Synaptotagmin C2B domain regulates Ca2+-triggered fusion in vitro: critical residues revealed by scanning alanine mutagenesis

Abstract

Synaptotagmin (syt) 1 is localized to synaptic vesicles, binds Ca2+, and regulates neuronal exocytosis. Syt 1 harbors two Ca2+-binding motifs referred to as C2A and C2B. In this study we examine the function of the isolated C2 domains of Syt 1 using a reconstituted, SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor)-mediated, fusion assay. We report that inclusion of phosphatidylethanolamine into reconstituted SNARE vesicles enabled isolated C2B, but not C2A, to regulate Ca2+-triggered fusion. The isolated C2B domain had a 6-fold lower EC50 for Ca2+-activated fusion than the intact cytosolic domain of Syt 1 (C2AB). Phosphatidylethanolamine increased both the rate and efficiency of C2AB- and C2B-regulated fusion without affecting their abilities to bind membrane-embedded syntaxin-SNAP-25 (t-SNARE) complexes. At equimolar concentrations, the isolated C2A domain was an effective inhibitor of C2B-, but not C2AB-regulated fusion; hence, C2A has markedly different effects in the fusion assay depending on whether it is tethered to C2B. Finally, scanning alanine mutagenesis of C2AB revealed four distinct groups of mutations within the C2B domain that play roles in the regulation of SNARE-mediated fusion. Surprisingly, substitution of Arg-398 with alanine, which lies on the opposite end of C2B from the Ca2+/membrane-binding loops, decreases C2AB t-SNARE binding and Ca2+-triggered fusion in vitro without affecting Ca2+-triggered interactions with phosphatidylserine or vesicle aggregation. In addition, some mutations uncouple the clamping and stimulatory functions of syt 1, suggesting that these two activities are mediated by distinct structural determinants in C2B.

FIGURE 1.

The isolated C2B domain of syt 1 is sufficient to regulate Ca²⁺-triggered membrane fusion. A, shown is a schematic diagram depicting the components of the in vitro fusion assay. B, fusion assays were carried out using donor v-SNARE vesicles, t-SNARE acceptor vesicles, and 10 μm of the C2AB, C2A, or C2B domain of syt 1. Components were incubated together for 20 min at 37 °C in the presence of 0.2 mm EGTA, followed by the addition of Ca²⁺ (arrow) to give a final free concentration of 1 mm. Fluorescence intensity was measured every minute for 60 min and normalized as described under “Experimental Procedures.” Reconstituted v- and t-SNARE vesicles were composed of either: 100% PC, 15%PS/85%PC, 30%PE/70%PC, or 15%PS/30%PE/55%PC. Shown are representative traces from three independent experiments. C, binding of each domain to t-SNARE vesicles was monitored using a co-flotation assay; t-SNARE vesicles used in the fusion assays were incubated with 10 μm C2AB, 30 μm C2A, or 30 μm C2B in the presence or absence of 1 mm Ca²⁺. Bound material co-floated through a density gradient and was analyzed by SDS-PAGE and Coomassie Blue staining. Shown is a representative gel of three independent experiments. Note: the line between the PS/PC and PS/PC/PE samples indicates the data were obtained from two different gels.

FIGURE 2.

Dose response of Ca²⁺-triggered fusion regulated by the C2AB, C2A, or C2B domains of syt 1. A, shown are representative traces (n ≥ 2 experiments) of Ca²⁺-triggered fusion between t- and v-SNARE vesicles as a function of C2AB, C2A, or C2B concentration. B, the normalized fluorescence intensity at 60 min was plotted as a function of the syt 1 domain concentration. The EC₅₀ for Ca²⁺-triggered fusion was 5.22 ± 1.1μm and 30.6 ± 1.0μm for C2AB and C2B, respectively. Similarly, the ability of each syt 1 domain to inhibit fusion in the presence of EGTA was plotting, and the IC₅₀ was determined to be 4.34 ± 1.5 μm for C2AB, 9.8 ± 1.3 μm for C2B, and >100 μm for C2A.

FIGURE 3.

Characterization of PE in the in vitro fusion assay. A, PS/PC/PE t- and v-SNARE vesicles were prepared as described, with either 0, 10, 20, 30, 40, or 50% PE, incubated with either 10 μm C2AB or 50 μm C2B, and analyzed in the fusion assay. B, the ability of each domain to bind t-SNAREs was monitored by flotation assays. PS-free vesicles containing 0, 10, 20, 30, 40, 50% PE were incubated with 10 μm C2AB or 50 μm C2B and floated through a density gradient in the presence or absence of 1 mm Ca²⁺. Vesicles were collected from the top of the gradient, separated by SDS-PAGE, and stained with Coomassie Blue. Shown are representative fusion traces, and a Coomassie-stained gel, from three separate experiments. C, shown are representative fusion traces (n = 2) in which 10 μm C2AB (left) or 50 μm C2B (right) were incubated with t- and v-SNARE vesicles with (shaded) and without 30% PE (clear).

