Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains.

The Ca(2+)-dependent oligomerization activity of the second C2 (C2B) domain of synaptotagmin I (Syt I) has been hypothesized to regulate neurotransmitter release. We previously showed that the cytoplasmic domains of several other Syt isoforms also show Ca(2+)-dependent oligomerization activity (Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180-28185), but little is known about the involvement of their C2 domains in Ca(2+)-dependent oligomerization. In this study, we analyzed the Ca(2+)-dependent oligomerization properties of the first (C2A) and the second C2 (C2B) domains of Syt VII. Unlike Syt I, both C2 domains of Syt VII contribute to Ca(2+)-dependent homo- and hetero-oligomerization with other isoforms. For instance, the Syt VII C2A domain Ca(2+)-dependently binds itself and the C2A domain of Syt VI but not its C2B domain, whereas the Syt VII C2B domain Ca(2+)-dependently binds itself and the C2B domain of Syt II but not its C2A domain. In addition, we showed by gel filtration that a single Syt VII C2 domain is sufficient to form a Ca(2+)-dependent multimer of very high molecular weight. Because of this "two handed" structure, the Syt VII cytoplasmic domain has been found to show the strongest Ca(2+)-dependent multimerization activity in the Syt family. We also identified Asn-328 in the C2B domain as a crucial residue for the efficient Ca(2+)-dependent switch for multimerization by site-directed mutagenesis. Our results suggest that Syt VII is a specific isoform that can cluster different Syt isoforms with two hands in response to Ca(2+).

Neurotransmitter release is achieved by a fusion of synaptic vesicles to presynaptic plasma membranes in response to a rapid increase in Ca 2ϩ ions (up to 200 M) entering through voltage-gated Ca 2ϩ channels (1,2). Synaptic vesicle proteins are then recycled from the plasma membrane by endocytosis to prepare for the next round of synaptic vesicle exocytosis. The synaptic vesicle cycle is now known to consist of at least four distinct steps: (i) docking of synaptic vesicles to active zones (docking step), (ii) an ATP-dependent priming step, (iii) actual fusion of synaptic vesicles to the presynaptic plasma membrane (fusion step), and (iv) recycling of synaptic vesicles by endocytosis (reviewed in Refs. 3 and 4). A variety of proteins involved in the regulation of each step of the synaptic vesicle cycle have already been identified (3,4). One of them, synaptotagmin I (Syt I), 1 an integral membrane protein of synaptic vesicles, is very interesting, because it regulates three distinct steps in the synaptic vesicle cycle (i.e. docking, fusion, and recycling).
Our previous study showed that Syt VII, which is involved in Ca 2ϩ -dependent lysosomal exocytosis in fibroblasts (31) and insulin secretion in pancreatic ␤-cells (32), has the strongest Ca 2ϩ -dependent self-oligomerization capacity in the Syt family (26,27). The Syt VII Ca 2ϩ -dependent self-oligomerization via the cytoplasmic domain occurs without tethering at the aminoterminal domain, whereas the Syt II Ca 2ϩ -dependent self-oligomerization via the C2B domain occurs only when the two molecules are preassembled at the amino-terminal domain (26). Although the Ca 2ϩ -dependent self-oligomerization of Syt I (or II) has been shown to be mediated by the C2B domain (22)(23)(24), no such information is available for other Syt isoforms, including Syt VII. In this study, we sought to determine the structural basis of Ca 2ϩ -dependent oligomerization of Syt VII. Unlike Syt I, both the C2A and C2B domains of Syt VII function as a Ca 2ϩ -dependent oligomerization site (i.e. Syt VII has "two hands"), and this explains its robust Ca 2ϩ -dependent oli-gomerization activity. We also showed that the single C2 domain is sufficient for Ca 2ϩ -dependent multimerization and identified Asn-328 in the C2B domain as a crucial residue for Ca 2ϩ -dependent multimerization by site-directed mutagenesis. Taq DNA polymerase and restriction enzymes were obtained from Toyobo Biochemicals (Tokyo, Japan). Polyclonal antibody against FLAG peptide was obtained from Zymed Laboratories Inc. (San Francisco, CA). Horseradish peroxidase (HRP)conjugated anti-T7 tag antibody and anti-T7 tag antibody-conjugated agarose were from Novagen (Madison, WI), and the 0.1 M EGTA (neutral pH) and 0.1 M CaCl 2 solutions were from Nacali Tesque (Kyoto, Japan). All other chemicals were commercial products of reagent grade. Solutions were made up in deionized water prepared with an Elix10 water purification system and Milli-Q Biocel A10 system (Millipore Corp., Bedford, MA).
