©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Functional Diversity of C2 Domains of Synaptotagmin Family
MUTATIONAL ANALYSIS OF INOSITOL HIGH POLYPHOSPHATE BINDING DOMAIN (*)

(Received for publication, August 30, 1995)

Mitsunori Fukuda (1) (2)(§) Toshio Kojima (1) (2) Jun Aruga (1) Michio Niinobe (2) (3) Katsuhiko Mikoshiba (1) (2)

From the  (1)Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan, the (2)Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, Japan, and the (3)Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Synaptotagmins I and II are inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate (IP(4)), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) binding proteins, which are thought to be essential for Ca-regulated exocytosis of neurosecretory vesicles. In this study, we analyzed the inositol high polyphosphate series binding site in the C2B domain by site-directed mutagenesis and compared the IP(4) binding properties of the C2B domains of multiple synaptotagmins (II-IV). The IP(4) binding domain of synaptotagmin II is characterized by a cluster of highly conserved, positively charged amino acids (321 GKRLKKKKTTVKKK 324). Among these, three lysine residues, at positions 327, 328, and 332 in the middle of the C2B domain, which is not conserved in the C2A domain, were found to be essential for IP(4) binding in synaptotagmin II. When these lysine residues were altered to glutamine, the IP(4) binding ability was completely abolished. The primary structures of the IP(4) binding sites are highly conserved among synaptotagmins I through IV. However, synaptotagmin III did not show significant binding ability, which may be due to steric hindrance by the C-terminal flanking region. These functional diversities of C2B domains suggest that not all synaptotagmins function as inositol high polyphosphate sensors at the synaptic vesicle.


INTRODUCTION

Synaptotagmin is an integral membrane protein of secretory vesicles, abundant in neural and some endocrine cells(1) . The structure of synaptotagmin is characterized by two copies of highly conserved repeats homologous to the C2 regulatory region of protein kinase C in the cytoplasmic domain(2) . The importance of the C2 domain of synaptotagmin in Ca-regulated exocytosis is shown by several studies. A recombinant protein of the C2A domain inhibited Ca-regulated exocytosis of a large dense core vesicle in PC12 cells(3) . Injection of basic peptides from the central region of both C2 domains into the squid giant presynaptic terminal also inhibited neurotransmitter release(4) . The studies of mutants of Caenorhabditis elegans(5) , Drosophila(6, 7) , and the knock-out mouse (8) also indicated that synaptotagmin plays a key role in Ca-regulated exocytosis.

In our previous study, we characterized synaptotagmin II as an IP(4)(^1)or rather an inositol-high polyphosphate series (IP(4), inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate, referred to as IHPS) binding protein(9) . Deletion analysis of synaptotagmin II clearly showed that about 30 amino acids of the central region of the C2B domain were essential for IHPS binding(10) . This binding domain includes a sequence corresponding to the squid Pep20 peptide, which is essential for neurotransmitter release(4) , suggesting that IHPS has some effect on neurotransmitter release. Our recent study of the squid giant presynapse supported this suggestion. Microinjection of the members of IHPS into the squid giant presynaptic terminal is shown to block synaptic transmission without affecting the presynaptic Ca current(11) , whereas inositol 1,4,5-trisphosphate shows no effect, suggesting that IHPS is a potent modulator of neurotransmitter release.

Recently, two novel isoforms designated synaptotagmins III and IV were isolated(12, 13) . Multiple isoforms of the proteins involving vesicular trafficking, such as the syntaxin family (14) and the synaptobrevin family(15, 16) , are not always functionally similar as indicated by in vitro protein interaction(17, 18) . Our interest is whether multiple synaptotagmin isoforms are functionally diversified or not.

In the present study, we first determined the essential residues for IP(4) binding in synaptotagmin II by site-directed mutagenesis. Then, we tested IP(4) binding to the C2B domains of synaptotagmins III and IV. Our data indicate that synaptotagmin III cannot bind IP(4) despite the presence of a putative IP(4) binding sequence. On the basis of these results, we discuss the functional differences in C2 domains in multiple synaptotagmin isoforms.


