Distinct Ca2+-dependent Properties of the First and Second C2-domains of Synaptotagmin I

Synaptotagmin I (SytI) is a synaptic vesicle protein that binds Ca2+ and is essential for fast, Ca2+-dependent neurotransmitter release in the hippocampus, suggesting that it serves as a Ca2+ sensor for exocytosis. Although SytI has two cytoplasmic C2-domains, only the first C2-domain was shown to exhibit Ca2+ regulation; it binds phospholipids and syntaxin in a Ca2+-dependent manner. By contrast, the second C2-domain is inactive in these assays and only binds putative interacting molecules in a Ca2+-independent manner. We have now discovered in a yeast two-hybrid screen for SytI-interacting molecules that the C2-domains of SytI interact with themselves. Using immobilized recombinant C2-domains from SytI and SytII, we found that only the second but not the first C2-domains of these synaptotagmins are capable of affinity-purifying native rat brain SytI and that this binding is Ca2+-dependent, suggesting that only the second C2-domain is capable of a Ca2+-triggered self-association. A relatively high Ca2+ concentration (>100 μM) is required for binding in the presence of Mg2+; Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+. Our data suggest that the second C2-domain of SytI is also a Ca2+-regulated domain similar to the first C2-domain but with distinct binding activities. Synaptotagmin I (SytI) is a synaptic vesicle protein that binds Ca2+ and is essential for fast, Ca2+-dependent neurotransmitter release in the hippocampus, suggesting that it serves as a Ca2+ sensor for exocytosis. Although SytI has two cytoplasmic C2-domains, only the first C2-domain was shown to exhibit Ca2+ regulation; it binds phospholipids and syntaxin in a Ca2+-dependent manner. By contrast, the second C2-domain is inactive in these assays and only binds putative interacting molecules in a Ca2+-independent manner. We have now discovered in a yeast two-hybrid screen for SytI-interacting molecules that the C2-domains of SytI interact with themselves. Using immobilized recombinant C2-domains from SytI and SytII, we found that only the second but not the first C2-domains of these synaptotagmins are capable of affinity-purifying native rat brain SytI and that this binding is Ca2+-dependent, suggesting that only the second C2-domain is capable of a Ca2+-triggered self-association. A relatively high Ca2+ concentration (>100 μM) is required for binding in the presence of Mg2+; Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+. Our data suggest that the second C2-domain of SytI is also a Ca2+-regulated domain similar to the first C2-domain but with distinct binding activities. INTRODUCTIONSynaptotagmin I (SytI) ( 1The abbreviations used are: SytI and SytIIsynaptotagmins I and IIY2Hyeast two-hybridGSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresis.) is a member of a family of neuronal proteins that is characterized by a short N-terminal intravesicular sequence, a single transmembrane region, and a large cytoplasmic sequence containing two C2-domains (reviewed in Südhof(1995)). Nine synaptotagmin isoforms have been described in mammals (Perin et al., 1990; Geppert et al., 1991;Wendland et al., 1991; Mizuta et al., 1994; Hilbush and Morgan, 1994; Li et al., 1995a; Craxton and Goedert, 1995; Hudson and Birnbaum, 1995), four of which are also found in non-neural tissues (Li et al., 1995a; Hudson and Birnbaum, 1995). At least three synaptotagmins (SytI, SytII, and SytIII) are synaptic vesicle proteins, of which SytIII is present throughout the brain whereas SytI and SytII show restricted complementary expression patterns with partially overlapping distributions (Ullrich et al., 1994). Mice in which the SytI gene has been mutated exhibit a lethal phenotype in which there is a selective loss of fast Ca2+-dependent neurotransmitter release in hippocampal synapses (Geppert et al., 1994). Spontaneous neurotransmitter release and neurotransmitter release evoked by Ca2+-independent mechanisms are normal, suggesting an