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. 2001 Mar 1;21(5):1421-33.
doi: 10.1523/JNEUROSCI.21-05-01421.2001.

synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo

J T Littleton  1 , J Bai, B Vyas, R Desai, A E Baltus, M B Garment, S D Carlson, B Ganetzky, E R Chapman
Affiliations

synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo

J T Littleton et al. J Neurosci. .

Abstract

Synaptotagmin has been proposed to function as a Ca(2+) sensor that regulates synaptic vesicle exocytosis, whereas the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is thought to form the core of a conserved membrane fusion machine. Little is known concerning the functional relationships between synaptotagmin and SNAREs. Here we report that synaptotagmin can facilitate SNARE complex formation in vitro and that synaptotagmin mutations disrupt SNARE complex formation in vivo. Synaptotagmin oligomers efficiently bind SNARE complexes, whereas Ca(2+) acting via synaptotagmin triggers cross-linking of SNARE complexes into dimers. Mutations in Drosophila that delete the C2B domain of synaptotagmin disrupt clathrin AP-2 binding and endocytosis. In contrast, a mutation that blocks Ca(2+)-triggered conformational changes in C2B and diminishes Ca(2+)-triggered synaptotagmin oligomerization results in a postdocking defect in neurotransmitter release and a decrease in SNARE assembly in vivo. These data suggest that Ca(2+)-driven oligomerization via the C2B domain of synaptotagmin may trigger synaptic vesicle fusion via the assembly and clustering of SNARE complexes.