MATERIALS AND METHODS

Purification of Cerebellar Synaptotagmin II

Mouse synaptotagmin II was purified by sequential column chromatography as described by Niinobe et al.(9) .

cDNA Cloning of Mouse Synaptotagmins III and IV

cDNA encoding synaptotagmin III (amino acids 1-587) from mouse cerebellum was amplified by the reverse transcriptase polymerase chain reaction (PCR) (LA-PCR kit; Takara Shuzo) (sense 5`-CCACTATGTCTGGGGACTAC-3` and antisense 5`-CCTCACTCTGAATTCTCTTT-3`, designed on the basis of rat sequences(12) ) for 40 cycles, each involving denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 3 min. The PCR product, purified on an agarose gel and extracted with a Geneclean kit (Bio 101), was directly inserted into pT7 Blue T-vector (Novagen). Both strands were completely sequenced by using a BcaBEST dideoxy sequencing kit (Takara Shuzo). As compared to rat synaptotagmin III(12) , seven amino acid substitutions and one deletion were found: Ile at position 24 was altered to Val (I24V), A135G, P137T, P157L, S248T, G254A, L445R, and Thr at position 287 was deleted. These changes are probably not due to PCR-induced errors because they were also found in other PCR products of synaptotagmin III by using three sets of different oligonucleotides (data not shown).

Mouse synaptotagmin IV cDNA (amino acids 1-425) was also obtained by reverse transcriptase-PCR from mouse cerebellum (sense 5`-ACATGGCTCCTATCACCACC-3` and antisense 5`-AGCTAACCATCACAGAGCAT-3` (13) ). Cycling conditions were the same as in the case of synaptotagmin III described above. The PCR product was subcloned into pT7 Blue T-vector and completely sequenced.

Preparation of Mutant GST Fusion Proteins

Construction, expression, and purification of the glutathione S-transferase (GST) fusion proteins were as described by Fukuda et al.(10) . GST fusion proteins of mouse synaptotagmins I and II used in this study were also prepared as described previously(10) . GST-STIII-C2A, -C2B, and -C2B(DeltaC) coded for amino acids 290-421, 425-549, and 425-501, respectively, of mouse synaptotagmin III. GST-STIV-C2A and -C2B coded for amino acids 151-281 and 281-408, respectively, of mouse synaptotagmin IV.

Site-directed mutagenesis of GST-STII-C2B (mut3, mut4, mut5, mut8, and mut9) was carried out with a mutan-K kit (Takara Shuzo) as described previously(10) . All other mutant GST fusion proteins including deletion mutants (mut13) were produced by two-step PCR techniques as follows(19) . In the case of GST-STII-C2B-mut1(K322Q), for example, the right and left halves of the C2B domain were separately amplified with two pairs of oligonucleotides (primer A, 5`-CGGATCCGAGAAGGAAGAGCCAGAGAA-3` and mutagenic primer C, 5`-GCTTAAGTCTCTGACCGTTC-3` (right half); mutagenic primer D: 5`-GCTTAAGAAGAAGAAGACGACAGT-3` and primer B, 5`-CGAATTCATGTCGGACCAGTGCCGCA-3` (left half)). The two resulting PCR fragments were digested with AflII (underlined), ligated to each other, and reamplified with primers A and B. The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin II (K322Q) was subcloned into the BamHI-EcoRI site of pGEX-2T and verified by DNA sequencing.

Since we were also able to introduce the AflII site into the C2B domain of synaptotagmin III without amino acid substitutions (Fig. 5B, arrowhead), we could produce the chimera from synaptotagmins II and III by two-step PCR techniques. GST-STII/III-C2B-a contains amino acids 267-323 of mouse synaptotagmin II followed by amino acids 480-549 of mouse synaptotagmin III. GST-STIII/II-C2B contains amino acids 425-479 of mouse synaptotagmin III followed by amino acids 324-393 of mouse synaptotagmin II. GST-STII/III-C2B-b, containing amino acids 267-344 of mouse synaptotagmin II followed by amino acids 500-549 of mouse synaptotagmin III, was also produced by the same techniques.


