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Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release.

Sakisaka T, Yamamoto Y, Mochida S, Nakamura M, Nishikawa K, Ishizaki H, Okamoto-Tanaka M, Miyoshi J, Fujiyoshi Y, Manabe T, Takai Y - J. Cell Biol. (2008)

Bottom Line: Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD).However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased.Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

ABSTRACT
Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex-mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

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Tomosyn-enhanced oligomerization of the SNARE complex in vivo. (A) Immunoblot analysis of the oligomerized SNARE complex from the brains of wild-type or tomosyn-deficient mice. Extracts of brains from wild-type and tomosyn-deficient mice were incubated with or without MBP-tomosyn for 16 h. Each sample was solubilized in the SDS sample buffer at RT or with boiling and subjected to SDS-PAGE followed by immunoblotting. Top, immunoblotting with anti–syntaxin-1 mAb; bottom, immunoblotting with anti–SNAP-25 mAb. Quantification of the relative oligomerization of the SNARE complex is shown in the right panel. KO, knockout; WT, wild type. Error bar represents SD. (B) Glycerol density gradient ultracentrifugation of brain extracts from wild-type and tomosyn-deficient mice. Extracts from wild-type and tomosyn-deficient mice were subjected to 10–40% glycerol density gradient ultracentrifugation. An aliquot of each fraction was solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Localization of SNAP-25 in the hippocampal CA3 region in wild-type and tomosyn-deficient mice. Adult mouse hippocampal sections were doubly stained with antitomosyn pAb and anti–SNAP-25 (SMI81) mAb. The results shown are representative of three independent experiments. SL, stratum lucidum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bars, 30 μm. (D) Localization of the SNARE proteins in the lipid raft fraction. The CSM fractions from wild-type and tomosyn-deficient mice were solubilized with Lubrol WX, and the lipid raft–enriched fractions were separated by sucrose gradient centrifugation as described previously (Vetrivel et al., 2004). Each fraction was boiled in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting. Flotillin-2 was used as a lipid raft marker. Quantification of the amount of protein in each fraction is shown in the right panel.
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fig2: Tomosyn-enhanced oligomerization of the SNARE complex in vivo. (A) Immunoblot analysis of the oligomerized SNARE complex from the brains of wild-type or tomosyn-deficient mice. Extracts of brains from wild-type and tomosyn-deficient mice were incubated with or without MBP-tomosyn for 16 h. Each sample was solubilized in the SDS sample buffer at RT or with boiling and subjected to SDS-PAGE followed by immunoblotting. Top, immunoblotting with anti–syntaxin-1 mAb; bottom, immunoblotting with anti–SNAP-25 mAb. Quantification of the relative oligomerization of the SNARE complex is shown in the right panel. KO, knockout; WT, wild type. Error bar represents SD. (B) Glycerol density gradient ultracentrifugation of brain extracts from wild-type and tomosyn-deficient mice. Extracts from wild-type and tomosyn-deficient mice were subjected to 10–40% glycerol density gradient ultracentrifugation. An aliquot of each fraction was solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Localization of SNAP-25 in the hippocampal CA3 region in wild-type and tomosyn-deficient mice. Adult mouse hippocampal sections were doubly stained with antitomosyn pAb and anti–SNAP-25 (SMI81) mAb. The results shown are representative of three independent experiments. SL, stratum lucidum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bars, 30 μm. (D) Localization of the SNARE proteins in the lipid raft fraction. The CSM fractions from wild-type and tomosyn-deficient mice were solubilized with Lubrol WX, and the lipid raft–enriched fractions were separated by sucrose gradient centrifugation as described previously (Vetrivel et al., 2004). Each fraction was boiled in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting. Flotillin-2 was used as a lipid raft marker. Quantification of the amount of protein in each fraction is shown in the right panel.