FIGURE 4.

The isolated C2A domain of syt 1 inhibits C2B-regulated fusion. A, representative traces of two separate experiments demonstrating the ability of C2A or C2B to inhibit C2AB regulated vesicle fusion. Ca²⁺-triggered fusion assays were performed with 10 μm C2AB and increasing concentrations of either C2A (left) or C2B (center), and the %F_max was plotted as a function of the isolated C2 domain concentration (right). These data indicate that high molar ratios of isolated C2A, but not isolated C2B, inhibit C2AB-regulated fusion. B, representative traces demonstrating the ability of C2A to inhibit C2AB or C2B-regulated fusion. The concentration of C2AB (left) and C2B (center) was maintained at 50 μm to observe robust C2B-regulated fusion. The efficacy of C2A-mediated inhibition was determined by plotting the %F_max for each domain as a function of C2A concentration (right).

FIGURE 5.

Scanning alanine mutagenesis reveals distinct regions of syt 1 that are important for Ca²⁺-triggered fusion. A, the %F_max at 60 min was determined and normalized to the wt activity and plotted for each mutant. The histogram shows the extent of Ca²⁺-stimulated (right) and Ca²⁺-independent (left) fusion for each mutant relative to wt C2AB (gray). The mean ± S.E. for each mutant was generated from ≥3 independent experiments. Significance was determined by one-way analysis of variance (* = p < 0.05, ** = p < 0.01, and *** = p < 0.001). B, to determine if there was a correlation between a mutant's ability to activate Ca²⁺-triggered fusion or their ability to clamp fusion in EGTA, these values were plotted against each other for each mutant. Select mutants are labeled to highlight their location on the plot.

FIGURE 6.

The R398A mutation inhibits the t-SNARE-binding activity of syt 1 in both the absence and presence of Ca²⁺. A, a representative t-SNARE binding experiment of selected mutants (n = 4) is shown. PS-free t-SNARE vesicles were incubated with 10 μm of each mutant in the presence or absence of Ca²⁺, floated through a density gradient, and analyzed by SDS-PAGE. The optical density of the syt and syntaxin bands was measured, and the ratio was used to determine the extent of binding (right). B, the -fold increase in syt 1 C2AB binding to t-SNAREs in response to Ca²⁺ was calculated for each mutant (left panel). These data are plotted against the relative extent of fusion (normalized to the amount of fusion observed using wt protein (right panel)).

FIGURE 7.

Ca²⁺-dependent PS-binding activity of syt 1 C2AB mutants. A, the PS-binding activity of wt and several mutant forms of syt 1 C2AB was measured using a glutathione S-transferase pulldown assay and plotted as a function of Ca²⁺ concentration. Mutants were grouped based on their relative changes in [Ca²⁺]_½. B, table summarizing the Hill slope and [Ca²⁺]_½ for each mutant tested.

FIGURE 8.

A model of the syt 1·SNARE·membrane complex showing the surfaces of C2B proposed to interact with t-SNAREs and membranes. A, shown is the NMR structure of the syt 1 C2B domain (PDB 1K5W) with Ca²⁺ ions modeled as white spheres. The substituted residues in the mutants that displayed decreased Ca²⁺-triggered fusion activity are highlighted as stick structures. B, close up of residues Thr-328 (cream) and Glu-341 (tan). C, close up of residues Lys-326 and Lys-327 (blue). D, close up of residues Asp-392 (red), Arg-398, Arg-399, and Arg-281 (aqua). E, a “lateral” view of the C2B domain modeled with space-filled side chains. F, an “axial” view of the C2B domain from the Ca²⁺-binding loops modeled with space-filled side chains to emphasize the alignment of the mutations on one face of C2B. G, a proposed model for C2AB interactions with the SNARE complex (red) during membrane fusion. In the absence of Ca²⁺, C2AB initiates contact with t-SNAREs via the C2B domain through the polybasic region, and Arg-398. Influx of Ca²⁺ tightens the interaction between C2AB and the SNARE complex while the Ca²⁺-binding loops of C2B penetrate into the fusion stalk and the Ca²⁺-binding loops of C2A penetrate the plasma membrane. All structural images were created using ICM BrowserPro (Molsoft, La Jolla, CA) and Adobe Illustrator CS. H, a functional model of syt during the initial stages of SNARE assembly and following the Ca²⁺ trigger.