Figure 5: IP(4) binding properties of multiple synaptotagmins. A, schematic representation of synaptotagmins II, III, and IV is shown at the top, where the transmembrane region (TM) and the two C2 repeats are shown. Below, construct of the chimera and deletion mutants of the GST fusion protein are shown. B, alignment of putative IP(4) binding domains of the synaptotagmin family. Residues that are identical in the three sequences are shaded. Residue numbers are given on both sides. Asterisks indicate the most essential residues for IP(4) binding to synaptotagmin II as determined from Fig. 2. Arrowhead indicates chimeric point between synaptotagmin II and III. C, [^3H]IP(4) binding to GST fusion proteins from C2 domains of synaptotagmins III and IV. GST fusion proteins (5 µg) were analyzed by [^3H]IP(4) binding assay as described under ``Materials and Methods.'' The data are means ± S.E. of three measurements, normalized to 100% for binding to GST-STII-C2B.




Figure 2: Mutational analysis of the IP(4) binding domain. A, the amino acid sequence (top) is the putative IP(4) binding domain of synaptotagmin II as determined previously(10) . Q indicates the substitution of Gln for Lys or Arg, and a dash indicates the deletion. Shaded boxes indicate the most important residues for IP(4) binding. B, mutant GST-STII-C2B (5 µg) were analyzed by [^3H]IP(4) binding assay as described under ``Materials and Methods.'' The data are means ± S.E. of three measurements, normalized to 100% for binding to GST-STII-C2B.



Production of Polyclonal Antibody

New Zealand White rabbits were immunized with purified GST-STII-C2A (amino acids 139-267) and GST-STII-C2B (amino acids 267-393) using Freund's or the RIBI adjuvant system. After absorption by GST-STII-C2B (or C2A) to remove the cross-reactive component, mainly anti-GST, the STII-C2A (or C2B) domain-specific antibody was affinity purified by Affi-Gel 10 immobilized with each antigen according to the manufacturer's notes.

Immunoblot analysis was carried out with these domain-specific antibodies as follows: proteins were transferred to a polyvinylidene difluoride membrane (Millipore), blocked with 2% skim milk, 0.05% Tween 20 in phosphate-buffered saline, incubated with anti-STII-C2A (or C2B) polyclonal antibody (2.3 µg/ml), and incubated with peroxidase-labeled goat IgG against rabbit IgG. Immunoreactive bands were visualized with 3,3`-diaminobenzidine.

Measurements of [H]IP Binding to GST Fusion Proteins

GST fusion proteins (5 µg) were incubated with 9.6 nM [^3H]IP(4) (DuPont NEN) in 50 µl of binding buffer (20 mM Tris-HCl (pH 8), 1 mM beta-mercaptoethanol) for 10 min at 4 °C. The sample was then mixed with 2 µl of 50 mg/ml -globulins and 52 µl of a solution containing 45% (w/v) PEG6000, 1 mM beta-mercaptoethanol, and 20 mM Tris-HCl (pH 8) and placed on ice for 5 min. The precipitate obtained by centrifugation at 10,000 times g for 5 min was solubilized in 500 µl of Solvable (DuPont NEN), and radioactivity was measured in Aquasol 2 (DuPont NEN) with a liquid scintillation counter(10) .

Inhibition of IP Binding to Synaptotagmin II by Specific Antibody

Purified synaptotagmin II (0.7 µg) or GST-STII-C2B (5 µg) was incubated with various concentrations of purified anti-STII-C2A, anti-STII-C2B, or normal rabbit IgG in 49 µl of binding buffer (20 mM Tris-HCl, pH 8, 1 mM beta-mercaptoethanol). After incubation for 30 min at 4 °C, [^3H]IP(4) (DuPont NEN) was added at a final concentration of 9.6 nM, and IP(4) binding assay was carried out as described previously(10) .

Other Procedures

Protein concentrations were determined by the Bio-Rad protein assay kit with bovine serum albumin used as a reference.


