Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

Lynch, Kara L.; Gerona, Roy; Kielar, Dana M.; Martens, Sascha; McMahon, Harvey T.; Martin, Thomas F.J.

doi:10.1091/mbc.e08-03-0235

Cited by 120 publications

(162 citation statements)

References 62 publications

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“…Although these hypothetical events shown in Figure 9 allow for SNARE-dependent zippering to closely approximate the two bilayers, the working model makes no specific predictions about molecular pathways by which the phospholipids themselves physically interchange between the two bilayers, and which additional proteins (e.g., Munc18, CAPS, and synaptotagmin) might facilitate this interchange and thus initiate fusion. In this regard, two recent reports discuss a specific mechanism by which membrane insertion of hydrophobic residues at the tips of the C2 domains of synaptotagmin could induce membrane buckling and thus lower the activation barrier for fusion (Lynch et al, 2008;Paddock et al, 2008). Note added in Proof.…”

Section: Discussionmentioning

confidence: 99%

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

Williams

Vicogne

Зайцева

et al. 2009

MBoC

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The juxtamembrane domain of vesicle-associated membrane protein (VAMP) 2 (also known as synaptobrevin2) contains a conserved cluster of basic/hydrophobic residues that may play an important role in membrane fusion. Our measurements on peptides corresponding to this domain determine the electrostatic and hydrophobic energies by which this domain of VAMP2 could bind to the adjacent lipid bilayer in an insulin granule or other transport vesicle. Mutation of residues within the juxtamembrane domain that reduce the VAMP2 net positive charge, and thus its interaction with membranes, inhibits secretion of insulin granules in ␤ cells. Increasing salt concentration in permeabilized cells, which reduces electrostatic interactions, also results in an inhibition of insulin secretion. Similarly, amphipathic weak bases (e.g., sphingosine) that reverse the negative electrostatic surface potential of a bilayer reverse membrane binding of the positively charged juxtamembrane domain of a reconstituted VAMP2 protein and inhibit membrane fusion. We propose a model in which the positively charged VAMP and syntaxin juxtamembrane regions facilitate fusion by bridging the negatively charged vesicle and plasma membrane leaflets.

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Section: Discussionmentioning

confidence: 99%

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

Williams

Vicogne

Зайцева

et al. 2009

MBoC

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“…Constructs were subcloned into tag red fluorescent protein-containing plasmids to monitor successful re-expression of CAPS by fluorescence. Studies of evoked dense-core vesicle exocytosis were conducted in PC12 cells expressing brain-derived neurotrophic factor-enhanced green fluorescent protein by total internal reflection fluorescence microscopy as previously described (22).…”

Section: Methodsmentioning

confidence: 99%

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Nojiri

Loyet²,

Klenchin

et al. 2009

Journal of Biological Chemistry

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CAPS (Ca 2؉ -dependent activator protein for secretion) functions in priming Ca 2؉ -dependent vesicle exocytosis, but the regulation of CAPS activity has not been characterized. Here we show that phosphorylation by protein kinase CK2 is required for CAPS activity. Dephosphorylation eliminated CAPS activity in reconstituting Ca 2؉ -dependent vesicle exocytosis in permeable and intact PC12 cells. Ser-5, -6, and -7 and Ser-1281 were identified by mass spectrometry as the major phosphorylation sites in the 1289 residue protein. Ser-5, -6, and -7 but not Ser-1281 to Ala substitutions abolished CAPS activity. Protein kinase CK2 phosphorylated CAPS in vitro at these sites and restored the activity of dephosphorylated CAPS. CK2 is the likely in vivo CAPS protein kinase based on inhibition of phosphorylation by tetrabromo-2-benzotriazole in PC12 cells and by the identity of in vivo and in vitro phosphorylation sites. CAPS phosphorylation by CK2 was constitutive, but the elevation of Ca 2؉ in synaptosomes increased CAPS Ser-5 and -6 dephosphorylation, which terminates CAPS activity. These results identify a functionally important N-terminal phosphorylation site that regulates CAPS activity in priming vesicle exocytosis.Regulated neurotransmitter secretion is central to intercellular communication in the nervous system. Two types of secretory vesicles mediate neurotransmitter release; that is, synaptic vesicles that release transmitters such as glutamate at synapses and dense-core vesicles that release modulatory transmitters and neuropeptides at non-synaptic sites. Both types of secretory vesicles are recruited to docking sites on the plasma membrane where they are primed to a ready release state to undergo fusion in response to Ca 2ϩ elevations. Many of the proteins that mediate the targeting, docking, priming, and Ca 2ϩ -dependent fusion of vesicles with the plasma membrane function in both synaptic vesicle and dense-core vesicle pathways (1). CAPS-1 2 (also known as Cadps1) is a 1289-residue protein that reconstitutes Ca 2ϩ -triggered dense-core vesicle exocytosis in permeable neuroendocrine cells at a priming step (2-4). CAPS is required for secretion of a subset of transmitters in Caenorhabditis elegans (5) and Drosophila melanogaster (6) and for priming dense-core vesicle exocytosis in neuroendocrine cells (7) and synaptic vesicle exocytosis in neurons (8). Vesicle priming reactions are extensively modulated during physiological demand (9), but mechanisms that regulate CAPS function remain to be identified.Reversible protein phosphorylation is a major mechanism for the regulation of cellular processes including vesicle exocytosis. Many proteins that function in evoked vesicle exocytosis are phosphoproteins (10, 11). The neuronal SNARE proteins syntaxin 1A, VAMP-2, and SNAP-25 are phosphorylated by several protein kinases in vitro (12)(13)(14). Protein kinase C and protein kinase A sites on SNAP-25 affect refilling rates and size, respectively, of the primed pool of vesicles in chromaffin cells (15,16). Several SNARE...