essential role for SytI only in the Ca2+-dependent last step of membrane fusion. Together with the Ca2+ binding properties of SytI (Li et al., 1995a, 1995b), these data suggest that SytI may serve as an exocytotic Ca2+ sensor.Based on the presence of C2-domains in SytI and the observation that the C2-domain confers Ca2+ regulation onto protein kinase C, it was speculated early on that SytI may be a Ca2+-binding protein (Perin et al., 1990). Indeed, experiments with purified SytI demonstrated that it binds Ca2+ and phospholipids (Brose et al., 1992) and that it undergoes a conformational change as a function of Ca2+ (Davletov and Südhof, 1994). Studies on recombinant C2-domains showed that the first C2-domain of SytI and of most but not all other synaptotagmins binds phospholipids as a function of Ca2+ (Davletov and Südhof, 1993; Chapman and Jahn, 1994; Ullrich et al., 1994; Li et al., 1995a). In addition, Ca2+ triggers binding of syntaxin to the first C2-domain of synaptotagmins with a Ca2+ dependence that is distinct from that of phospholipid binding, implying the presence of two Ca2+-binding sites in a single C2-domain (Li et al., 1995a, 1995b). Surprisingly, the second C2-domain of all synaptotagmins is inactive in these assays despite a high degree of sequence homology. Furthermore, the Ca2+-dependent binding properties of the native cytoplasmic domain of purified brain SytI containing both C2-domains has the same properties as the recombinant single first C2-domain (Li et al., 1995a, 1995b). Together these data suggest that the known Ca2+ binding properties of SytI can be entirely accounted for by its first C2-domain alone and raised the possibility that the second C2-domain may not represent a Ca2+-binding domain. This possibility was supported by the Ca2+-independent interactions of the second C2-domains of synaptotagmins with clathrin AP2 (Zhang et al., 1994; Li et al., 1995a) and with polyanions such as polyinositol phosphates (Fukuda et al., 1994).We now report the results of a yeast two-hybrid interaction screen for proteins binding to the C2-domains of SytI. Unexpectedly, SytI itself was identified as an interacting partner. In vitro binding assays demonstrated a Ca2+-dependent self-interaction of SytI that is specific for its second C2-domain. These data suggest that in addition to the first C2-domain, the second C2-domain of SytI is a Ca2+-regulated domain. However, the two C2-domains have distinct Ca2+-regulated properties, suggesting a functional diversification of C2-domains in synaptotagmins.EXPERIMENTAL PROCEDURESYeast Two-hybrid (Y2H) Screens and Interaction AssaysA bait vector (pBTM116p65-8) encoding the cytoplasmic domains of SytI starting with the first C2-domain was constructed by cloning the 0.9-kilobase pair SmaI-SalI fragment from pGEX65-8 (Davletov et al., 1993) into the same sites of pBTM116 (Vojtek et al., 1993). This results in a vector expressing a LexA-fusion protein with SytI starting at residue 120. A cDNA library was constructed in the NotI site of Y2H prey vector pVP16 (Vojtek et al., 1993) from poly(A)+-enriched rat brain RNA using the Life Technologies, Inc. Choice system. Y2H screens were performed essentially as described (Fields and Song, 1989; Vojtek et al., 1993; Hata and Südhof, 1995) by sequentially transfecting yeast strain L40 with the bait vector and the cDNA library using the lithium acetate method (Schiestl and Gietz, 1989). Transformants were plated on selection plates lacking histidine, uracil, tryptophan, lysine, and leucine but containing 2.5 mM 3-amino triazol. Positive clones were picked after 4-6 days of incubation at 30°C, and the β-galactosidase activity of the clones was assayed on a nitrocellulose filter. Extrachromosomal DNA from clones that grew in the absence of histidine and were β-galactosidase positive was isolated using the glass bead method (Ward, 1990). Prey plasmids were rescued in Escherichia coli HB101 cells by electroporation and selection on M9 plates containing 50 mg/liter proline and 0.1 g/liter ampicillin. 