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Figures

Fig. 1.
Fig. 1.
Interaction of synaptotagmin with assembled SNARE complexes. A,Left panel,The “midi” SNARE complex is SDS-resistant. Midi SNARE complex (1.5 μg) was dissociated into its component parts (residues 1–96 of synaptobrevin, 1–206 of SNAP-25, and 180–262 of syntaxin) by boiling in SDS sample buffer. Middle panels, Increasing concentrations of synaptotagmin were incubated with midi complexes (2 μm) in the presence of EGTA or Ca2+ in a 75 μl reaction volume. Synaptotagmin binding was assayed by coimmunoprecipitation using anti-synaptobrevin antibodies. Proteins were separated by SDS-PAGE and visualized with Coomassie blue. Forty percent of the bound material was loaded onto the gel.Right panel, Coimmunoprecipitated synaptotagmin was quantified by densitometry. The level of binding in EGTA (open circles) and Ca2+ (closed circles) was normalized to the maximum level of binding and plotted versus [synaptotagmin]. In the presence of Ca2+, the EC50 was 1.7 μm; at saturation the stoichiometry was 0.5 mol of synaptotagmin per mole of midi complex. B,Left panel, Synaptotagmin (3 μm) was mixed with midi–SNARE complex (2 μm) in 75 μl of HBS–0.5% Triton X-100 plus EGTA (2 mm) or the indicated concentration of Ca2+ for 2 hr at 4°C. SNARE complexes were immunoprecipitated with an anti-synaptobrevin antibody. Proteins were separated by SDS-PAGE and stained with Coomassie blue. Forty percent of the bound material was loaded onto the gel;total corresponds to 10% of the binding reaction.Right panel, Coimmunoprecipitated synaptotagmin was quantified by densitometry, normalized, and plotted versus the free Ca2+ concentration. The [Ca2+]1/2 was ∼100 μm.C, Synaptotagmin-midi–SNARE complex formation was monitored as described in B in the presence of the indicated divalent cations (1 mm Mg2+; 200 μm Ca2+, Ba2+, Sr2+). The synaptotagmin and midi–SNARE complex concentrations were 2 μm. Synaptotagmin binding was normalized (binding in 2 mm EGTA and 200 μmCa2+ were set at 0 and 100% binding, respectively), and the means from triplicate determinations are plotted. Error bars represent the SD from triplicate determinations.
Fig. 2.
Fig. 2.
Synaptotagmin facilitates SNARE complex assemblyin vitro. A, Recombinant his6-syntaxin, his6-SNAP-25B, and his6-synaptobrevin were incubated in the presence and absence of recombinant synaptotagmin in 2 mm EGTA (−Ca2+) or 1 mm Ca2+(+Ca2+) for 0, 5, or 15 min at room temperature. SDS-resistant 7S SNARE-complex formation was assayed by subjecting the samples to SDS-PAGE, without previous boiling (except where indicated), and immunoblotting with anti-syntaxin antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence. 7S denotes an SDS-resistant complex consisting of syntaxin, SNAP-25, and synaptobrevin.dimer denotes the trace formation of disulfide-bonded SNARE complex dimers that form under these conditions.B, The optical densities of 7S complexes from the +Ca2+ lanes in A are plotted versus time of incubation. C, Assembly experiments were performed as described in A for 2 hr but in the absence of DTT, except where indicated. Assembly reactions were conducted with (+) or without (−) synaptotagmin in either 2 mm EGTA, 1 mm Mg2+, 1 mmCa2+, or 1 mm Mg2+plus 1 mm Ca2+. As controls, samples were prepared that lacked either synaptobrevin, syntaxin, or SNAP-25. As a further control, samples were boiled before analysis. Samples were analyzed by immunoblotting with a mixture of anti-synaptobrevin and anti-synaptotagmin antibodies. Ca2+ and synaptotagmin enhanced the formation of SDS-resistant dimers. These dimers are disulfide-linked and are dissociated by DTT. SDS-resistant 7S complex formation only occurs in the presence of all three SNAREs, and complexes are dissociated by boiling.
Fig. 3.
Fig. 3.
Oligomerized synaptotagmin binds to assembled SNARE complexes. A, GST and GST–synaptotagmin were immobilized on beads (15 μg per data point) and assayed for binding to midi–SNARE complexes (2 μm) in 2 mm EGTA (−Ca2+) or 1 mm Ca2+(+Ca2+) in 150 μl of HBS using a cosedimentation assay, as described in Materials and Methods. To leave SNARE complexes intact, samples were subjected to SDS-PAGE without previous boiling. Coomassie staining revealed only low levels of SNARE binding to immobilized synaptotagmin in either condition. Immobilized synaptotagmin was then preincubated with soluble synaptotagmin (10 μm) in EGTA or Ca2+. Beads were washed three times to remove unbound soluble synaptotagmin, and the soluble- immobilized synaptotagmin oligomers were assayed for binding to midi–SNARE complexes. Twenty-five percent of the bound material was loaded onto the gel; the left two lanes correspond to 0.3 and 0.5 μg of soluble synaptotagmin and midi–SNARE complex, respectively. Coomassie staining revealed efficient binding of soluble synaptotagmin to immobilized synaptotagmin. Furthermore, midi–SNARE complexes efficiently bound to the soluble-immobilized synaptotagmin oligomers. These results demonstrate that synaptotagmin, which has oligomerized, is capable of binding SNARE complexes. *Denotes proteolytic fragments from GST–synaptotagmin. Note, Ca2+ induces a shift in the