Miscellaneous Procedures-Cotransfection of pEF-T7-Syts and pEF-FLAG-Syts into COS-7 cells was carried out (5 ϫ 10 5 cells the day before transfection/10-cm dish) by the DEAE-dextran method, as described previously (33). Proteins were solubilized with a buffer containing 1% Triton X-100, 250 mM NaCl, 50 mM HEPES-KOH, pH 7.2, 0.1 mM phenylmethylsulfonyl fluoride, 10 M leupeptin, and 10 M pepstatin A at 4°C for 1 h. T7-Syts were immunoprecipitated by anti-T7 tag antibody-conjugated agarose in the presence of various concentrations of Ca 2ϩ or 2 mM EGTA, as described previously (26). SDS-polyacrylamide gel electrophoresis and immunoblotting analyses were also performed, as described previously (33). The blots shown in this paper are representative of at least three independent experiments. Immunoreactive bands on x-ray film (X-Omat AR, Kodak) were captured by Gel Print 2000i/VGA (Bio Image) and analyzed with Basic Quantifier Software (version 1.0) (Bio Image). Multiple sequence alignment of the mouse synaptotagmin family was performed by using the ClustalW program, as described previously (38).

RESULTS
Mapping of the Domain Responsible for Ca 2ϩ -dependent Selfoligomerization of Synaptotagmin VII-In our previous study we showed that the Syt VII cytoplasmic domain alone is sufficient for Ca 2ϩ -dependent self-oligomerization activity (26,27). To identify the domain responsible for the Ca 2ϩ -dependent self-oligomerization of Syt VII, we produced various Syt VII deletion mutants and tagged them with T7 (see Fig. 1A). Each T7-tagged mutant was coexpressed with the FLAG-Syt VII cytoplasmic domain (Syt VII-cyto) in COS-7 cells, and the associations between the T7-and FLAG-tag proteins were evaluated by immunoprecipitation, as described previously (33). Briefly, T7-Syt proteins were immunoprecipitated by anti-T7 tag antibody-conjugated agarose in the presence and absence of 500 M Ca 2ϩ . The co-immunoprecipitated FLAG-Syt-cyto proteins were first detected by anti-FLAG rabbit polyclonal antibody (Fig. 1B, upper panel), and the same blot was then stripped and reprobed with HRP-conjugated anti-T7 tag antibodies (Fig. 1B, lower panel). Surprisingly, with the exception of T7-Syt VII-cyto⌬C2AB (spacer domain alone), all the deletion mutants interacted with FLAG-Syt VII-cyto only in the presence of Ca 2ϩ , indicating that each of the C2 domains functions as an acceptor for the Ca 2ϩ -dependent self-oligomerization. By contrast, Syt I Ca 2ϩ -dependent self-oligomerization has been shown to be mediated by the C2B domain alone and not by the C2A domain (22,28).
Ca 2ϩ -dependent Homo-and Hetero-oligomerization Properties of the C2 Domains of Synaptotagmin VII-To investigate whether a single C2 domain alone is sufficient for the Ca 2ϩ -dependent self-oligomerization capacity of Syt VII, Ca 2ϩ -dependent association between T7-and FLAG-Syt VII-C2A (or C2B) was investigated as described above. As shown in Fig. 2A, both C2 domains showed Ca 2ϩ -dependent self-oligomerization activity, although some fractions (about 40%) of the C2A domain self-oligomerized even in the absence of Ca 2ϩ . Because the entire cytoplasmic domain of Syt VII did not show Ca 2ϩ -inde-pendent self-oligomerization activity ( Fig. 1B and Ref. 26), the Ca 2ϩ -independent self-oligomerization of the Syt VII C2A domain observed may be an artifact, probably induced by removing the C2A domain from the whole protein. By contrast, no hetero-oligomerization activity was detected between the C2A and C2B domains ( Fig. 2A).