RESULTS

Inhibition of IP Binding by Specific Antibody against the C2B Domain

Our previous study, using bacterially expressed GST fusion proteins, indicated that the C2A and C2B domains of synaptotagmins I and II are different in terms of phospholipid and inositol polyphosphate binding properties(10) . First, we tried to check whether IP(4) exclusively binds to the C2B domain of purified cerebellar synaptotagmin II before detailed mutational analysis of the IP(4) binding domain. For this purpose, we tried to raise domain-specific antibodies against C2A or C2B, taking advantage of the functional difference between the two C2 domains. However, antibody against the C2A domain of synaptotagmin II (referred to as anti-STII-C2A) slightly cross-reacted with the C2B domain despite preabsorption with GST-STII-C2B. Antibody against the C2B domain of synaptotagmin II (anti-STII-C2B) showed hardly any cross-reaction with the C2A domain (Fig. 1A). These two antibodies can recognize the cerebellar synaptotagmin II as well as GST fusion proteins on SDS-polyacrylamide gel electrophoresis (Fig. 1A).


Figure 1: Effect of specific antibodies against the C2 domain on IP(4) binding to synaptotagmin II. A, purified GST-STII-C2A (a, 0.5 µg/lane), GST-STII-C2B (b, 0.5 µg/lane), and solubilized mouse cerebellar membrane proteins (c, 5 µg/lane) were analyzed by SDS-polyacrylamide gel electrophoresis. Proteins were visualized by amido black (left), and immunoblotting was carried out with a polyclonal antibody raised against each C2 domain to check its specificity (middle and right). Note that anti-STII-C2A slightly reacted with GST-STII-C2B, whereas anti-STII-C2B specifically recognized GST-STII-C2B. Numbers on the left indicate positions of molecular weight markers. B, [^3H]IP(4) binding activity of GST-STII-C2B (5 µg); C, purified synaptotagmin II (0.7 µg) in the presence of various concentrations of IgG: anti-STII-C2A (open circles), anti-STII-C2B (closed circles), and normal rabbit IgG (triangles). The data are means ± S.E. of three measurements.



By use of these two antibodies, we confirmed that the C2B domain was the authentic IP(4) binding domain in synaptotagmin II purified from mice cerebella. In the presence of the anti-STII-C2B antibody, the IP(4) binding activity of both GST-STII-C2B and purified synaptotagmin II was completely inhibited dose dependently (Fig. 1, B and C). In contrast to anti-STII-C2B, the effect of anti-STII-C2A antibody was very weak. The reduction of IP(4) binding activity by anti-STII-C2A antibody may be due to the cross-reactivity with the C2B domain (Fig. 1, A and B). Although anti-STII-C2A antibody has weak inhibitory effect on IP(4) binding to synaptotagmin II, this antibody inhibits Ca/phospholipid binding to the C2A domain of synaptotagmin II. (^2)The normal rabbit IgG had almost no effect.

Mutational Analysis of the IP Binding Domain of Synaptotagmin II

Since the IP(4) binding domain of synaptotagmin II is characterized by a cluster of positively charged amino acids (8 Lys and 1 Arg), which are highly conserved from C. elegans to human(10) , we tried to investigate the role of positively charged amino acids in IP(4) binding activity. For this purpose, we produced a series of 13 mutant GST-STII-C2Bs, referred to as mut1-13, and tested them for IP(4) binding activity (Fig. 2, A and B). In the case of substitution of one amino acid (mut1-8), these mutants were divided into three groups on the basis of their IP(4) binding activity: 50% reduction of IP(4) binding activity (mut5, -6, and -7), 20% reduction of IP(4) binding activity (mut3, -4, and -8), and no change (mut1 and -2). These results indicate that Lys at positions 327, 328, and 332 (shaded boxes in Fig. 2A) are most important residues for IP(4) binding ability. When two of these important Lys residues were altered to Gln (mut9-11), the IP(4) binding activity was reduced to below 30%, whereas a double mutation other than in the important residues, such as K322Q and K333Q, still generates more than 60% binding (data not shown). mut12, which carries three amino acid substitutions for three important residues, and mut13, in which the deleted sequence almost completely corresponds to the squid Pep20(4) , did not show significant IP(4) binding. It is interesting that these three Lys residues are not conserved in the C2A domain of synaptotagmin II (shaded boxes in Fig. 3). To examine whether these substitutions are a major cause of the loss of the IP(4) binding ability of the C2A domain, we produced another mutant, GST-STII-C2B (K327Y, K328E, and K332H) as illustrated in Fig. 3. As expected, this mutant had lost its IP(4) binding ability (Fig. 2B).


