Synaptotagmin-1 utilizes membrane bending and SNARE binding to drive fusion pore expansion.
In regulated vesicle exocytosis, SNARE protein complexes drive membrane fusion to connect the vesicle lumen with the extracellular space. The triggering of fusion pore formation by Ca(2+) is mediated by specific isoforms of synaptotagmin (Syt), which employ both SNARE complex and membrane binding. Ca(2+) also promotes fusion pore expansion and Syts have been implicated in this process but the mechanisms involved are unclear. We determined the role of Ca(2+)-dependent Syt-effector interactions in fusion pore expansion by expressing Syt-1 mutants selectively altered in Ca(2+)-dependent SNARE binding or in Ca(2+)-dependent membrane insertion in PC12 cells that lack vesicle Syts. The release of different-sized fluorescent peptide-EGFP vesicle cargo or the vesicle capture of different-sized external fluorescent probes was used to assess the extent of fusion pore dilation. We found that PC12 cells expressing partial loss-of-function Syt-1 mutants impaired in Ca(2+)-dependent SNARE binding exhibited reduced fusion pore opening probabilities and reduced fusion pore expansion. Cells with gain-of-function Syt-1 mutants for Ca(2+)-dependent membrane insertion exhibited normal fusion pore opening probabilities but the fusion pores dilated extensively. The results indicate that Syt-1 uses both Ca(2+)-dependent membrane insertion and SNARE binding to drive fusion pore expansion.
Lynch
Kara L
KL
Department of Biochemistry, University of Wisconsin, Madison, WI 53706, USA.
Gerona
Roy R L
RR
Kielar
Dana M
DM
Martens
Sascha
S
McMahon
Harvey T
HT
Martin
Thomas F J
TF
D000818:Animals; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry / Q000378:metabolism; D005089:Exocytosis / Q000502:physiology*; D008561:Membrane Fusion / Q000502:physiology*; D009154:Mutation; D016716:PC12 Cells; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D050600:SNARE Proteins / Q000378:metabolism*; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050863:Synaptotagmin I / Q000235:genetics / Q000378:metabolism*
D011993:Recombinant Fusion Proteins; D050600:SNARE Proteins; D050863:Synaptotagmin I; D002118:Calcium
BMC Genomics. 2007 Jul 31;8:259
17672888
J Biol Chem. 2002 Feb 15;277(7):4601-4
11751925
Nature. 2003 Aug 21;424(6951):943-7
12931189
J Cell Biol. 2006 Oct 23;175(2):201-7
17043140
Neuron. 2002 Jan 17;33(2):287-99
11804575
Genes Dev. 2002 Apr 15;16(8):948-58
11959843
J Neurosci. 2006 Nov 29;26(48):12556-65
17135417
J Physiol. 2004 Oct 15;560(Pt 2):413-28
15297569
FEBS Lett. 1995 Apr 24;363(3):221-5
7737406
Nat Struct Mol Biol. 2006 Apr;13(4):323-30
16565726
Nat Rev Mol Cell Biol. 2002 Jul;3(7):498-508
12094216
Cell. 1994 Nov 18;79(4):717-27
7954835
Nature. 2007 Nov 29;450(7170):676-82
18046404
Science. 2001 Nov 2;294(5544):1111-5
11691996
J Biol Chem. 2004 Dec 10;279(50):52677-84
15456748
Biochemistry. 2006 Aug 15;45(32):9668-74
16893168
Biochem Biophys Res Commun. 2006 Aug 18;347(1):76-82
16808897
Science. 2004 Apr 9;304(5668):289-92
15016962
Science. 2002 Aug 23;297(5585):1349-52
12193788
Trends Biochem Sci. 2004 Mar;29(3):143-51
15003272
J Cell Sci. 2005 Sep 15;118(Pt 18):4271-82
16141231
Nature. 1992 Mar 5;356(6364):60-3
1538782
J Cell Biol. 2001 Sep 17;154(6):1117-23
11551981
J Mol Biol. 2007 Mar 30;367(3):848-63
17320903
Nature. 1997 Oct 2;389(6650):509-12
9333242
Proc Natl Acad Sci U S A. 2002 Feb 5;99(3):1665-70
11805296
FEBS Lett. 2005 Sep 26;579(23):5241-6
16165130
Biochim Biophys Acta. 2003 Aug 18;1641(2-3):167-73
12914957
Neuron. 1999 Oct;24(2):363-76
10571230
J Biol Chem. 2000 Mar 3;275(9):6328-36
10692432
J Biol Chem. 2001 Aug 31;276(35):33027-35
11402049
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3992-7
18308938
Mol Cell Biol. 2005 Oct;25(19):8693-702
16166648
J Biol Chem. 1998 Jun 26;273(26):15879-82
9632630
J Membr Biol. 2004 May 1;199(1):1-14
15366419
Proc Natl Acad Sci U S A. 2005 Dec 20;102(51):18664-9
16352718
Biotechniques. 1992 Jun;12(6):870-4, 876-9
1642895
J Neurosci. 2005 Aug 17;25(33):7601-14
16107647
Neuron. 2004 Mar 25;41(6):929-42
15046725
Annu Rev Neurosci. 2004;27:509-47
15217342
Curr Opin Neurobiol. 1996 Jun;6(3):350-7
8794084
Neuron. 2007 May 24;54(4):567-81
17521570
J Chromatogr. 1967 May;28(1):69-81
6048444
Neuron. 2002 May 16;34(4):599-611
12062043
Proc Natl Acad Sci U S A. 2008 Mar 11;105(10):3998-4003
18308932
FEBS Lett. 1995 Apr 24;363(3):217-20
7737405
Mol Biol Cell. 2007 Dec;18(12):4957-68
17914059
J Cell Sci. 2007 Feb 15;120(Pt 4):617-27
17264148
Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):2070-5
12538853
Trends Cell Biol. 2006 Jul;16(7):339-50
16698267
Cell. 1998 Mar 20;92(6):759-72
9529252
Cell. 2005 Nov 4;123(3):375-82
16269330
Nucleic Acids Res. 2000 Aug 15;28(16):E78
10931937
J Physiol. 2006 Jan 15;570(Pt 2):295-307
16293646
Science. 2007 May 25;316(5828):1205-8
17478680
Nature. 1993 Jun 10;363(6429):554-8
8505984
Trends Cell Biol. 2006 Aug;16(8):413-20
16839766
Nature. 1987 Aug 27-Sep 2;328(6133):814-7
2442614
Biochemistry. 1998 Sep 8;37(36):12395-403
9730811
J Cell Biol. 2003 Jul 21;162(2):199-209
12860971
Annu Rev Biophys Biomol Struct. 2006;35:135-60
16689631