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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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Different affinities for SNAP-25 and syntaxin-1 between the N-terminal WD-40 repeat domain and the C-terminal VLD. (A) Molecular structures of tomosyn-ΔVLD and VLD. (B) Binding of syntaxin-1 and SNAP-25 to the VLD of tomosyn. Quantification of the amount of syntaxin-1 bound to VLD is shown in the right panel. No binding of syntaxin-1 and SNAP-25 to MBP was shown in the middle panel (negative control). (C) Binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD. Quantification of the amount of syntaxin-1 bound to MBP-tomosyn-ΔVLD is shown in the right panel. For B and C, syntaxin-1, SNAP-25, or the mixture of syntaxin-1 and SNAP-25 were incubated with the indicated MBP-fusion protein immobilized on amylose beads in the presence or absence of VAMP-2. After the beads were extensively washed, proteins bound to the beads were solubilized in the SDS sample buffer with boiling. The bound proteins were subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb, anti–SNAP-25 mAb, or anti–VAMP-2 mAb. (B and C) Error bars represent SD. (D) Formation of the oligomerized SNARE complex in the supernatant fraction of the MBP-tomosyn-ΔVLD beads. Syntaxin-1, SNAP-25, and VAMP-2 were incubated with MBP-tomosyn-ΔVLD immobilized on amylose beads for the indicated periods of time. After centrifugation, the proteins in the supernatant fraction were soIubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb.
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fig4: Different affinities for SNAP-25 and syntaxin-1 between the N-terminal WD-40 repeat domain and the C-terminal VLD. (A) Molecular structures of tomosyn-ΔVLD and VLD. (B) Binding of syntaxin-1 and SNAP-25 to the VLD of tomosyn. Quantification of the amount of syntaxin-1 bound to VLD is shown in the right panel. No binding of syntaxin-1 and SNAP-25 to MBP was shown in the middle panel (negative control). (C) Binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD. Quantification of the amount of syntaxin-1 bound to MBP-tomosyn-ΔVLD is shown in the right panel. For B and C, syntaxin-1, SNAP-25, or the mixture of syntaxin-1 and SNAP-25 were incubated with the indicated MBP-fusion protein immobilized on amylose beads in the presence or absence of VAMP-2. After the beads were extensively washed, proteins bound to the beads were solubilized in the SDS sample buffer with boiling. The bound proteins were subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb, anti–SNAP-25 mAb, or anti–VAMP-2 mAb. (B and C) Error bars represent SD. (D) Formation of the oligomerized SNARE complex in the supernatant fraction of the MBP-tomosyn-ΔVLD beads. Syntaxin-1, SNAP-25, and VAMP-2 were incubated with MBP-tomosyn-ΔVLD immobilized on amylose beads for the indicated periods of time. After centrifugation, the proteins in the supernatant fraction were soIubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb.

Mentions: Tomosyn has at least two domains: an N-terminal WD-40 repeat domain and a C-terminal VLD (Fujita et al., 1998). It has been shown that the VLD binds syntaxin-1 in the same manner as the SNARE domain and thereby competes with VAMP-2 in the formation of the SNARE complex (Pobbati et al., 2004). However, this competing activity of the VLD does not explain the activity of tomosyn to enhance the oligomerization of the SNARE complex. Therefore, we further characterized the possible interaction of tomosyn with the SNARE proteins. We first made an MBP-fused VLD (MBP-VLD; Fig. 4 A) and examined the interaction between the MBP-VLD and SNARE proteins. Syntaxin-1 and SNAP-25 bound to MBP-VLD as previously reported (Fig. 4 B; Yokoyama et al., 1999). VAMP-2 did not bind to MBP-VLD. Syntaxin-1 and SNAP-25 each enhanced the binding of the other to MBP-VLD by threefold, presumably reflecting the formation of a SNARE complex–like structure. VAMP-2 did not affect the binding of syntaxin-1 and SNAP-25 to MBP-VLD. We next made a VLD-deficient mutant of tomosyn (tomosyn-ΔVLD) as an MBP fusion protein and examined the interaction between MBP-tomosyn-ΔVLD and the SNARE proteins. Syntaxin-1 or SNAP-25 bound to MBP-tomosyn-ΔVLD (Fig. 4 C); however, VAMP-2 did not bind to MBP-tomosyn-ΔVLD. Neither syntaxin-1 nor SNAP-25 enhanced the binding of the other to MBP-tomosyn-ΔVLD. VAMP-2 decreased the binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD and induced the oligomerization of the SNARE complex (Fig. 4 D). These results indicate that the large N-terminal WD-40 repeat domain of tomosyn and the C-terminal VLD have different affinities for syntaxin-1 and SNAP-25.