14 million yeast transformants with the cDNA library were screened, and 42 clones positive upon retransformation were isolated and sequenced using the dideoxy chain termination method.Construction of Expression VectorsThe recombinant GST-synaptotagmin fusion proteins used were synthesized from the following expression plasmids in the vector pGEX-KG (Guan and Dixon, 1991) encoding the following residues of SytI and SytII (Perin et al., 1990; Geppert et al., 1991): pGEX65-4 (GSTSytIC2-A), residues 140-267; pGEX65-9 (GSTSytIC2-B), residues 266-421; pGEX65-8 (GSTSytIC2-A/B), residues 120-421; pGEX1071/1081 (GSTSytIIC2-A), residues 141-268; pGEX1153/1159 and pGEX1153/1159Y312N (GSTSytIIC2-B wild type and mutant, respectively), residues 266-399; pGEX1071/1152 and pGEX1071/1152Y312N (GSTIISytC2-A/B wild type and mutant, respectively), residues 141-422. Recombinant proteins were purified on glutathione-agarose and used immobilized on glutathione-agarose without elution. Amounts of proteins used were standardized based on Coomassie Blue-stained SDS gels.SytI Binding to Recombinant ProteinsOne frozen rat brain (Pelfreeze) was homogenized in 11 ml of 4 mM HEPES-NaOH pH 7.4 containing 0.1 g/liter phenylmethylsulfonyl fluoride. The homogenate was extracted for 4 h at 4°C after addition of 11 ml of 4 mM HEPES-NaOH pH 7.4, 0.1 g/liter phenylmethylsulfonyl fluoride, 0.2 M NaCl, 2% Nonidet P-40, 2 mM EDTA. Insoluble material was removed from the extract by centrifugation (30 min at 100,000 × g), and MgCl2 was added to the supernatant to 3.5 mM final concentration. 1-ml aliquots of the supernatant were incubated overnight at 4°C with glutathione-agarose beads (GSH beads) containing 5-10 μg of immobilized GST-fusion proteins with either 3.5 mM CaCl2 or 5 mM EGTA. The GSH beads were centrifuged, washed 5 times in the respective incubation buffers, and resuspended in 120 μl of SDS-PAGE sample buffer, and 40 μl were analyzed by SDS-PAGE and immunoblotting using antibodies against synaptotagmin I (Cl604.4; kind gift of Dr. R. Jahn, Yale University), syntaxin I (HPC-1), or AP2 (M11AC1; kind gift of M. Robinson, Cambridge). For the experiments testing the effects of various cations, identical procedures were used except that the extraction was performed in the absence of EDTA, no MgCl2 was added to the extract, and the incubations with the GSH beads and washing steps were carried out with buffers containing 1 mM EGTA, CaCl2, MgCl2, BaCl2, or SrCl2. The Ca2+ titration experiments were performed similarly except that the different indicated Ca2+ concentrations were used in the incubation buffers.Miscellaneous ProceduresSDS-PAGE and immunoblotting were performed using standard procedures and antibodies described previously (Laemmli, 1970; Johnston et al., 1989; Li et al., 1995a). Protein assays were performed with the Bio-Rad kit.RESULTSYeast Two-hybrid Screens for Synaptotagmin I Interacting ProteinsWe screened 14 million yeast colonies transformed with a rat brain cDNA library with a bait construct encoding the cytoplasmic C2-domains of SytI. 42 clones were isolated that demonstrated β-galactosidase activation after retransformation of the prey plasmids into yeast. Sequencing revealed that most of these clones encoded either novel proteins or proteins unlikely to interact with SytI physiologically (such as mucin apoprotein; data not shown). One positive clone (pPrey820), however, encoded SytI itself, starting at residue 120 immediately N-terminal to the first C2-domain and containing its complete C terminus (data not shown). This clone resulted in high β-galactosidase levels upon co-transformation with the SytI bait construct, confirming an interaction in the Y2H assay.Ca2+-dependent Binding of the Second C2-domain of SytI to Endogenous Brain SytIThe Y2H result suggested that the C2-domains of SytI can interact with themselves. To obtain independent evidence for such an interaction and to localize the interacting sequences, we purified GST-fusion proteins encoding either the first or the second or