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“…The effect of PI(4,5)P 2 on the concentration dependence of the dichroic ratio was likely due to an enhanced binding of the synaptotagmin C2 domains to bilayers containing PI(4,5)P 2 . An increased membrane and Ca 2ϩ affinity for C2AB in the presence of PI(4,5)P 2 has been observed (25)(26)(27), and estimates based upon fluorescence correlation spectroscopy 3 indicate that the membrane binding of C2AB was increased by 3-5-fold by the incorporation of 1 mol % PI(4,5)P 2 into PC:PS (3:1) bilayers.…”

Section: Synaptotagmin 1 C2 Domains Alter the Acyl Chain Order Of Popmentioning

confidence: 98%

Synaptotagmin 1 Modulates Lipid Acyl Chain Order in Lipid Bilayers by Demixing Phosphatidylserine

Lai

Tamm

Ellena

et al. 2011

Journal of Biological Chemistry

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Synaptotagmin 1 (syt1) functions as the Ca 2؉ sensor in neuronal exocytosis, and it has been proposed to act by modulating lipid bilayer curvature. Here we examine the effect of the two C2 domains (C2A and C2B) of syt1 on membrane lipid order and lateral organization. In mixtures of phosphatidylcholine and phosphatidylserine (PS), attenuated total internal reflection Fourier transform infrared spectroscopy indicates that a fragment containing both domains (C2AB) or C2B alone disorders the lipid acyl chains, whereas the C2A domain has little effect upon chain order. Two observations suggest that these changes reflect a demixing of PS. First, the changes in acyl chain order are reversed at higher protein concentration; second, selective lipid deuteration demonstrates that the changes in lipid order are associated only with the PS component of the bilayer. Independent evidence for lipid demixing is obtained from fluorescence selfquenching of labeled lipid and from natural abundance 13 C NMR, where heteronuclear single quantum correlation spectra reveal Ca 2؉ -dependent chemical shift changes for PS, but not for phosphatidylcholine, in the presence of the syt1 C2 domains. The ability of syt1 to demix PS is observed in a range of lipid mixtures that includes cholesterol, phosphatidylethanolamine, and varied PS content. These data suggest that syt1 might facilitate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptors)-mediated membrane fusion by phase separating PS, a process that is expected to locally buckle bilayers and disorder lipids due to the curvature tendencies of PS.

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Synaptotagmin-1 Utilizes Membrane Bending and SNARE Binding to Drive Fusion Pore Expansion

Cited by 120 publications

References 62 publications

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

Evidence that Electrostatic Interactions between Vesicle-associated Membrane Protein 2 and Acidic Phospholipids May Modulate the Fusion of Transport Vesicles with the Plasma Membrane

CAPS Activity in Priming Vesicle Exocytosis Requires CK2 Phosphorylation

Synaptotagmin 1 Modulates Lipid Acyl Chain Order in Lipid Bilayers by Demixing Phosphatidylserine

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