In our previous study, we showed that the Syt VII cytoplasmic domain can interact with various Syt isoforms in a Ca 2ϩdependent manner (26,27). Syt VII preferentially interacts with Syts V, VI, and X, weakly with Syts I, II, and IX, but not essentially with Syts III, IV, VIII, and XI (26,27). To determine which C2 domains of Syt VII are involved in hetero-oligomerization with other Syt isoforms, FLAG-Syt VII-C2A (or -C2B) was coexpressed with T7-Syts II, IV, VI, and VIII cytoplasmic domains in COS-7 cells, and their associations were evaluated as described above. Surprisingly, Syt II-cyto weakly interacted with both C2 domains of Syt VII in the presence of Ca 2ϩ , whereas Syt-VI-cyto interacted only with the Syt VII C2A domain in the presence of Ca 2ϩ (Fig. 2B). By contrast, Syt IV-cyto and Syt VIII-cyto did not interact with either C2 domain of Syt VII, consistent with the findings in a previous report (27). To further specify the domains of Syt II (or Syt VI) that act as an acceptor for Ca 2ϩ -dependent hetero-oligomerization with Syt VII, each C2 domain of Syt II (or Syt VI) with a T7-tag was similarly coexpressed with FLAG-Syt VII-cyto in COS-7 cells. As shown in Fig. 2C, the Syt II C2B and Syt VI C2A domains were found to be essential for Ca 2ϩ -dependent hetero-oligomerization with Syt VII. Thus, the contribution of the C2 domains to Syt Ca 2ϩ -dependent oligomerization differs among isoforms.
Because the cytoplasmic domain of Syt VII showed Ca 2ϩ -dependent self-oligomerization activity with an EC 50 value of about 150 M Ca 2ϩ , we investigated the Ca 2ϩ dependence of the self-oligomerization activity of each of the C2 domains of Syt VII (Fig. 3A). The EC 50 values for self-oligomerization by the Syt VII C2A and C2B domains were ϳ150 M Ca 2ϩ and 250 M Ca 2ϩ , respectively ( Fig. 3B; Syt VII-C2A, closed squares; Syt VII-C2B, open squares). Thus, the EC 50 values for selfoligomerization with the isolated C2 domains and the full cytoplasmic domain are essentially the same.
Identification of the Amino Acid Residues Responsible for the Ca 2ϩ -dependent Self-oligomerization of Synaptotagmin VII-In the next set of experiments we sought to identify the amino acid residues responsible for the robust Ca 2ϩ -dependent self-oligomerization of Syt VII. First, we made sequence comparisons among mouse Syts I-XI C2 domains in a search for specific amino acid residues found only in Syt VII and not in other Syt isoforms. We found one Asn residue that was only present in the C2 domains of Syt VII (Asn-195 in the C2A domain and Asn-328 in the C2B domain of Syt VII), with the corresponding positions of Syts I-VI and VIII-XI being occupied by Thr (Fig. 4A, asterisk). These Asn residues of Syt VII are located between the ␤4 and ␤5 strands of the eightstranded ␤-sandwich structure of the C2 domain and are in close proximity to the C2B effector domain responsible for inositol polyphosphate, AP-2 binding, and self-oligomerization of Syt I (two Lys residues in Fig. 4A, number signs (##)) (24,29,35,39). To examine the involvement of these Asn residues in the selfoligomerization of Syt VII, we performed site-directed mutagenesis, and obtained Syt VII-C2A(N195T) and -C2B(N328T)

FIG. 1. Mapping of the domain responsible for Ca 2؉ -dependent self-oligomerization of synaptotagmin VII.
A, schematic representation of deletion mutants of Syt VII. The transmembrane domain (TM) and two C2 domains are represented by an open box and hatched boxes, respectively. Systematic deletions were made from the amino and carboxyl termini. The self-oligomerizing activity of each mutant is indicated after its name (ϩ or Ϫ, respectively) and was determined on the basis of the results shown in B. Amino acid numbers are given on both sides. B, Ca 2ϩ -dependent oligomerization of various T7-Syt VII deletion mutants with FLAG-Syt VII-cyto. pEF-T7-Syts and pEF-FLAG-Syt VII-cyto were cotransfected into COS-7 cells. The proteins expressed were solubilized with 1% Triton X-100 and immunoprecipitated by anti-T7 tag antibody-conjugated agarose, as described previously (33). Co-immunoprecipitated FLAG-Syts were first detected by anti-FLAG rabbit antibody (5 g/ml, upper panel). The same blot was stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure loading of the same amounts of T7-Syt proteins (lower panel). Note that, except for Syt VII-cyto⌬C2AB, all deletion mutants showed Ca 2ϩ -dependent oligomerization activity, indicating that all of the C2 domains are involved in oligomerization. The positions of the molecular weight markers (ϫ10 Ϫ3 ) are shown on the left. Mock, transfection with a control vector (pEF-BOS).