both C2-domains of SytI and SytII. Recombinant proteins were immobilized on glutathione-agarose beads, and binding of endogenous brain SytI to the immobilized fusion proteins was measured as a function of Ca2+. Fusion proteins containing both C2-domains or containing only the second C2-domain of either SytI or SytII efficiently affinity-purified SytI from total brain (Fig. 1). Strong binding was observed only in the presence of Ca2+, whereas in the absence of Ca2+ weak binding was present. By contrast, the first C2-domains of SytI and of SytII were unable to bind. A point mutation in the second C2-domain of SytII that corresponds to a mutation in Drosophila synaptotagmin, which impairs synaptotagmin function (DiAntonio and Schwarz, 1994), had no effect on Ca2+-dependent binding. Together these results suggest that the C2-domains of synaptotagmins self-associate as a function of Ca2+ via their second but not their first C2-domains. Since the low binding observed in the absence of Ca2+ appears to be sufficient for an interaction observed in the Y2H assay, it is possible to identify Ca2+-dependent binding proteins for SytI using the Ca2+-independent Y2H screen.Divalent Cation Specificity of Synaptotagmin Self-interactionPrevious studies on the first C2-domains of synaptotagmins demonstrated that Sr2+ and Ba2+ but not Mg2+ can substitute for Ca2+ in activating phospholipid binding, although with a much lower affinity (Davletov and Südhof, 1993; Li et al., 1995b). We therefore tested the effects of different divalent cations on the binding of SytI to the second C2-domain of SytI (Fig. 2). Mg2+ was unable to trigger binding and in fact inhibited binding compared with that observed in the absence of divalent cations (Fig. 2). By contrast, both Sr2+ and Ba2+ were capable of activating binding, with Ba2+ having the lowest effect. Parallel incubations with GST alone demonstrated that the binding observed was dependent on the SytI-fusion protein and not due to divalent cation-dependent aggregation of SytI in the homogenate (lower panel in Fig. 2).Figure 2:Cation specificity of the binding of SytI to the recombinant second C2-domain of SytI. A recombinant GST-fusion protein with the second C2-domain of SytI (top panel labeled GSTSytIC2-B) or recombinant GST alone (bottom panel labeled GST) was immobilized on glutathione beads and incubated with solubilized brain homogenates containing either 1 mM EGTA, no additions, or 1 mM of the indicated divalent cations. Beads were washed in the incubation buffers, and bound proteins were analyzed by SDS-PAGE and immunoblotting. The asterisk identifies the position of full-length SytI; the 40-kDa band observed in lanes 3 and 6 containing high levels of bound SytI represents the major proteolytic product of SytI (Perin et al., 1991). Note that addition of Mg2+ slightly suppresses binding. Numbers on the left indicate positions of molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Ca2+ Dependence of Second C2-domain BindingTo test the Ca2+ concentration dependence of the binding of brain SytI to the second C2-domains of SytI or SytII, we incubated brain homogenates with immobilized GST-C2-domain fusion proteins at different Ca2+ concentrations and analyzed binding of SytI, syntaxin I, and clathrin AP2 to the immobilized proteins as a function of Ca2+ (Fig. 3). As shown earlier (Zhang et al., 1994; Li et al., 1995a), binding of AP2 and syntaxin I to the second C2-domain was Ca2+-independent. By contrast, SytI binding was enhanced by Ca2+ with an identical concentration dependence for the two C2-domains tested, with half-maximal binding at approximately 250 μM free Ca2+. This Ca2+ concentration dependence resembles that of syntaxin I binding to the first C2-domain of SytI and SytII (Li et al., 1995a). Note that these experiments were carried out in the presence of 3.5 mM Mg2+ and the apparent Ca2+ affinity may be higher in the absence of Mg2+.Figure 3:Ca2+ dependence of SytI binding to the second C2-domains of SytI and SytII. Recombinant GST-fusion proteins of the second C2-domains of SytI and SytII (GSTSytIC2-B and GSTSytIIC2-B, respectively) were immobilized and incubated with brain homogenates in Mg2+-containing buffers with the Ca2+ concentrations indicated on top of the panels (E = EGTA). Beads were washed extensively in the incubation buffers. Bound proteins were analyzed by immunoblotting for the clathrin assembly protein AP2, SytI, and syntaxin I. Numbers on the left indicate positions of molecular weight markers.