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)

Different affinities for SNAP-25 and syntaxin-1 between the N-terminal WD-40 repeat domain and the C-terminal VLD. (A) Molecular structures of tomosyn-ΔVLD and VLD. (B) Binding of syntaxin-1 and SNAP-25 to the VLD of tomosyn. Quantification of the amount of syntaxin-1 bound to VLD is shown in the right panel. No binding of syntaxin-1 and SNAP-25 to MBP was shown in the middle panel (negative control). (C) Binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD. Quantification of the amount of syntaxin-1 bound to MBP-tomosyn-ΔVLD is shown in the right panel. For B and C, syntaxin-1, SNAP-25, or the mixture of syntaxin-1 and SNAP-25 were incubated with the indicated MBP-fusion protein immobilized on amylose beads in the presence or absence of VAMP-2. After the beads were extensively washed, proteins bound to the beads were solubilized in the SDS sample buffer with boiling. The bound proteins were subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb, anti–SNAP-25 mAb, or anti–VAMP-2 mAb. (B and C) Error bars represent SD. (D) Formation of the oligomerized SNARE complex in the supernatant fraction of the MBP-tomosyn-ΔVLD beads. Syntaxin-1, SNAP-25, and VAMP-2 were incubated with MBP-tomosyn-ΔVLD immobilized on amylose beads for the indicated periods of time. After centrifugation, the proteins in the supernatant fraction were soIubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb.
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fig4: Different affinities for SNAP-25 and syntaxin-1 between the N-terminal WD-40 repeat domain and the C-terminal VLD. (A) Molecular structures of tomosyn-ΔVLD and VLD. (B) Binding of syntaxin-1 and SNAP-25 to the VLD of tomosyn. Quantification of the amount of syntaxin-1 bound to VLD is shown in the right panel. No binding of syntaxin-1 and SNAP-25 to MBP was shown in the middle panel (negative control). (C) Binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD. Quantification of the amount of syntaxin-1 bound to MBP-tomosyn-ΔVLD is shown in the right panel. For B and C, syntaxin-1, SNAP-25, or the mixture of syntaxin-1 and SNAP-25 were incubated with the indicated MBP-fusion protein immobilized on amylose beads in the presence or absence of VAMP-2. After the beads were extensively washed, proteins bound to the beads were solubilized in the SDS sample buffer with boiling. The bound proteins were subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb, anti–SNAP-25 mAb, or anti–VAMP-2 mAb. (B and C) Error bars represent SD. (D) Formation of the oligomerized SNARE complex in the supernatant fraction of the MBP-tomosyn-ΔVLD beads. Syntaxin-1, SNAP-25, and VAMP-2 were incubated with MBP-tomosyn-ΔVLD immobilized on amylose beads for the indicated periods of time. After centrifugation, the proteins in the supernatant fraction were soIubilized in the SDS sample buffer at RT and subjected to SDS-PAGE followed by immunoblotting with anti–syntaxin-1 mAb.
Mentions: Tomosyn has at least two domains: an N-terminal WD-40 repeat domain and a C-terminal VLD (Fujita et al., 1998). It has been shown that the VLD binds syntaxin-1 in the same manner as the SNARE domain and thereby competes with VAMP-2 in the formation of the SNARE complex (Pobbati et al., 2004). However, this competing activity of the VLD does not explain the activity of tomosyn to enhance the oligomerization of the SNARE complex. Therefore, we further characterized the possible interaction of tomosyn with the SNARE proteins. We first made an MBP-fused VLD (MBP-VLD; Fig. 4 A) and examined the interaction between the MBP-VLD and SNARE proteins. Syntaxin-1 and SNAP-25 bound to MBP-VLD as previously reported (Fig. 4 B; Yokoyama et al., 1999). VAMP-2 did not bind to MBP-VLD. Syntaxin-1 and SNAP-25 each enhanced the binding of the other to MBP-VLD by threefold, presumably reflecting the formation of a SNARE complex–like structure. VAMP-2 did not affect the binding of syntaxin-1 and SNAP-25 to MBP-VLD. We next made a VLD-deficient mutant of tomosyn (tomosyn-ΔVLD) as an MBP fusion protein and examined the interaction between MBP-tomosyn-ΔVLD and the SNARE proteins. Syntaxin-1 or SNAP-25 bound to MBP-tomosyn-ΔVLD (Fig. 4 C); however, VAMP-2 did not bind to MBP-tomosyn-ΔVLD. Neither syntaxin-1 nor SNAP-25 enhanced the binding of the other to MBP-tomosyn-ΔVLD. VAMP-2 decreased the binding of syntaxin-1 and SNAP-25 to MBP-tomosyn-ΔVLD and induced the oligomerization of the SNARE complex (Fig. 4 D). These results indicate that the large N-terminal WD-40 repeat domain of tomosyn and the C-terminal VLD have different affinities for syntaxin-1 and SNAP-25.

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