mutants. As expected, the C2B(N328T) mutant had completely lost Ca 2ϩ -dependent oligomerization activity (Fig. 4B, right  panel), although some fractions of C2B(N328T) mutant pro-teins showed Ca 2ϩ -independent oligomerization activity with the wild-type C2B domain. By contrast, the N198T mutation had no effect on Ca 2ϩ -dependent oligomerization by the C2A domain (Fig. 4B, left panel). Similar experiments were performed by using the full cytoplasmic domain carrying the N195T or N328T mutation (Fig. 4C), and interestingly, the N328T mutation completely abolished the Ca 2ϩ -dependent self-oligomerization activity (i.e. Ca 2ϩ -independent self-oligomerization), even when the C2A domain was intact.
To further investigate the importance of Asn-328 of the C2B domain in Ca 2ϩ -dependent self-oligomerization, we mutated this residue to other amino acids with different side-chain structures (N328A, N328L, N328D, N328K, N328P). As shown in Fig. 4D, except for the N328P mutant, self-oligomerization of these mutants was also stimulated by Ca 2ϩ (to an extent similar to that of the wild-type protein), but the Ca 2ϩ -independent self-oligomerization activity was significantly increased. By contrast, the N328P mutant resulted in the loss of Ca 2ϩ -dependent oligomerization, the same as the N328T mutation. These results indicate that Asn-328 in the C2B domain is associated with efficient Ca 2ϩ -dependent self-oligomerization.
A Single C2 Domain Is Sufficient for Ca 2ϩ -dependent Multimerization of Synaptotagmin VII-Using a dominant negative approach, Chapman and co-workers (28) recently showed that Ca 2ϩ -dependent Syt I oligomerization is involved in fusion of secretory vesicles in PC12 cells and proposed that Ca 2ϩ -de-

FIG. 3. Ca 2؉ dependence of synaptotagmin VII C2A (or C2B) domain self-oligomerization.
A, pEF-T7-Syt VII-C2A (or -C2B) and -FLAG-Syt VII-C2A (or -C2B) were cotransfected into COS-7 cells. The proteins expressed were solubilized with 1% Triton X-100 and immunoprecipitated by anti-T7 tag antibody-conjugated agarose in the presence of various concentrations of Ca 2ϩ , as described previously (26). Co-immunoprecipitated FLAG-Syts were first detected with anti-FLAG rabbit antibody (5 g/ml, upper panels). The same blot was then stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure loading of the same amounts of T7-Syt proteins (lower panels). pendent Syt clustering may regulate opening and/or expansion of a "fusion pore." Nevertheless, it remains unknown how many Syt molecules cluster in response to Ca 2ϩ , even though such information is quite important in evaluating whether Syt molecules can regulate the fusion pore. To address this, we used gel filtration to estimate the molecular weight of each Syt VII C2 domain in the presence and absence of 1 mM Ca 2ϩ (see "Experimental Procedures" for details). In the absence of Ca 2ϩ , the Syt VII-C2B proteins were eluted as a single peak (Fig. 5, top  panel, fractions 75-80), and their apparent molecular mass was estimated to be ϳ10 kDa. Because the calculated molecular weight of T7-Syt VII-C2B is 17,451 and Syt VII-C2B did not form oligomers in the immunoprecipitation assay ( Fig. 2A), we concluded that Syt VII-C2B is present as a monomer in the absence of Ca 2ϩ (Fig. 5, top panel). By contrast, in the presence of 1 mM Ca 2ϩ , Syt VII-C2B proteins were eluted as a single peak with a very high molecular weight (Fig. 5, second panel, fractions [31][32][33][34][35][36] that was identical to the elution profile of blue dextran (M r 2,000,000), an indicator of the void volume of the column (Fig. 5, bottom). Thus, we were unable to determine the exact number of the C2B domain clustered by Ca 2ϩ . The molecular weight shift induced by Ca 2ϩ probably reflects direct clustering of the Syt VII C2B domain itself, and it is most unlikely to be mediated by certain adapter proteins (i.e. Ca 2ϩdependent binding partner of Syts, such as syntaxin I) (40), because after Coomassie Brilliant Blue R-250 staining of the gel we were unable to observe any bands other than Syt molecules and the anti-T7-tag antibody used for immunoprecipitation, and syntaxin I is not expressed in COS-7 cells (data not shown). We therefore concluded that the Syt VII-C2B domain forms a multimer in the presence of Ca 2ϩ . We also evaluated the apparent molecular weight of Syt VII-C2B(N328T) mutant protein by using the same gel filtration column. Interestingly, Syt VII-C2B(N328T) proteins form multimers irrespective of the presence of Ca 2ϩ (Fig. 5, fifth and sixth panels).