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DISCUSSIONSytI is a Ca2+-binding protein of synaptic vesicles that is essential for fast Ca2+-dependent neurotransmitter release from hippocampal neurons (Brose et al., 1992; Geppert et al., 1994), suggesting that it serves as the exocytotic Ca2+ sensor. Previous studies demonstrated that the first C2-domain of SytI serves as a Ca2+-dependent phospholipid- and syntaxin-binding domain (Davletov and Südhof, 1993, 1994; Li et al., 1995a, 1995b). The second C2-domain is inactive in these assays but binds AP2 and polyanions in a Ca2+-independent manner (Zhang et al., 1994; Fukuda et al., 1994). The phospholipid and syntaxin binding properties of a cytoplasmic fragment from SytI containing both C2-domains are identical to that of the single recombinant first C2-domain (Li et al., 1995a, 1995b). Together these results suggested that SytI may perform its Ca2+ sensor function primarily via its first C2-domain whereas the second C2-domain may have a distinct function. We now demonstrate that the second C2-domain also has a Ca2+-dependent activity suggestive of a Ca2+-binding domain. It mediates the Ca2+-dependent aggregation of SytI and SytII with a Ca2+ concentration dependence that mirrors the Ca2+ dependence of neurotransmitter release if the experiments are performed in the presence of physiological concentrations of Mg2+. These data suggest a model of SytI whereby both C2-domains of SytI can serve as Ca2+ binding modules with distinct functions (Fig. 4).Figure 4:Domain model of SytI and its binding activities. SytI binds phospholipids and syntaxin in a Ca2+-dependent manner via its first C2-domain and self-associates in a Ca2+-dependent manner via its second C2-domain. In addition, the N-terminal domains of synaptotagmin I self-associate in a Ca2+-independent manner via an undetermined sequence, and the C-terminal domains also bind AP2, polyanions such as polyinositol phosphates (IP4-6), and neurexins in a Ca2+-independent manner (see text for references).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Even in the absence of Ca2+, SytI is not a monomer but a multimer (Perin et al., 1991). The basic unit of this multimer is an SDS-resistant dimer that can be detected by SDS-PAGE, and this multimerization is mediated by sequences N-terminal to the two C2-domains (Brose et al., 1992) (Fig. 4). Our current demonstration of a Ca2+-dependent binding of SytI to itself via its second C2-domain raises the possibility that during nerve terminal depolarization and Ca2+ influx, SytI multimers may be cross-linked by Ca2+ into large superstructures. The function of such a superstructure in fusion is unknown, but it is conceivable that it would aid in forming a pore that during membrane fusion and assembly of a of interactions has been described for not all of which may be physiologically the point of of SytI, it that Ca2+-regulated are binding activities. that the physiological of an interaction is the of the binding on these two the interactions of SytI with and itself to be the most to be The recombinant second C2-domain of SytI also binds syntaxin (see Fig. and phospholipids and in a Ca2+-independent manner. However, the complete C2-domain fragment from native SytI by proteolytic is phospholipid binding and syntaxin binding are dependent on Ca2+, and Ca2+-independent binding is observed (Li et al., 1995a, 1995b). other binding were demonstrated for synaptotagmin I that are Ca2+-independent and binding of neurexins and Although no in data to the physiological of