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Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis.

Darios F, Wasser C, Shakirzyanova A, Giniatullin A, Goodman K, Munoz-Bravo JL, Raingo J, Jorgacevski J, Kreft M, Zorec R, Rosa JM, Gandia L, Gutiérrez LM, Binz T, Giniatullin R, Kavalali ET, Davletov B - Neuron (2009)

Bottom Line: Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication.Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin.Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

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Sphingosine Inhibits Interaction of the Cytoplasmic Part of Synaptobrevin with Vesicular Membranes(A) Protein profiles of purified synaptic vesicles (SV; 30 μg protein) and synaptobrevin liposomes (2 μg protein) visualized in a Coomassie-stained SDS-PAGE gel. Synaptotagmin, synaptophysin and synaptobrevin are prominent synaptic vesicle markers.(B) Liposomes containing synaptobrevin (0.1 μg) were incubated in the presence of soluble syntaxin/SNAP-25 heterodimer (0.3 μg) and increasing concentrations of sphingosine. Immunoblot shows the transition of monomeric synaptobrevin into the SNARE complex. Note that a proportion of reconstituted synaptobrevin resides in the liposomal interior (not shown) and therefore is not available for syntaxin/SNAP-25 binding.(C) The cytoplasmic domain of synaptobrevin (aa 1–96), immobilized on glutathione beads via a GST tag, exhibits robust binding of phosphatidylcholine/phosphatidylserine liposomes labeled by the fluorescent dye DiO. Sphingosine reduces the ability of the cytoplasmic part of synaptobrevin to bind the phospholipid membrane with an EC50 of 6 μM. Error bars represent SEM, n = 5.(D) Graph showing increase in absorbance of liposomal solution at 350 nm upon addition of 2.5 μM GST-synaptobrevin (GST-Syb). This increase is immediately reversed upon addition of sphingosine (Sph, 50 μM, red curve).(E) 2.5 μM GST-C2A domain of synaptotagmin-1 induces an increase in absorbance of liposome solution in the presence of 1 mM free calcium. This effect is insensitive to the addition of 50 μM sphingosine (Sph). Vertical drops in absorbance in (D) and (E) are due to dilution of reactions.(F) Limited proteolysis of synaptic vesicles by V8 protease uncovers sensitivity of synaptobrevin, but not of synaptophysin, to sphingosine, as assessed by immunoblotting. Sphingosine-1-phosphate, ceramide, or sphingomyelin (all 50 μM) did not significantly affect proteolysis of the synaptic vesicle proteins.(G) Schematic showing sphingosine-mediated relief of the cytoplasmic part of synaptobrevin from inhibition by the vesicular membrane, a step necessary for further interaction with the syntaxin/SNAP-25 heterodimer. Ternary SNARE complex formation leads to vesicle fusion with the plasma membrane.
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fig7: Sphingosine Inhibits Interaction of the Cytoplasmic Part of Synaptobrevin with Vesicular Membranes(A) Protein profiles of purified synaptic vesicles (SV; 30 μg protein) and synaptobrevin liposomes (2 μg protein) visualized in a Coomassie-stained SDS-PAGE gel. Synaptotagmin, synaptophysin and synaptobrevin are prominent synaptic vesicle markers.(B) Liposomes containing synaptobrevin (0.1 μg) were incubated in the presence of soluble syntaxin/SNAP-25 heterodimer (0.3 μg) and increasing concentrations of sphingosine. Immunoblot shows the transition of monomeric synaptobrevin into the SNARE complex. Note that a proportion of reconstituted synaptobrevin resides in the liposomal interior (not shown) and therefore is not available for syntaxin/SNAP-25 binding.(C) The cytoplasmic domain of synaptobrevin (aa 1–96), immobilized on glutathione beads via a GST tag, exhibits robust binding of phosphatidylcholine/phosphatidylserine liposomes labeled by the fluorescent dye DiO. Sphingosine reduces the ability of the cytoplasmic part of synaptobrevin to bind the phospholipid membrane with an EC50 of 6 μM. Error bars represent SEM, n = 5.(D) Graph showing increase in absorbance of liposomal solution at 350 nm upon addition of 2.5 μM GST-synaptobrevin (GST-Syb). This increase is immediately reversed upon addition of sphingosine (Sph, 50 μM, red curve).(E) 2.5 μM GST-C2A domain of synaptotagmin-1 induces an increase in absorbance of liposome solution in the presence of 1 mM free calcium. This effect is insensitive to the addition of 50 μM sphingosine (Sph). Vertical drops in absorbance in (D) and (E) are due to dilution of reactions.(F) Limited proteolysis of synaptic vesicles by V8 protease uncovers sensitivity of synaptobrevin, but not of synaptophysin, to sphingosine, as assessed by immunoblotting. Sphingosine-1-phosphate, ceramide, or sphingomyelin (all 50 μM) did not significantly affect proteolysis of the synaptic vesicle proteins.(G) Schematic showing sphingosine-mediated relief of the cytoplasmic part of synaptobrevin from inhibition by the vesicular membrane, a step necessary for further interaction with the syntaxin/SNAP-25 heterodimer. Ternary SNARE complex formation leads to vesicle fusion with the plasma membrane.