Similar results were obtained with the Syt VII-C2A domain (Fig. 5, third and fourth panels), although in the absence of Ca 2ϩ the Syt VII-C2A domain eluted as a broad peak. Because the calculated molecular weight of T7-Syt VII-C2A is 16,667 and Syt VII-C2A formed Ca 2ϩ -independent oligomers in the immunoprecipitation assay ( Fig. 2A), this peak probably consists of the Syt VII-C2A dimer and monomer. DISCUSSION In the present study we analyzed the Ca 2ϩ -dependent multimerization properties of the C2 domains of Syt VII to determine why Syt VII is the only member of the Syt family to exhibit robust Ca 2ϩ -dependent oligomerization activity (26), why Syt VII is able to interact with various Syt molecules (27), and how many Syt VII molecules cluster in response to Ca 2ϩ . The results presented in this paper indicate that both C2 domains of Syt VII contribute to Ca 2ϩ -dependent homo-and/or hetero-multimerization, with slightly different affinities for Ca 2ϩ (EC 50 value for C2A domain self-multimerization of about 150 M, and for the C2B domain self-multimerization, of about 250 M). This finding is in considerable contrast to the results obtained for Syt I or II, because only the C2B domain of Syts I and II is involved in Ca 2ϩ -dependent self-oligomerization (22)(23)(24). The Syt VII C2A domain efficiently interacts with itself and the Syt VI C2A domain, and weakly with the C2B domain of Syt II, but cannot interact with its C2B domain. Similarly, the Syt VII C2B domain interacts with itself and weakly with the C2B domain of Syt II, but not with its C2A domain. The Ca 2ϩ -dependent homo-and hetero-oligomerization properties of Syt VII are summarized in Fig. 2D. Because of this two handed structure of Syt VII, it can oligomerize more efficiently than other Syt isoforms and can interact with various Syt isoforms without being tethered at the amino-terminal domain (26,27).
Sequence alignment of the Syt family and site-directed mu- FIG. 4. Identification of the amino acid residues responsible for the Ca 2؉ -dependent self-oligomerization of synaptotagmin VII. A, alignment of the central region of two C2 domains of mouse synaptotagmins I-XI (33). Residues, half of whose sequences were conserved, are shown against a black background. The number signs (#) indicate the position of the Lys residues that are only conserved in the C2B domain (35) and are essential for inositol 1,3,4,5-tetrakisphosphate binding, AP-2 binding, and self-oligomerization (so-called "C2B effector domain") (24,29,35,39). The asterisk indicates the Asn residues that are only conserved in the Syt VII C2 domains. The location of the ␤-strands is indicated by arrows (41,42). The amino acid numbers are indicated on the right. B, pEF-T7-Syt-C2A (or -C2B) and -FLAG-Syt-C2A (or -C2B) mutants were cotransfected into COS-7 cells. C and D, pEF-T7/FLAG-Syt VII-cyto mutants were cotransfected into COS-7 cells. The proteins expressed were solubilized with 1% Triton X-100 and immunoprecipitated by anti-T7 tag antibody-conjugated agarose, as described previously (33). Co-immunoprecipitated FLAG-Syts were first detected by anti-FLAG rabbit antibody (5 g/ml, upper panels). The same blots were stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure loading of the same amounts of T7-Syt proteins (lower panels). The positions of the molecular weight markers (ϫ10 Ϫ3 ) are shown on the left. Note that the N328T (in C) and N328P (in D) mutations resulted in the loss of Ca 2ϩ -dependent self-oligomerization of Syt VII and that the N328A, N328L, N328D, and N328K mutations increased the Ca 2ϩ -independent self-oligomerization of Syt VII in D.