mobility of synaptotagmin that has not been boiled. Therefore, soluble and GST–synaptotagmin are indicated with double arrows.B, SNARE complexes do not inhibit synaptotagmin oligomerization. GST (12 μg per data point) and GST–synaptotagmin (8 μg per data point) were immobilized on beads. Soluble synaptotagmin (1.5 μm; +) and midi–SNARE complex (6 μm; +) or the indicated [SNARE complex] were incubated with the beads in 2 mm EGTA (−) or 1 mmCa2+ (+) for 1.5 hr. Samples were also prepared that lacked SNARE complexes (−) or soluble synaptotagmin (−). Bound material was boiled in SDS sample buffer and subjected to SDS-PAGE. Twenty-five percent of the bound material was loaded onto the gel; total corresponds to the mixture of 0.3 and 0.7 μg of soluble synaptotagmin and midi–SNARE complex. Gels were stained with Coomassie blue to visualize bound synaptotagmin. Staining of disassembled SNARE complexes was poor, therefore SNARE binding was detected by immunoblotting with anti-SNAP-25 and anti-syntaxin antibodies. Immunoreactive bands were visualized using enhanced chemiluminescence.
Fig. 4.
Fig. 4.
Mutations in the C2B domain ofDrosophila synaptotagmin I. A, Alignment of the C2B domain sequence surrounding the Y364N change found in theAD3 mutant (DiAntonio and Schwartz, 1994). The five putative Ca2+ ligands are highlighted ingray, whereas the AD3 change is indicated in black. Y364 is conserved among all synaptotagmin isoforms from C. elegans to humans. B,Predicted structure of the AD1 and AD3 mutant proteins based on the crystal structure of synaptotagmin III (see Fig. 9 for details). The location of the Y to N change in AD3 is indicated by thearrow. The AD1 mutations result in a premature stop codon deleting the C2B domain. C, The electrophysiological defects observed in AD3 andAD1 heteroallelic combinations (Littleton et al., 1994) are plotted against the responses of the control cn bw sp line. Recordings were made in 0.4 or 6.0 mmCa2+ in Jan's Ringer's solution. Excitatory junctional potential (EJP) amplitude at muscle fiber 6 in segments A3–A5 is plotted vs the extracellular Ca2+ concentration. At low Ca2+, both AD1 and AD3 exhibit a profound block in evoked secretion. At higher Ca2+ levels, the defects inAD3 mutants can be partially rescued, whereasAD1 mutants continue to have dramatically abnormal synaptic responses. These EJP responses have not been corrected for nonlinear summation. Thus, both synaptotagminmutants still have significant defects compared with control responses even in high calcium, where the control responses already saturated at these calcium levels. Dominant defects from the AD1 andAD3 alleles when paired with a wild-type allele of synaptotagmin have not been observed (Littleton et al., 1994).
Fig. 5.
Fig. 5.
Ultrastructural analysis of stimulated synapses in C2B mutants. Ultrastructural defects in control cn(A), AD3 cn/T11 cn(B), and AD1 cn/T41 cn(C) photoreceptor synapses were examined by driving photoreceptors with constant light stimulation for 10 min, followed by rapid fixation. Both AD1 andAD3 mutants lack the on–off transients measured during ERG recordings in the retina (shown on the right), demonstrating that synaptic transmission is disrupted at these photoreceptor synapses. AD1 mutants show a decrease in the overall number of synaptic vesicles, whereas AD3synapses do not show a depletion of synaptic vesicles, but rather a defect in the ability of docked synaptic vesicles to fuse. Quantification of vesicles per photoreceptor synapse for each of the genotypes was: AD1 cn/T41 cn, 25 ± 14 SD;AD1 cn/T7 cn, 27 ± 20 SD; AD3 cn/T11 cn, 88 ± 28 SD; cn controls, 96 ± 37 SD. Quantification of vesicles per T-bar for each of the genotypes was:AD1 cn/T41 cn, 1.4 ± 0.9 SD; AD1 cn/T7 cn, 1.9 ± 0.9 SD; AD3 cn/T11 cn, 2.6 ± 1.4 SD; cn controls, 2.3 ± 0.9 SD.
Fig. 6.
Fig. 6.
Synaptotagmin AD1 mutants fail to bind AP-2.A, Ca2+-dependent phospholipid binding of immobilized recombinant wild-type (WT), AD3, or AD1 synaptotagmin I proteins. Both AD1 and AD3 recombinant proteins showed robust Ca2+-stimulated phospholipid binding. Phospholipid binding assays were conducted as previously described (Littleton et al., 1999). B, Binding of recombinant syntaxin (5 μm) and native AP-2 α-adaptin (0.2 mg ofDrosophila head membranes) to 30 μg of recombinant WT, AD3, or AD1 Drosophila synaptotagmins in 2 mm EGTA or 1 mm Ca2+ for 2 hr at 4°C. For detection of recombinant syntaxin binding to synaptotagmins, Western analysis with the monoclonal anti-syntaxin antisera 8C3 was performed. For analysis of AP-2 binding, fly head membranes were prepared as previously described (Littleton et al., 1998), and AP-2 binding was detected with a polyclonal antibody generated against α-adaptin (Gonzalez-Gaitan and Jackle, 1996). Immunoreactive bands were visualized by enhanced chemiluminescence. Both AD1 and AD3 mutant proteins showed Ca2+-dependent binding to syntaxin. However, only AD3 showed an interaction with AP-2.
Fig. 7.
Fig. 7.