Mentions: Since synaptobrevin resides in synaptic vesicle membrane among a dense network of proteins (Takamori et al., 2006), we probed whether sphingosine acts on the synaptobrevin/membrane interface, or targets synaptobrevin interaction with other vesicular proteins. We reconstituted full-length recombinant synaptobrevin into phospholipid liposomes containing phosphatidylcholine and phosphatidylserine (3:1 molar ratio). Figure 7A shows Coomassie-stained protein content of pure synaptic vesicles and reconstituted proteoliposomes; in the latter case, synaptobrevin is distributed equally in the outer and inner leaflets of liposomal membrane as assessed by trypsinolysis (Hu et al., 2002). Similar to vesicular synaptobrevin, liposomal synaptobrevin was inactive for interaction with syntaxin and SNAP-25 (Figure 7B), demonstrating that phospholipid membrane is sufficient for synaptobrevin restriction. Revealingly, sphingosine was sufficient to overcome synaptobrevin restriction in liposomes when added in a similar concentration range to that required for activation of synaptobrevin in synaptic vesicles (Figures 7B and 1C). In contrast, when we incorporated syntaxin/SNAP-25 into liposomes and tested SNARE assembly in the presence of a soluble part of synaptobrevin, sphingosine did not enhance the ability of this membrane-free synaptobrevin to form SNARE complex with syntaxin and SNAP-25 (Figure S4). Thus, membrane-embedded syntaxin/SNAP-25 heterodimers are available for SNARE assembly and sphingosine appears to affect specifically synaptobrevin in lipid membranes.


Sphingosine facilitates SNARE complex assembly and activates synaptic vesicle exocytosis.

Darios F, Wasser C, Shakirzyanova A, Giniatullin A, Goodman K, Munoz-Bravo JL, Raingo J, Jorgacevski J, Kreft M, Zorec R, Rosa JM, Gandia L, Gutiérrez LM, Binz T, Giniatullin R, Kavalali ET, Davletov B - Neuron (2009)

