Determinants of synaptobrevin regulation in membranes

Abstract

Neuronal exocytosis is driven by the formation of SNARE complexes between synaptobrevin 2 on synaptic vesicles and SNAP-25/syntaxin 1 on the plasma membrane. It has remained controversial, however, whether SNAREs are constitutively active or whether they are down-regulated until fusion is triggered. We now show that synaptobrevin in proteoliposomes as well as in purified synaptic vesicles is constitutively active. Potential regulators such as calmodulin or synaptophysin do not affect SNARE activity. Substitution or deletion of residues in the linker connecting the SNARE motif and transmembrane region did not alter the kinetics of SNARE complex assembly or of SNARE-mediated fusion of liposomes. Remarkably, deletion of C-terminal residues of the SNARE motif strongly reduced fusion activity, although the overall stability of the complexes was not affected. We conclude that although complete zippering of the SNARE complex is essential for membrane fusion, the structure of the adjacent linker domain is less critical, suggesting that complete SNARE complex assembly not only connects membranes but also drives fusion.

Figure 1.

Schematic drawing of the domain arrangement of synaptobrevin, highlighting the C-terminal end. The SNARE motif (black box) encompasses the region from amino acid (aa) 28 till aa 84 (layer + 8), followed by a short linker (aa 85-96) and a transmembrane region (aa 97-116). The alignment shows that the membrane-proximal tryptophan residues (white letters/black background) are highly conserved across species, irrespective of the proteins being involved in constitutive or regulated exocytosis. Residues that have been deleted in experiments are indicated by brackets. Layers 6, 7, and 8 are highlighted. For the alignment secretory R-SNAREs from the following species were included: Schizosaccharomyces pombe, ScPo; Saccharomyces cerevisiae, SaCe; Hirudo medicinalis, HiMe; Caenorhabditis elegans, CaEl; Strongylocentrotus purpuratus, StPu; Danio rerio, DaRe; Rattus norvegicus, RaNo; Homo sapiens, HoSa; Xenopus laevis, XeLa; Drosophila melanogaster, DrMe.

Figure 2.

Substitution of the membrane proximal tryptophans 89 and 90 with serine does not alter the efficiency of synaptobrevin to enter SNARE complexes or mediate fusion of liposomes. (A) Complex formation, monitored by the appearance of heat-resistant bands after SDS-PAGE and Coomassie Blue staining. Liposomes were reconstituted with either mutant or wild-type synaptobrevin (with approximately 200, 100, and 200 pmol of synaptobrevin, SyxH3 and SNAP-25, respectively). Note that the complex containing Synaptobrevin^{W89S W90S} migrates somewhat faster in SDS-PAGE, probably indicating a difference in the amount of bound SDS. (B) Complex formation, monitored by FRET. Synaptobrevin (both wild-type and W89S W90S) labeled at position 61 with Oregon Green was reconstituted in liposomes (∼100 nM final conc.) and incubated with SyxH3 labeled at position 225 with Texas Red (SyxH3^225TR; final conc. ∼300 nM). On addition of SNAP-25 (1.28 μM) donor fluorescence decreased, indicating complex formation. Addition of SNAP-25 in the absence of SyxH3 does not cause a measurable change in the FRET signal (not shown). Addition of soluble unlabeled synaptobrevin (2.5 μM) effectively competed with the labeled synaptobrevin for complex formation. For normalization, the minimum value of each trace was subtracted from its respective trace, and every data point of the trace was divided by its starting value. Similar results were obtained with the FRET pairs Syb^28OG/SyxH3^197TR, Syb^28OG/SNAP-25^130TR, and Syb^79OG/SNAP-25^84TR (data not shown). (C) Complex formation monitored by fluorescence anisotropy, using Syb^61OG (both wild-type and W89S W90S) containing liposomes as in B. Anisotropy increased when unlabeled soluble syntaxin and SNAP-25 were added. Again, excess soluble synaptobrevin (5 μM) blocks the increase in anisotropy. SNAP-25, alone, does not show any measurable increase in anisotropy (not shown). For normalization, the minimum value of each trace was subtracted from its respective trace. (D) SNARE-mediated lipid mixing monitored by the lipid-dequenching assay as previously described (Schuette et al., 2004). Liposomes reconstituted with both Syb^{W89S W90S} fused as effectively as Syb^wt with syntaxin 1 liposomes in the presence of SNAP-25. The fusion efficiency was not different when syntaxin 1/SNAP-25 binary complex was stabilized on the liposome (not shown). [NBD: N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl]. (E) SNARE mediates lipid mixing monitored by the lipid-dequenching assay when the SyxH3/SNAP-25 complex was stabilized by Syb49–96. Note that Syb^{W89S W90S} and Syb^wt reconstituted liposomes fused with equal efficiency.

Figure 3.

