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. 1997 Feb 4;94(3):997-1001.
doi: 10.1073/pnas.94.3.997.

Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses

G Schiavo  1 , G Stenbeck, J E Rothman, T H Söllner
Affiliations

Binding of the synaptic vesicle v-SNARE, synaptotagmin, to the plasma membrane t-SNARE, SNAP-25, can explain docked vesicles at neurotoxin-treated synapses

G Schiavo et al. Proc Natl Acad Sci U S A. .

Abstract

Neurotransmitter release requires the specific docking of synaptic vesicles to the presynaptic plasma membrane followed by a calcium-triggered fusion event. Herein we report a previously unsuspected interaction of the synaptic vesicle protein and likely calcium sensor synaptotagmin with the plasma membrane t-SNARE SNAP-25. This interaction appears to resolve the apparent paradox that synaptic vesicles are capable of docking even when VAMP (vesicle-associated membrane protein) or syntaxin is cleaved or deleted and suggests that two species of v-SNAREs (VAMP and synaptotagmin) and two species of t-SNAREs (SNAP-25 and syntaxin) interact to functionally dock synaptic vesicles.

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Figures

Figure 3
Figure 3
SNAP-25 promotes the assembly of the SNARE complex on tagmin. GST–tagmin was incubated alone (lane 1) or with His6–SNAP-25 (lanes 3, 6–8, 10, and 13–15) and/or full-length syntaxin 1B (lanes 2, 5, 6, 8, 9, 12, 13, and 15) and/or VAMP–His6 (lanes 4, 5, 7, 8, 11, 12, 14, and 15) in the presence of 2 mM EGTA (A) or 200 μM free calcium (B). The empty arrow indicates a contamination of GST from the recombinant syntaxin 1B preparation that interacts with glutathione–agarose beads. No nonspecific binding of SNARE proteins was observed by incubating them with GST-containing beads under the same conditions.
Figure 4
Figure 4
SNAP-25 in complex with tagmin is cleaved by botulinum neurotoxins. (A) His6–SNAP-25 prebound to GST–tagmin was incubated with buffer only (lane 2) or with activated BoNT/A (lane 3) or activated BoNT/E (lane 4). Under these conditions, the BoNT-generated amino-terminal fragments of SNAP-25 remain bound to tagmin. Botulinum neurotoxin treatment does not abolish the de novo formation of the tagmin/SNAP-25 complex (B). GST–tagmin was incubated with His6–SNAP-25 (lane 5) or His6–SNAP-25 pretreated with activated BoNT/A (lane 6) or activated BoNT/E (lane 7) in the presence of 200 μM calcium.
Figure 2
Figure 2
Native as well as recombinant tagmin binds SNAP-25 in a saturable and stoichiometric manner. Native tagmin immunopurified from brain cortex (A) and the recombinant cytoplasmic domain of tagmin (B) were incubated with increasing amounts of His6–SNAP-25 in the presence or absence of 200 μM free calcium as indicated. As control, beads containing the anti-tagmin antibody used for immunoprecipitation (15) (A, Control Ab) or GST (B, GST Control) were incubated with the maximal amount of His6–SNAP-25 (32 μg) in the presence or absence of calcium.
Figure 1
Figure 1
Native tagmin is associated with SNARE proteins in brain extract. Tagmin was immunopurified from brain cortex detergent extract in association with SNARE proteins, as revealed by Western blot analysis (A) using specific antibodies against syntaxin (lane 1), SNAP-25 (lane 2), and VAMP (lane 3). The analysis of the Coomassie blue-stained gels (B) reveals that an excess of SNAP-25 over syntaxin and VAMP is associated with tagmin (lane 4). For comparison, a sample containing only anti-tagmin antibody incubated in the absence of brain extract is shown in lane 5 (the asterisk indicates the position of the antibody L chain).
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Comment in

  • SNAREs and regulated vesicle exocytosis.
    Goda Y. Goda Y. Proc Natl Acad Sci U S A. 1997 Feb 4;94(3):769-72. doi: 10.1073/pnas.94.3.769. Proc Natl Acad Sci U S A. 1997. PMID: 9023331 Free PMC article. Review. No abstract available.

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