Sphingosine Inhibits Interaction of the Cytoplasmic Part of Synaptobrevin with Vesicular Membranes(A) Protein profiles of purified synaptic vesicles (SV; 30 μg protein) and synaptobrevin liposomes (2 μg protein) visualized in a Coomassie-stained SDS-PAGE gel. Synaptotagmin, synaptophysin and synaptobrevin are prominent synaptic vesicle markers.(B) Liposomes containing synaptobrevin (0.1 μg) were incubated in the presence of soluble syntaxin/SNAP-25 heterodimer (0.3 μg) and increasing concentrations of sphingosine. Immunoblot shows the transition of monomeric synaptobrevin into the SNARE complex. Note that a proportion of reconstituted synaptobrevin resides in the liposomal interior (not shown) and therefore is not available for syntaxin/SNAP-25 binding.(C) The cytoplasmic domain of synaptobrevin (aa 1–96), immobilized on glutathione beads via a GST tag, exhibits robust binding of phosphatidylcholine/phosphatidylserine liposomes labeled by the fluorescent dye DiO. Sphingosine reduces the ability of the cytoplasmic part of synaptobrevin to bind the phospholipid membrane with an EC50 of 6 μM. Error bars represent SEM, n = 5.(D) Graph showing increase in absorbance of liposomal solution at 350 nm upon addition of 2.5 μM GST-synaptobrevin (GST-Syb). This increase is immediately reversed upon addition of sphingosine (Sph, 50 μM, red curve).(E) 2.5 μM GST-C2A domain of synaptotagmin-1 induces an increase in absorbance of liposome solution in the presence of 1 mM free calcium. This effect is insensitive to the addition of 50 μM sphingosine (Sph). Vertical drops in absorbance in (D) and (E) are due to dilution of reactions.(F) Limited proteolysis of synaptic vesicles by V8 protease uncovers sensitivity of synaptobrevin, but not of synaptophysin, to sphingosine, as assessed by immunoblotting. Sphingosine-1-phosphate, ceramide, or sphingomyelin (all 50 μM) did not significantly affect proteolysis of the synaptic vesicle proteins.(G) Schematic showing sphingosine-mediated relief of the cytoplasmic part of synaptobrevin from inhibition by the vesicular membrane, a step necessary for further interaction with the syntaxin/SNAP-25 heterodimer. Ternary SNARE complex formation leads to vesicle fusion with the plasma membrane.
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fig7: Sphingosine Inhibits Interaction of the Cytoplasmic Part of Synaptobrevin with Vesicular Membranes(A) Protein profiles of purified synaptic vesicles (SV; 30 μg protein) and synaptobrevin liposomes (2 μg protein) visualized in a Coomassie-stained SDS-PAGE gel. Synaptotagmin, synaptophysin and synaptobrevin are prominent synaptic vesicle markers.(B) Liposomes containing synaptobrevin (0.1 μg) were incubated in the presence of soluble syntaxin/SNAP-25 heterodimer (0.3 μg) and increasing concentrations of sphingosine. Immunoblot shows the transition of monomeric synaptobrevin into the SNARE complex. Note that a proportion of reconstituted synaptobrevin resides in the liposomal interior (not shown) and therefore is not available for syntaxin/SNAP-25 binding.(C) The cytoplasmic domain of synaptobrevin (aa 1–96), immobilized on glutathione beads via a GST tag, exhibits robust binding of phosphatidylcholine/phosphatidylserine liposomes labeled by the fluorescent dye DiO. Sphingosine reduces the ability of the cytoplasmic part of synaptobrevin to bind the phospholipid membrane with an EC50 of 6 μM. Error bars represent SEM, n = 5.(D) Graph showing increase in absorbance of liposomal solution at 350 nm upon addition of 2.5 μM GST-synaptobrevin (GST-Syb). This increase is immediately reversed upon addition of sphingosine (Sph, 50 μM, red curve).(E) 2.5 μM GST-C2A domain of synaptotagmin-1 induces an increase in absorbance of liposome solution in the presence of 1 mM free calcium. This effect is insensitive to the addition of 50 μM sphingosine (Sph). Vertical drops in absorbance in (D) and (E) are due to dilution of reactions.(F) Limited proteolysis of synaptic vesicles by V8 protease uncovers sensitivity of synaptobrevin, but not of synaptophysin, to sphingosine, as assessed by immunoblotting. Sphingosine-1-phosphate, ceramide, or sphingomyelin (all 50 μM) did not significantly affect proteolysis of the synaptic vesicle proteins.(G) Schematic showing sphingosine-mediated relief of the cytoplasmic part of synaptobrevin from inhibition by the vesicular membrane, a step necessary for further interaction with the syntaxin/SNAP-25 heterodimer. Ternary SNARE complex formation leads to vesicle fusion with the plasma membrane.
Mentions: Since synaptobrevin resides in synaptic vesicle membrane among a dense network of proteins (Takamori et al., 2006), we probed whether sphingosine acts on the synaptobrevin/membrane interface, or targets synaptobrevin interaction with other vesicular proteins. We reconstituted full-length recombinant synaptobrevin into phospholipid liposomes containing phosphatidylcholine and phosphatidylserine (3:1 molar ratio). Figure 7A shows Coomassie-stained protein content of pure synaptic vesicles and reconstituted proteoliposomes; in the latter case, synaptobrevin is distributed equally in the outer and inner leaflets of liposomal membrane as assessed by trypsinolysis (Hu et al., 2002). Similar to vesicular synaptobrevin, liposomal synaptobrevin was inactive for interaction with syntaxin and SNAP-25 (Figure 7B), demonstrating that phospholipid membrane is sufficient for synaptobrevin restriction. Revealingly, sphingosine was sufficient to overcome synaptobrevin restriction in liposomes when added in a similar concentration range to that required for activation of synaptobrevin in synaptic vesicles (Figures 7B and 1C). In contrast, when we incorporated syntaxin/SNAP-25 into liposomes and tested SNARE assembly in the presence of a soluble part of synaptobrevin, sphingosine did not enhance the ability of this membrane-free synaptobrevin to form SNARE complex with syntaxin and SNAP-25 (Figure S4). Thus, membrane-embedded syntaxin/SNAP-25 heterodimers are available for SNARE assembly and sphingosine appears to affect specifically synaptobrevin in lipid membranes.

Bottom Line: Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication.Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin.Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

View Article: PubMed Central - PubMed

Affiliation: MRC Laboratory of Molecular Biology, Cambridge CB2 0QH, UK.

ABSTRACT
Synaptic vesicles loaded with neurotransmitters fuse with the plasma membrane to release their content into the extracellular space, thereby allowing neuronal communication. The membrane fusion process is mediated by a conserved set of SNARE proteins: vesicular synaptobrevin and plasma membrane syntaxin and SNAP-25. Recent data suggest that the fusion process may be subject to regulation by local lipid metabolism. Here, we have performed a screen of lipid compounds to identify positive regulators of vesicular synaptobrevin. We show that sphingosine, a releasable backbone of sphingolipids, activates synaptobrevin in synaptic vesicles to form the SNARE complex implicated in membrane fusion. Consistent with the role of synaptobrevin in vesicle fusion, sphingosine upregulated exocytosis in isolated nerve terminals, neuromuscular junctions, neuroendocrine cells and hippocampal neurons, but not in neurons obtained from synaptobrevin-2 knockout mice. Further mechanistic insights suggest that sphingosine acts on the synaptobrevin/phospholipid interface, defining a novel function for this important lipid regulator.

Show MeSH
Related in: MedlinePlus