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Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis.

Walter AM, Wiederhold K, Bruns D, Fasshauer D, Sørensen JB - J. Cell Biol. (2010)

Bottom Line: Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca(2+).In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration.Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Mechanism of Exocytosis, Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.

ABSTRACT
Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca(2+). It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin-SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca(2+)-triggered C-terminal assembly and membrane fusion.

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In vitro binding of syb-2 to a syntaxin–SNAP-25 acceptor complex. (A) Fluorescence anisotropy measurements of labeled syb-2 mutants and WT protein binding to the ΔN complex. Comparison of two N-terminal mutants (LATA and VAVA) with WT syb-2 (control) and a C-terminal mutant (L84A) is shown. An N-terminally truncated syb-2 (Δ32–35, deletion of aa 32–35) displays greatly decreased binding kinetics (note the axis break on the abscissa; 100 nM synaptobrevin was added to 500 nM ΔN complex). (B) A plot of the observed binding rates of the synaptobrevin WT protein (control trace) and the LATA and VAVA mutants against the different concentrations (conc.) of the acceptor ΔN complex allows calculation of the association rate (slope of linear fits; values given in Biophysical characterization of mutants).
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fig2: In vitro binding of syb-2 to a syntaxin–SNAP-25 acceptor complex. (A) Fluorescence anisotropy measurements of labeled syb-2 mutants and WT protein binding to the ΔN complex. Comparison of two N-terminal mutants (LATA and VAVA) with WT syb-2 (control) and a C-terminal mutant (L84A) is shown. An N-terminally truncated syb-2 (Δ32–35, deletion of aa 32–35) displays greatly decreased binding kinetics (note the axis break on the abscissa; 100 nM synaptobrevin was added to 500 nM ΔN complex). (B) A plot of the observed binding rates of the synaptobrevin WT protein (control trace) and the LATA and VAVA mutants against the different concentrations (conc.) of the acceptor ΔN complex allows calculation of the association rate (slope of linear fits; values given in Biophysical characterization of mutants).

Mentions: As expected, alanine substitutions at either the C- or the N-terminal end of the complex did not eliminate the ability of syb-2 to create ternary SNARE complexes. Circular dichroism (CD) spectroscopy revealed that assembled complexes were only slightly destabilized as compared with wild-type (WT) complexes (Fig. S1 A). With the intention of testing how our mutants affect the kinetics of SNARE complex assembly in vitro, we used fluorescence anisotropy measurements and analyzed the speed of synaptobrevin binding to the ΔN complex. For these experiments, we used syntaxin-1 lacking the N-terminal Habc domain. The Habc domain is not part of the SNARE bundle itself, but it reduces the overall speed of assembly by inducing an intermittent shift to the closed conformation of syntaxin-1 (Dulubova et al., 1999; Margittai et al., 2003). The absence of this domain is not expected to change the mechanism of assembly itself. We found that destabilization of the two most N-terminal layers (LATA mutant) of the SNARE complex reduced the speed of synaptobrevin binding to the acceptor complex, whereas no effect could be seen by mutations at the C-terminal end (L84A; Fig. 2 A). Titrating with increasing amounts of ΔN complex (Fig. 2 B), we estimated an association rate constant of 235,000 M−1 s−1 (pseudo first-order rate constant) for WT synaptobrevin, which is in reasonable agreement with recent findings (Pobbati et al., 2006). This rate was decreased fivefold by the LATA mutant (44,000 M−1 s−1) but was essentially unchanged for the VAVA mutant (263,000 M−1 s−1). Thus, apparently, the integrity of layers −7 and −6 is essential for rapid binding in vitro. This was confirmed by testing the deletion mutant Δ32–35, in which 4 aa, including these two layer residues, were deleted; this mutant displayed very slow binding kinetics (<3,000 M−1 s−1, which could not be measured accurately because of the lack of saturation; Fig. 2 A). In contrast, deleting the entire C-terminal half of the SNARE motif (Syb1–52, a deletion of 47 aa) only decreased the binding rate to 123,000 M−1 s−1 (Wiederhold and Fasshauer, 2009). Thus, there can be little doubt (also in light of previous in vitro data [Pobbati et al., 2006]) that the N-terminal end serves to initiate binding of synaptobrevin to the syntaxin–SNAP-25 dimer in vitro.


Synaptobrevin N-terminally bound to syntaxin-SNAP-25 defines the primed vesicle state in regulated exocytosis.

