997 Oct 31;272(44):28036-41 PMID: 9346956
  52. Ca2+-dependent and -independent interactions of the isoforms of the alpha1A subunit of brain Ca2+ channels with presynaptic SNARE proteins.
    Proc Natl Acad Sci U S A. 1997 Dec 23;94(26):14782-6 PMID: 9405690
  53. Crystal structure of a calcium-phospholipid binding domain from cytosolic phospholipase A2.
    J Biol Chem. 1998 Jan 16;273(3):1596-604 PMID: 9430701
  54. SNAREpins: minimal machinery for membrane fusion.
    Cell. 1998 Mar 20;92(6):759-72 PMID: 9529252
  55. Direct interaction of a Ca2+-binding loop of synaptotagmin with lipid bilayers.
    J Biol Chem. 1998 May 29;273(22):13995-4001 PMID: 9593749
  56. Ca2+ binding to synaptotagmin: how many Ca2+ ions bind to the tip of a C2-domain?
    EMBO J. 1998 Jul 15;17(14):3921-30 PMID: 9670009
  57. Temperature-sensitive paralytic mutations demonstrate that synaptic exocytosis requires SNARE complex assembly and disassembly.
    Neuron. 1998 Aug;21(2):401-13 PMID: 9728921
  58. Mechanism of phospholipid binding by the C2A-domain of synaptotagmin I.
    Biochemistry. 1998 Sep 8;37(36):12395-403 PMID: 9730811
  59. Lipid binding ridge on loops 2 and 3 of the C2A domain of synaptotagmin I as revealed by NMR spectroscopy.
    J Biol Chem. 1998 Oct 2;273(40):25659-63 PMID: 9748232
  60. Structure of the protein kinase Cbeta phospholipid-binding C2 domain complexed with Ca2+.
    Structure. 1998 Nov 15;6(11):1395-405 PMID: 9817842
  61. Delineation of the oligomerization, AP-2 binding, and synprint binding region of the C2B domain of synaptotagmin.
    J Biol Chem. 1998 Dec 4;273(49):32966-72 PMID: 9830048
  62. Calcium-dependent oligomerization of synaptotagmins I and II. Synaptotagmins I and II are localized on the same synaptic vesicle and heterodimerize in the presence of calcium.
    J Biol Chem. 1999 Jan 1;274(1):59-66 PMID: 9867811
  63. SNARE complex formation is triggered by Ca2+ and drives membrane fusion.
    Cell. 1999 Apr 16;97(2):165-74 PMID: 10219238
  64. The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.
    J Biol Chem. 1999 Jun 25;274(26):18290-6 PMID: 10373432
  65. Syntaxin 1A interacts with multiple exocytic proteins to regulate neurotransmitter release in vivo.
    Neuron. 1999 Jul;23(3):593-605 PMID: 10433270
Article Info
Journal
The Journal of cell biology
Abbr.
J Cell Biol
ISSN
0021-9525
DOI
10.1083/jcb.150.5.1125
Published
2000-09-04
Pages
1125-36
Language
English
Region
United States
NLM ID
0375356
PMCID
PMC2175261
Subset
IM
Grants
NIDDK NIH HHS · T35 DK062709 · United States
NIDDK NIH HHS · DK40428 · United States
NIDDK NIH HHS · DK25861 · United States
NIGMS NIH HHS · GM 56827-01 · United States
External Links
PubMed source DOI: 10.1083/jcb.150.5.1125 PMC Full Text View journal details
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