Synaptotagmin AD3 mutants decrease SNARE complex assembly in vivo.A, Enlarged image of an active zone inAD3/T11 mutant photoreceptor terminals demonstrating docked vesicles (arrowheads) under a T-bar that have not fused. B, 7S complexes from 10 control (CS) or synaptotagmin AD3/T11 mutants were isolated. Syntaxin is present in a 35 kDa monomeric form and in a 73 kDa complex with SNAP-25 and synaptobrevin in wild-type flies. A severe reduction in the amount of 7S complex was found inAD3/T11 synaptotagmin mutants. C, Both wild-type and AD3 recombinant synaptotagmins are able to bind SNARE complexes in a Ca2+-stimulated manner. Either 3 μm wild-type (sytWT) or AD3 mutant synaptotagmin (sytAD3) was incubated with 3 μm midi–SNARE complex for 1.5 hr in either 2 mm EGTA (E) or 1 mmCa2+. Midi–SNARE complex was immunoprecipitated, and samples were separated by SDS-PAGE and stained with Coomassie blue. As a control, samples were prepared that lacked midi–SNARE complex and immunoprecipitating antibodies. Thirty percent of the immunoprecipitated material was loaded onto the gel; total corresponds to 6% of the binding reaction. Note: the asteriskindicates a proteolytic fragment present in preparations of soluble AD3 mutant rat synaptotagmin. D, Both wild-type and AD3 synaptotagmins are able to bind the mammalian synprint peptide. Ten micrograms of GST or GST fused to the cytoplasmic domain of WT or AD3 mutant synaptotagmin were immobilized on beads and incubated with 1 μm T7-tagged synprint for 2 hr in 2 mm EGTA (E) or 1 mm Ca2+. Samples were washed, and bound material was subjected to SDS-PAGE and immunoblot analysis using an anti-T7 tag antibody and enhanced chemiluminescence. Twelve percent of the bound material was loaded onto the gel; total corresponds to 3.5% of the binding reaction.
Fig. 8.
Fig. 8.
The AD3 mutation blocks Ca2+-driven conformational changes within the C2B domain of synaptotagmin and disrupts Ca2+-triggered oligomerization activity.A, Crystal structure of the C2B domain of synaptotagmin III. This image was modified from Sutton et al. (1999); the structure of the C2B domain of synaptotagmin I has not been reported, however, all known C2B domains share similar structures. The tyrosine that is mutated to an asparagine in the AD3 mutant allele of Drosophila synaptotagmin is indicated, as are five putative Ca2+ ligands and a single bound Mg2+ ion. B, The C2B domain of WT (GST-C2BWT) and AD3 (GST-C2BAD3) rat synaptotagmin Ib were immobilized as a GST fusion proteins (20 μg/data point) and subjected to limited proteolysis in the presence of 2 mm EGTA (−) or 1 mmCa2+ (+) at the indicated [chymotrypsin] for 60 min at rt. Samples were boiled in SDS sample buffer, analyzed by SDS PAGE, and stained with Coomassie blue. C,Ca2+-triggered synaptotagmin oligomerization is impaired by the AD3 mutation. Eight micrograms of GST or GST fused to the cytoplasmic domain of wild-type (sytWT) or AD3 mutant (sytAD3) Drosophilasynaptotagmin was immobilized on beads. Beads were incubated with 1.5 μm soluble WT or AD3 mutant Drosophilasynaptotagmin for 1.5 hr in 150 μl of TBS plus 0.5% Triton X-100 and either 2 mm EGTA (−), 1 mmCa2+ (+), or the indicated concentration of Ca2+. Beads were washed three times with binding buffer and boiled in SDS sample buffer. Three percent of the soluble synaptotagmin from the binding assay (left two lanes) and 25% of the bound material (remaining lanes) were subjected to SDS-PAGE and visualized by staining with Coomassie blue.Syt, Cytoplasmic domain of synaptotagmin I. Note: theasterisk indicates a proteolytic fragment present in preparations of GST-fused AD3 mutant synaptotagmin. D,Data from two oligomerization assays (as described in C) were quantified by densitometry, normalized to the pixel intensity in the “total” lanes, and plotted versus the free [Ca2+]. Closed circles, Wild-type synaptotagmin; open circles, AD3 synaptotagmin.E, Soluble Drosophila synaptotagmin I (5 μm) was incubated with 30 μg of either wild-type synaptotagmin IV, AD3 synaptotagmin IV, or synaptotagmin IV containing a KK to AA substitution (Chapman et al., 1998) at amino acids 385 and 386. Binding of synaptotagmin I was visualized by Western analysis with anti-synaptotagmin I DSYT2 antisera (Littleton et al., 1993). ● denotes binding reactions lacking soluble synaptotagmin.
Fig. 9.
Fig. 9.
Model depicting the synaptotagmin-SNARE complex. A, The core of the SNARE complex, the Habc domain of syntaxin, the cytoplasmic domain of synaptotagmin, and a simulated lipid bilayer were modified from Sutton et al. (1998),Fernandez et al. (1998), Sutton et al. (1999), and Heller et al. (1993), respectively, and rendered using MOLSCRIPT (Kraulis, 1991). The regions that interact are indicated with brackets; both C2 domains of synaptotagmin are required for high affinity binding to the base of the SNARE complex (Chapman et al., 1995; Davis et al., 1999; Gerona et al., 2000). The transmembrane anchors of syntaxin, synaptobrevin, and synaptotagmin were generated by molecular modeling.B, Model for synaptotagmin-mediated assembly and clustering of SNARE complexes. One synaptotagmin can interact with two SNARE complexes; this interaction is depicted as two grooves within synaptotagmin that bind and assemble SNAREs (which are shown in an “end view” in which each strand of the four-helix bundle is depicted as a quarter of a circle).
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