Figure 3: Comparison of putative IHPS binding domains. Alignment of the central region of two C2 domains of mouse synaptotagmin II according to (2) . The top is the sequence of the C2A domain corresponding to the IHPS binding domain (middle(10) ). Shaded boxes indicate the most important residues for IP(4) binding as shown in Fig. 2. The bottom sequence is that of the mutant GST-STII-C2B (K327Y, K328E, and K332H). Amino acid residue numbers are shown on both sides.



To evaluate the mutational effect on IP(4) binding activity, Scatchard analysis of IP(4) binding to mutant GST fusion proteins was performed (Fig. 4A), and the dissociation constants (K(d)) and B(max) are shown in Fig. 4B. In the case of one mutation, the K(d) values of these mutants were also divided into three groups: almost no change (mut1 and -2), twice as high (mut3, -4, and -8), and three to four times as high as that of normal GST-STII-C2B (mut5, -6, and -7), while B(max) remained unchanged (mut5-9) or increased slightly (mut1-4). GST-mut9-11, double mutations of important residues, showed still lower affinity for IP(4), below one-fifth of that of GST-STII-C2B, and the B(max) value of GST-mut9 and -mut11 was two-thirds of that of the GST-STII-C2B. All these data suggest that three Lys residues at positions 327, 328, and 332 of synaptotagmin II are responsible for the high affinity binding to IP(4), but Lys at position 322 and Arg at position 323 have no effect.


Figure 4: IP(4) binding properties of mutant GST-STII-C2Bs. A, Scatchard analysis of IP(4) binding to mutant GST-STII-C2B: mut2 (closed circles), mut3 (closed squares), mut6 (open circles), mut10 (open squares), mut11 (triangles). Dashed line indicates the normal GST-STII-C2B as previously reported(10) . B, parameters of IP(4) binding to mutant GST-STII-C2Bs. dash, not determined because of lack of IP(4) binding activity.



The B(max) values of these mutants shown in Fig. 4B indicate substoichiometric binding of IP(4) to GST fusion proteins. This substoichiometric binding was also observed in the case of purified cerebellar synaptotagmin II previously described(9) . This may result from instability of an IP(4) sensitive form of synaptotagmin II because purified synaptotagmin II easily loses its IP(4) binding by repetitive freezing and thawing or short storage at 4 °C.

IP Binding Activity of Synaptotagmins III and IV

Recently, two other mammalian isoforms of synaptotagmin, named synaptotagmin III and IV, have been described(12, 13) . To test whether IP(4) binding is an intrinsic property of the whole family, we cloned mouse synaptotagmins III and IV and constructed GST fusion proteins of each C2 domain. GST-STIII-C2A and -STIV-C2A did not show IP(4) binding activity like that of GST-STII-C2A. It is surprising that GST-STIV-C2B can bind IP(4) but GST-STIII-C2B cannot, although the putative IP(4) binding domain was highly conserved in both synaptotagmins III and IV (Fig. 5C). This incapability of synaptotagmin III to bind IP(4) was also observed even when the entire cytoplasmic region (amino acids 76-587) or the region from C2A domain to C-terminal region (amino acids 290-587) with GST was used (data not shown).