binding, AP2 binding may be physiologically AP2 with synaptic vesicles and et al., 1992) and synaptic vesicle is in synaptotagmin in et al., 1995). INTRODUCTIONSynaptotagmin I (SytI) ( 1The abbreviations used are: SytI and SytIIsynaptotagmins I and IIY2Hyeast two-hybridGSTglutathione S-transferasePAGEpolyacrylamide gel electrophoresis.) is a member of a family of neuronal proteins that is characterized by a short N-terminal intravesicular sequence, a single transmembrane region, and a large cytoplasmic sequence containing two C2-domains (reviewed in Südhof(1995)). Nine synaptotagmin isoforms have been described in mammals (Perin et al., 1990; Geppert et al., 1991;Wendland et al., 1991; Mizuta et al., 1994; Hilbush and Morgan, 1994; Li et al., 1995a; Craxton and Goedert, 1995; Hudson and Birnbaum, 1995), four of which are also found in non-neural tissues (Li et al., 1995a; Hudson and Birnbaum, 1995). At least three synaptotagmins (SytI, SytII, and SytIII) are synaptic vesicle proteins, of which SytIII is present throughout the brain whereas SytI and SytII show restricted complementary expression patterns with partially overlapping distributions (Ullrich et al., 1994). Mice in which the SytI gene has been mutated exhibit a lethal phenotype in which there is a selective loss of fast Ca2+-dependent neurotransmitter release in hippocampal synapses (Geppert et al., 1994). Spontaneous neurotransmitter release and neurotransmitter release evoked by Ca2+-independent mechanisms are normal, suggesting an essential role for SytI only in the Ca2+-dependent last step of membrane fusion. Together with the Ca2+ binding properties of SytI (Li et al., 1995a, 1995b), these data suggest that SytI may serve as an exocytotic Ca2+ sensor.Based on the presence of C2-domains in SytI and the observation that the C2-domain confers Ca2+ regulation onto protein kinase C, it was speculated early on that SytI may be a Ca2+-binding protein (Perin et al., 1990). Indeed, experiments with purified SytI demonstrated that it binds Ca2+ and phospholipids (Brose et al., 1992) and that it undergoes a conformational change as a function of Ca2+ (Davletov and Südhof, 1994). Studies on recombinant C2-domains showed that the first C2-domain of SytI and of most but not all other synaptotagmins binds phospholipids as a function of Ca2+ (Davletov and Südhof, 1993; Chapman and Jahn, 1994; Ullrich et al., 1994; Li et al., 1995a). In addition, Ca2+ triggers binding of syntaxin to the first C2-domain of synaptotagmins with a Ca2+ dependence that is distinct from that of phospholipid binding, implying the presence of two Ca2+-binding sites in a single C2-domain (Li et al., 1995a, 1995b). Surprisingly, the second C2-domain of all synaptotagmins is inactive in these assays despite a high degree of sequence homology. Furthermore, the Ca2+-dependent binding properties of the native cytoplasmic domain of purified brain SytI containing both C2-domains has the same properties as the recombinant single first C2-domain (Li et al., 1995a, 1995b). Together these data suggest that the known Ca2+ binding properties of SytI can be entirely accounted for by its first C2-domain alone and raised the possibility that the second C2-domain may not represent a Ca2+-binding domain. This possibility was supported by the Ca2+-independent interactions of the second C2-domains of synaptotagmins with clathrin AP2 (Zhang et al., 1994; Li et al., 1995a) and with polyanions such as polyinositol phosphates (Fukuda et al., 1994).We now report the results of a yeast two-hybrid interaction screen for proteins binding to the C2-domains of SytI. Unexpectedly, SytI itself was identified as an interacting partner. In vitro binding assays demonstrated a Ca2+-dependent self-interaction of SytI that is specific for its second C2-domain. These data suggest that in addition to the first C2-domain, the second C2-domain of SytI is a Ca2+-regulated domain. However, the two C2-domains have distinct Ca2+-regulated properties, suggesting a functional diversification of C2-domains in