Walter AM, Wiederhold K, Bruns D, Fasshauer D, Sørensen JB - J. Cell Biol. (2010)

In vitro binding of syb-2 to a syntaxin–SNAP-25 acceptor complex. (A) Fluorescence anisotropy measurements of labeled syb-2 mutants and WT protein binding to the ΔN complex. Comparison of two N-terminal mutants (LATA and VAVA) with WT syb-2 (control) and a C-terminal mutant (L84A) is shown. An N-terminally truncated syb-2 (Δ32–35, deletion of aa 32–35) displays greatly decreased binding kinetics (note the axis break on the abscissa; 100 nM synaptobrevin was added to 500 nM ΔN complex). (B) A plot of the observed binding rates of the synaptobrevin WT protein (control trace) and the LATA and VAVA mutants against the different concentrations (conc.) of the acceptor ΔN complex allows calculation of the association rate (slope of linear fits; values given in Biophysical characterization of mutants).
© Copyright Policy - openaccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2819690&req=5

fig2: In vitro binding of syb-2 to a syntaxin–SNAP-25 acceptor complex. (A) Fluorescence anisotropy measurements of labeled syb-2 mutants and WT protein binding to the ΔN complex. Comparison of two N-terminal mutants (LATA and VAVA) with WT syb-2 (control) and a C-terminal mutant (L84A) is shown. An N-terminally truncated syb-2 (Δ32–35, deletion of aa 32–35) displays greatly decreased binding kinetics (note the axis break on the abscissa; 100 nM synaptobrevin was added to 500 nM ΔN complex). (B) A plot of the observed binding rates of the synaptobrevin WT protein (control trace) and the LATA and VAVA mutants against the different concentrations (conc.) of the acceptor ΔN complex allows calculation of the association rate (slope of linear fits; values given in Biophysical characterization of mutants).
Mentions: As expected, alanine substitutions at either the C- or the N-terminal end of the complex did not eliminate the ability of syb-2 to create ternary SNARE complexes. Circular dichroism (CD) spectroscopy revealed that assembled complexes were only slightly destabilized as compared with wild-type (WT) complexes (Fig. S1 A). With the intention of testing how our mutants affect the kinetics of SNARE complex assembly in vitro, we used fluorescence anisotropy measurements and analyzed the speed of synaptobrevin binding to the ΔN complex. For these experiments, we used syntaxin-1 lacking the N-terminal Habc domain. The Habc domain is not part of the SNARE bundle itself, but it reduces the overall speed of assembly by inducing an intermittent shift to the closed conformation of syntaxin-1 (Dulubova et al., 1999; Margittai et al., 2003). The absence of this domain is not expected to change the mechanism of assembly itself. We found that destabilization of the two most N-terminal layers (LATA mutant) of the SNARE complex reduced the speed of synaptobrevin binding to the acceptor complex, whereas no effect could be seen by mutations at the C-terminal end (L84A; Fig. 2 A). Titrating with increasing amounts of ΔN complex (Fig. 2 B), we estimated an association rate constant of 235,000 M−1 s−1 (pseudo first-order rate constant) for WT synaptobrevin, which is in reasonable agreement with recent findings (Pobbati et al., 2006). This rate was decreased fivefold by the LATA mutant (44,000 M−1 s−1) but was essentially unchanged for the VAVA mutant (263,000 M−1 s−1). Thus, apparently, the integrity of layers −7 and −6 is essential for rapid binding in vitro. This was confirmed by testing the deletion mutant Δ32–35, in which 4 aa, including these two layer residues, were deleted; this mutant displayed very slow binding kinetics (<3,000 M−1 s−1, which could not be measured accurately because of the lack of saturation; Fig. 2 A). In contrast, deleting the entire C-terminal half of the SNARE motif (Syb1–52, a deletion of 47 aa) only decreased the binding rate to 123,000 M−1 s−1 (Wiederhold and Fasshauer, 2009). Thus, there can be little doubt (also in light of previous in vitro data [Pobbati et al., 2006]) that the N-terminal end serves to initiate binding of synaptobrevin to the syntaxin–SNAP-25 dimer in vitro.

Bottom Line: Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca(2+).In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration.Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch.

View Article: PubMed Central - HTML - PubMed

Affiliation: Molecular Mechanism of Exocytosis, Department of Membrane Biophysics, Max Planck Institute for Biophysical Chemistry, D-37077 Göttingen, Germany.

ABSTRACT
Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)-dependent exocytosis pathway at an intermediate "cocked" state, from which fusion can be triggered by Ca(2+). It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin-SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca(2+)-triggered C-terminal assembly and membrane fusion.

Show MeSH
Related in: MedlinePlus