Effects of small deletions either within the SNARE motif or within the linker between the SNARE motif and the transmembrane region on liposome fusion rates. Fusion was monitored by lipid dequenching as described in Figure 2D. (A) Fusion was unaffected when the residues in the linker (indicated in Figure 1) between the SNARE motif and transmembrane region of synaptobrevin were deleted. (B) Deletion of C-terminal SNARE motif residues (indicated in Figure 1) of synaptobrevin retarded fusion. Traces are representative of at least three independent experiments.

Figure 4.

Calmodulin binds to soluble synaptobrevin in a calcium-dependent manner but does not influence SNARE-mediated lipid mixing. Calmodulin binding to synaptobrevin was monitored by fluorescence emission of membrane-proximal tryptophan residues of synaptobrevin. (A) SNARE-mediated lipid mixing assay monitored by the lipid dequenching assay, as in Figure 2D. Ca²⁺/Calmodulin (1 μM) did not influence the rate of SNARE-mediated lipid mixing. Calcium (100 μM) alone had no effect on fusion (not shown). (B) Ca²⁺/Calmodulin binds synaptobrevin (Syb1–96). Tryptophan fluorescence emission derived from Syb 1–96 (1 μM) remained unchanged when calmodulin (1 μM) was added. Fluorescence emission increased and was slightly blue-shifted when calcium (100 μM) was added, which was reversed upon addition of EGTA (1 mM).

Figure 5.

SNARE complex formation on synaptic vesicles, monitored by SDS-PAGE and immunoblotting for synaptobrevin. SyxH3 (40 pmol) and SNAP-25 (100 pmol) were incubated with 5.6 μg of purified synaptic vesicles for 4 h in 50 μl volume. The samples were analyzed for the presence of SDS-resistant SNARE complexes. (A) Complex formation on synaptic vesicles resulted in an almost complete shift of synaptobrevin to an SDS-resistant band of higher molecular weight (Ternary complex). Treatment of vesicles with the light chain of tetanus toxin (TeNT) before addition of SNAP-25 and SyxH3 cleaved nearly the entire pool of synaptobrevin (left lanes), documenting that it is completely accessible. (B) Efficiency of SNARE complex formation on synaptic vesicles does not change in the presence of increasing amounts of calcium. Synaptic vesicles were incubated with SyxH3 and SNAP-25 for 30 min in the presence of the indicated Ca²⁺ concentrations. Note that under these conditions, complex formation is not completed (see also Figure 6); thus the differences in calcium concentrations also did not alter the assembly rate.

Figure 6.

The rates of SNARE complex (TC) assembly on synaptic vesicles and on synaptobrevin liposomes are comparable. Syx^225OG and SNAP-25 were incubated with synaptobrevin liposomes or synaptic vesicles (see Figure 5 legend). SNARE complex formation, measured by the appearance of SDS-resistant bands, was monitored by immunoblotting for synaptobrevin (left panels) or by measuring fluorescence derived from syntaxin (right panels). To ensure that the reactions are completely arrested at the end of the incubation, SDS-containing sample buffer was added, and the samples were immediately shock-frozen and thawed only immediately before SDS-PAGE. For quantitation, the intensity of the bands was determined, corrected for background, and plotted against incubation time. LAU, luminescence arbitrary units.

Figure 7.

Synaptobrevin is displaced from synaptophysin upon formation of SNARE complexes. (A–C) An enriched vesicle fraction (LP2, 50 μg of protein) was incubated or not (as indicated) with 50 μg fluorescently labeled Syx^225TR, 200 μg unlabeled SyxH3 and 500 μg SNAP-25 for 2 h, followed by solubilization in Triton X-100 and immunoprecipitation for either synaptophysin (Syp, A) or synaptobrevin (B). All samples were analyzed by SDS-PAGE. LP2 fraction instead of purified synaptic vesicles was used because of the larger yield suitable for this experiment. (A) Immunoblotting for synaptobrevin shows that the amount of synaptobrevin coprecipitating with synaptophysin is reduced in the presence of SyxH3 and SNAP-25. (B) Conversely, immunoblotting for synaptophysin shows that the amount of synaptophysin coprecipitating with synaptobrevin is reduced in the presence of SNAREs. Note that in both cases the efficiency of antigen immunoprecipitation is comparable. (C) SNARE complexes (visualized by fluorescence of Syx^225TR) coprecipitated with synaptobrevin but not with synaptophysin. (D) Disappearance of the synaptophysin–synaptobrevin complex in the presence of unlabeled SyxH3 and SNAP-25, monitored by cross-linking with DSS, a bifunctional reagent. In the absence of the SNAREs, cross-linking results in the appearance of a band of ∼55 kDa (*) that is recognized by both synaptophysin- (D) and synaptobrevin-specific (not shown) antibodies and thus represents a heterodimer (Edelmann et al., 1995).