Mentions: Because the tomosyn deficiency facilitated synaptic transmission, we examined whether the formation of the SNARE complex was affected by tomosyn deficiency. For this purpose, we took advantage of the fact that the assembled SNARE complex is resistant to an SDS sample buffer at RT (Hayashi et al., 1994). Brain lysates from tomosyn-deficient and wild-type mice were solubilized in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 and anti–SNAP-25 mAbs. Because tomosyn was known to inhibit the formation of the SNARE complex by competing with VAMP-2 (Fujita et al., 1998; Pobbati et al., 2004), we expected that the formation of the SNARE complex would be increased in tomosyn-deficient mice. Unexpectedly, the amount of the assembled SNARE complex in tomosyn-deficient mice was reduced compared with wild-type mice (Fig. 2 A). The molecular mass of the immunoblot band was apparently larger (∼110 kD) than that of the expected single ternary SNARE complex (∼50 kD). Because a previous study has suggested that the single ternary SNARE complex can form an oligomeric structure and exists as a star-shaped particle with three to four bundles in vivo (Rickman et al., 2005), we assumed that the higher molecular mass bands might represent an oligomerized SNARE complex. Glycerol density gradient ultracentrifugation confirmed that the higher molecular mass bands sedimented in fractions with the mass of the oligomerized SNARE complex (Fig. 2 B). Furthermore, the oligomerized SNARE complex was restored by the addition of recombinant maltose-binding protein (MBP)–tomosyn to brain extracts from tomosyn-deficient mice (Fig. 2 A, top). These results indicate that tomosyn enhances the oligomerization of the SNARE complex.


Dual inhibition of SNARE complex formation by tomosyn ensures controlled neurotransmitter release.

Sakisaka T, Yamamoto Y, Mochida S, Nakamura M, Nishikawa K, Ishizaki H, Okamoto-Tanaka M, Miyoshi J, Fujiyoshi Y, Manabe T, Takai Y - J. Cell Biol. (2008)

