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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 vitro. (A) Reconstitution of tomosyn-induced oligomerization of the SNARE complex. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence or absence of the indicated amounts of MBP-tomosyn for 12 h under reducing conditions (1 mM DTT). The proteins were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1, anti–VAMP-2, or anti–SNAP-25 mAbs or anti-MBP pAb. (B) Analysis of molecular mass of the oligomerized SNARE complex by density gradient ultracentrifugation. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence of MBP-tomosyn for 12 h. The sample was subjected to a 10–30% glycerol density gradient. Aliquots of each gradient fraction were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Morphology of the oligomerized SNARE complex. Rotary shadowing electron micrographs of the SNARE complexes recovered in gradient fractions 11 and 17. Quantification of the number of legs in the SNARE complex is shown in the right panel. The results shown are representative of three independent experiments.
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fig3: Tomosyn-enhanced oligomerization of the SNARE complex in vitro. (A) Reconstitution of tomosyn-induced oligomerization of the SNARE complex. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence or absence of the indicated amounts of MBP-tomosyn for 12 h under reducing conditions (1 mM DTT). The proteins were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1, anti–VAMP-2, or anti–SNAP-25 mAbs or anti-MBP pAb. (B) Analysis of molecular mass of the oligomerized SNARE complex by density gradient ultracentrifugation. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence of MBP-tomosyn for 12 h. The sample was subjected to a 10–30% glycerol density gradient. Aliquots of each gradient fraction were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Morphology of the oligomerized SNARE complex. Rotary shadowing electron micrographs of the SNARE complexes recovered in gradient fractions 11 and 17. Quantification of the number of legs in the SNARE complex is shown in the right panel. The results shown are representative of three independent experiments.

Mentions: Next, we examined the ability of tomosyn to either enhance the formation of the SNARE complex or the oligomerization of the SNARE complex in a cell-free assay system using recombinant proteins. Recombinant MBP-tomosyn was incubated with syntaxin-1 lacking the transmembrane domain (referred to as syntaxin-1 hereafter), SNAP-25, and VAMP-2 lacking the transmembrane (referred to as VAMP-2 hereafter) for 12 h under reducing conditions. The reaction was terminated by the addition of the SDS sample buffer at RT, and each sample was subjected to SDS-PAGE followed by immunoblotting. The recombinant tomosyn protein shifted a 50-kD immunoreactive band to molecular masses of 90 and 130 kD in a dose-dependent manner (Fig. 3 A), suggesting that tomosyn enhanced the oligomerization of the SNARE complex. An intense band for MBP-tomosyn was detected at a higher molecular mass than the oligomerized SNARE complex, indicating that tomosyn was not incorporated into the SDS-resistant oligomerized SNARE complex. Characterization of the proteins extracted from the 130-kD band suggestive of the oligomerized SNARE complex and the 50-kD band suggestive of the monomeric SNARE complex revealed that both bands were composed of syntaxin-1, SNAP-25, and VAMP-2 at a ratio of 1:1:1, suggesting that the high molecular weight reaction product represents the oligomerized SNARE complex (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805150/DC1).


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 vitro. (A) Reconstitution of tomosyn-induced oligomerization of the SNARE complex. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence or absence of the indicated amounts of MBP-tomosyn for 12 h under reducing conditions (1 mM DTT). The proteins were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1, anti–VAMP-2, or anti–SNAP-25 mAbs or anti-MBP pAb. (B) Analysis of molecular mass of the oligomerized SNARE complex by density gradient ultracentrifugation. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence of MBP-tomosyn for 12 h. The sample was subjected to a 10–30% glycerol density gradient. Aliquots of each gradient fraction were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Morphology of the oligomerized SNARE complex. Rotary shadowing electron micrographs of the SNARE complexes recovered in gradient fractions 11 and 17. Quantification of the number of legs in the SNARE complex is shown in the right panel. The results shown are representative of three independent experiments.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2568027&req=5

fig3: Tomosyn-enhanced oligomerization of the SNARE complex in vitro. (A) Reconstitution of tomosyn-induced oligomerization of the SNARE complex. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence or absence of the indicated amounts of MBP-tomosyn for 12 h under reducing conditions (1 mM DTT). The proteins were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1, anti–VAMP-2, or anti–SNAP-25 mAbs or anti-MBP pAb. (B) Analysis of molecular mass of the oligomerized SNARE complex by density gradient ultracentrifugation. Syntaxin-1, SNAP-25, and VAMP-2 were incubated in the presence of MBP-tomosyn for 12 h. The sample was subjected to a 10–30% glycerol density gradient. Aliquots of each gradient fraction were solubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb. (C) Morphology of the oligomerized SNARE complex. Rotary shadowing electron micrographs of the SNARE complexes recovered in gradient fractions 11 and 17. Quantification of the number of legs in the SNARE complex is shown in the right panel. The results shown are representative of three independent experiments.
Mentions: Next, we examined the ability of tomosyn to either enhance the formation of the SNARE complex or the oligomerization of the SNARE complex in a cell-free assay system using recombinant proteins. Recombinant MBP-tomosyn was incubated with syntaxin-1 lacking the transmembrane domain (referred to as syntaxin-1 hereafter), SNAP-25, and VAMP-2 lacking the transmembrane (referred to as VAMP-2 hereafter) for 12 h under reducing conditions. The reaction was terminated by the addition of the SDS sample buffer at RT, and each sample was subjected to SDS-PAGE followed by immunoblotting. The recombinant tomosyn protein shifted a 50-kD immunoreactive band to molecular masses of 90 and 130 kD in a dose-dependent manner (Fig. 3 A), suggesting that tomosyn enhanced the oligomerization of the SNARE complex. An intense band for MBP-tomosyn was detected at a higher molecular mass than the oligomerized SNARE complex, indicating that tomosyn was not incorporated into the SDS-resistant oligomerized SNARE complex. Characterization of the proteins extracted from the 130-kD band suggestive of the oligomerized SNARE complex and the 50-kD band suggestive of the monomeric SNARE complex revealed that both bands were composed of syntaxin-1, SNAP-25, and VAMP-2 at a ratio of 1:1:1, suggesting that the high molecular weight reaction product represents the oligomerized SNARE complex (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200805150/DC1).

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