tagenesis enabled us to identify Asn-328 in the C2B domain as a crucial residue for Ca 2ϩ -dependent self-multimerization by Syt VII. This Asn residue is found to be conserved in human, rat, and mouse Syt VII, but the corresponding position in other mouse Syt isoforms and Syts from other species is occupied by Thr (data not shown). Thus, the strong Ca 2ϩ -dependent multimerization properties are probably retained, at least in mammals.
Recent crystallographic analysis of C2 domains of Syt I, phospholipase C␦1 (PLC␦1), and cytosolic phospholipase A2 (cPLA2) showed the C2 domains to be composed of a common eight-stranded anti-parallel ␤-sandwich consisting of fourstranded ␤-sheets, although their structures have been classified into two groups based on their topology (e.g. synaptotagmin I C2A domain with type I topology and PLC␦1 C2 domain with type II topology) (41)(42)(43)(44)(45). Three flexible loops (named loops 1-3) protrude from the tip of the ␤-sandwich structure, and two of them (loops 1 and 3) are involved in Ca 2ϩ binding in the Syt I-C2A domain, all three of which are loops in the PLC␦1-and cPLA2-C2A domains (41)(42)(43)(44)(45). Interestingly, Asn-677 (PLC␦1) and Asn-65 (cPLA2) in loop 2 bind Ca 2ϩ , and these residues correspond closely to the Syt VII Asn-328 residue located in loop 2, which is formed by ␤-4 and ␤-5 strands (Fig. 4A), suggesting a role of Asn-328 in direct Ca 2ϩ binding or indirect stabilization of Ca 2ϩ binding to loops 1 and 3. Because the Ca 2ϩ -binding sites of the two C2 domains of Syt III face each other (41) and some fractions of the isolated C2A domain appear to form dimers in the absence of Ca 2ϩ (Fig. 5), it is tempting to speculate that the Asn-328 residue of Syt VII is also involved in Ca 2ϩ -dependent self-oligomerization of the C2A domain.
Mutation of Asn-328 to Pro or Thr resulted in the loss of Ca 2ϩ -dependent multimerization, and these mutants showed Ca 2ϩ -independent multimerization (Figs. 4C, 4D, and 5), whereas mutation of Asn-328 to Ala, Leu, Asp, or Lys had no significant effect on Ca 2ϩ -dependent self-multimerization but Ca 2ϩ -independent self-oligomerization activity was significantly increased (Fig. 4D). If the Asn-328 residue serves as an interface for multimerization (i.e. a major site for multimerization), mutation of Asn-328 to other amino acids with different side-chain structures should result in loss of self-oligomerization activity. However, we could not obtain a mutant completely lacking self-multimerization activity by mutation of Asn-328, suggesting that the Asn-328 residue is crucial for controlling the Ca 2ϩ dependence of self-multimerization rather than for multimerization itself. Indeed, the Syt I-C2B effector domain responsible for Ca 2ϩ -dependent oligomerization is located in the middle of the ␤-4 strand (side of the ␤-sandwich structure that is distinct from the putative Ca 2ϩ -binding site) (24,29). Thus, we hypothesize that Ca 2ϩ binding to the apex of the ␤-sandwich structure (loops 1-3) may change electrostatic charges or induce structural changes, which would allow the putative C2B effector domain of Syt VII (middle of the ␤-4 strand located at the side of the ␤-sandwich structure) to participate in self-multimerization. Further three-dimensional studies are required to elucidate how Asn-328 of Syt VII regulates Ca 2ϩ -dependent self-oligomerization of C2 domains.
In summary, we showed that both the C2A and C2B domains of Syt VII contribute to Ca 2ϩ -dependent multimerization. Because of this two handed structure, Syt VII can cluster heterogeneous Syt isoforms (Syts I, II, V, VI, VII, IX, and X) in response to Ca 2ϩ , and this Ca 2ϩ -dependent Syt multimer may regulate vesicular exocytosis.