Tomosyn-enhanced oligomerization of the SNARE complex in vivo. (A) Immunoblot analysis of the oligomerized SNARE complex from the brains of wild-type or tomosyn-deficient mice. Extracts of brains from wild-type and tomosyn-deficient mice were incubated with or without MBP-tomosyn for 16 h. Each sample was solubilized in the SDS sample buffer at RT or with boiling and subjected to SDS-PAGE followed by immunoblotting. Top, immunoblotting with anti–syntaxin-1 mAb; bottom, immunoblotting with anti–SNAP-25 mAb. Quantification of the relative oligomerization of the SNARE complex is shown in the right panel. KO, knockout; WT, wild type. Error bar represents SD. (B) Glycerol density gradient ultracentrifugation of brain extracts from wild-type and tomosyn-deficient mice. Extracts from wild-type and tomosyn-deficient mice were subjected to 10–40% glycerol density gradient ultracentrifugation. An aliquot of each fraction was solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Localization of SNAP-25 in the hippocampal CA3 region in wild-type and tomosyn-deficient mice. Adult mouse hippocampal sections were doubly stained with antitomosyn pAb and anti–SNAP-25 (SMI81) mAb. The results shown are representative of three independent experiments. SL, stratum lucidum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bars, 30 μm. (D) Localization of the SNARE proteins in the lipid raft fraction. The CSM fractions from wild-type and tomosyn-deficient mice were solubilized with Lubrol WX, and the lipid raft–enriched fractions were separated by sucrose gradient centrifugation as described previously (Vetrivel et al., 2004). Each fraction was boiled in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting. Flotillin-2 was used as a lipid raft marker. Quantification of the amount of protein in each fraction is shown in the right panel.
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fig2: Tomosyn-enhanced oligomerization of the SNARE complex in vivo. (A) Immunoblot analysis of the oligomerized SNARE complex from the brains of wild-type or tomosyn-deficient mice. Extracts of brains from wild-type and tomosyn-deficient mice were incubated with or without MBP-tomosyn for 16 h. Each sample was solubilized in the SDS sample buffer at RT or with boiling and subjected to SDS-PAGE followed by immunoblotting. Top, immunoblotting with anti–syntaxin-1 mAb; bottom, immunoblotting with anti–SNAP-25 mAb. Quantification of the relative oligomerization of the SNARE complex is shown in the right panel. KO, knockout; WT, wild type. Error bar represents SD. (B) Glycerol density gradient ultracentrifugation of brain extracts from wild-type and tomosyn-deficient mice. Extracts from wild-type and tomosyn-deficient mice were subjected to 10–40% glycerol density gradient ultracentrifugation. An aliquot of each fraction was solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Localization of SNAP-25 in the hippocampal CA3 region in wild-type and tomosyn-deficient mice. Adult mouse hippocampal sections were doubly stained with antitomosyn pAb and anti–SNAP-25 (SMI81) mAb. The results shown are representative of three independent experiments. SL, stratum lucidum; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Bars, 30 μm. (D) Localization of the SNARE proteins in the lipid raft fraction. The CSM fractions from wild-type and tomosyn-deficient mice were solubilized with Lubrol WX, and the lipid raft–enriched fractions were separated by sucrose gradient centrifugation as described previously (Vetrivel et al., 2004). Each fraction was boiled in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting. Flotillin-2 was used as a lipid raft marker. Quantification of the amount of protein in each fraction is shown in the right panel.
Mentions: Because the tomosyn deficiency facilitated synaptic transmission, we examined whether the formation of the SNARE complex was affected by tomosyn deficiency. For this purpose, we took advantage of the fact that the assembled SNARE complex is resistant to an SDS sample buffer at RT (Hayashi et al., 1994). Brain lysates from tomosyn-deficient and wild-type mice were solubilized in the SDS sample buffer and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 and anti–SNAP-25 mAbs. Because tomosyn was known to inhibit the formation of the SNARE complex by competing with VAMP-2 (Fujita et al., 1998; Pobbati et al., 2004), we expected that the formation of the SNARE complex would be increased in tomosyn-deficient mice. Unexpectedly, the amount of the assembled SNARE complex in tomosyn-deficient mice was reduced compared with wild-type mice (Fig. 2 A). The molecular mass of the immunoblot band was apparently larger (∼110 kD) than that of the expected single ternary SNARE complex (∼50 kD). Because a previous study has suggested that the single ternary SNARE complex can form an oligomeric structure and exists as a star-shaped particle with three to four bundles in vivo (Rickman et al., 2005), we assumed that the higher molecular mass bands might represent an oligomerized SNARE complex. Glycerol density gradient ultracentrifugation confirmed that the higher molecular mass bands sedimented in fractions with the mass of the oligomerized SNARE complex (Fig. 2 B). Furthermore, the oligomerized SNARE complex was restored by the addition of recombinant maltose-binding protein (MBP)–tomosyn to brain extracts from tomosyn-deficient mice (Fig. 2 A, top). These results indicate that tomosyn enhances the oligomerization of the SNARE complex.

Bottom Line: Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD).However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased.Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

View Article: PubMed Central - PubMed

Affiliation: Division of Membrane Dynamics, Department of Physiology and Cell Biology, Kobe University Graduate School of Medicine, Kobe 650-0017, Japan.

ABSTRACT
Neurotransmitter release from presynaptic nerve terminals is regulated by soluble NSF attachment protein receptor (SNARE) complex-mediated synaptic vesicle fusion. Tomosyn inhibits SNARE complex formation and neurotransmitter release by sequestering syntaxin-1 through its C-terminal vesicle-associated membrane protein (VAMP)-like domain (VLD). However, in tomosyn-deficient mice, the SNARE complex formation is unexpectedly decreased. In this study, we demonstrate that the N-terminal WD-40 repeat domain of tomosyn catalyzes the oligomerization of the SNARE complex. Microinjection of the tomosyn N-terminal WD-40 repeat domain into neurons prevented stimulated acetylcholine release. Thus, tomosyn inhibits neurotransmitter release by catalyzing oligomerization of the SNARE complex through the N-terminal WD-40 repeat domain in addition to the inhibitory activity of the C-terminal VLD.

Show MeSH
Related in: MedlinePlus