To compare the putative IP(4) binding domains of synaptotagmins II and III (Fig. 5B), two Lys residues were replaced by Arg in synaptotagmin III (amino acid positions 477 and 482). Especially, Arg at position 482 is a corresponding residue which is important for IP(4) binding ability in synaptotagmin II (Fig. 2). To investigate the effect of this substitution, two mutant GST fusion proteins were produced (GST-STIII-C2B (R482K) and GST-STII-C2B (K327R)). The R482K mutation could not rescue the IP(4) binding activity of GST-STIII-C2B, and the K327R mutation had no effect on IP(4) binding of GST-STII-C2B (Fig. 5C). To further determine which region causes the difference in IP(4) binding activity between synaptotagmins II and III, we constructed three chimera proteins from synaptotagmins II and III, named GST-STII/III-C2B-a, -b, and -STIII/II-C2B (Fig. 5A). GST-STIII/II-C2B showed strong IP(4) binding activity (65% of that of GST-STII-C2B), whereas the others showed very weak activity (below 15% of that of GST-STII-C2B), indicating that the loss of IP(4) binding activity was mainly caused by one-third of the C-terminal of the C2B domain of synaptotagmin III (Fig. 5C). This was confirmed by the facts that GST-STIII-C2B(DeltaC), in which one-third of the C-terminal of the C2B domain of synaptotagmin III was deleted, showed significant IP(4) binding activity (30% of that of GST-STII-C2B, Fig. 5C). These findings suggest that the IP(4) binding property varies in multiple synaptotagmins, although all of them basically possess the IP(4) binding domain.


DISCUSSION

The present experiments using specific antibodies against the C2A and C2B domain demonstrate that the C2B domain of synaptotagmin II is the IP(4) binding domain. Success in producing these specific antibodies provided us important information: the C2A and C2B domains have different structures and functions. This was supported by three recent studies: 1) Ca-dependent phospholipid binding of the C2A domain and Ca-independent phospholipid binding of the C2B domain(10, 21) ; 2) inositol polyphosphate binding to the C2B domain(10) ; and 3) clathrin-assembly protein AP-2 binding to the C2B domain(22, 23) .

Mutational analysis of the IP(4) binding domain of GST-STII-C2B clearly indicated that the numbers and positions of the positively charged amino acids, especially Lys at positions 327, 328, and 332, were responsible for high affinity IP(4) binding. These three Lys residues were conserved in the C2B but not in the C2A domain. Based on the recently reported three-dimensional structure of the C2A domain, these Lys residues are located in the beta4 strand of the C2 key(28) . However, more detailed structural analysis of the C2B domain will be required to state the IP(4) bound form of the C2B domain.

The amino acid sequences of the central region of the C2B domain are highly conserved among different species (4, 5, 24, 25) as well as isoforms (Fig. 5B). Nevertheless, synaptotagmin III cannot bind IP(4). Chimeric and mutational analysis suggested that the putative IP(4) binding domain of synaptotagmin III (amino acid positions 470-501) has the potential to bind IP(4), but one-third of the C-terminal of the C2B domain prevented IP(4) from binding to this synaptotagmin. The meaning of this functional difference in IP(4) binding to the synaptotagmin family is not clear. However, it is possible that inositol polyphosphate cannot modulate synaptotagmin III-protein interaction, such as C2B-AP2 binding, which may be involved in synaptic vesicle endocytosis(22, 23) .

On the basis of the available data, we propose that multiple isoforms of synaptotagmins do not always have the same roles in synaptic function. First of all, synaptotagmins I and II are thought to be functionally equivalent because of their high degree of sequence similarities (above 87% identity in the C2 domain), the conserved neurexin binding sequence, which was not in synaptotagmins III and IV (22, 26) , and complementary distribution(22, 27) . Second, synaptotagmin III is distinguished from other isoforms (I, II, and IV) in respect of the lack of inositol polyphosphate binding to the C2B domain and the unique structural feature, namely, the insertion of about 150 amino acids between the transmembrane and the C2 domains (Fig. 5A and (12) ). Third, the C2A domain of synaptotagmin IV lacked Ca-dependent phospholipid binding properties when liposomes consisting of phosphatidylserine and phosphatidylcholine (1:2.5, w/w) were used(22) . Furthermore, the protein interaction of synaptotagmin IV may be different from that of synaptotagmins I and II based on the recent observation that a novel protein that specifically interacts with synaptotagmin IV but not with synaptotagmins I and II was isolated by a yeast two-hybrid system (29) .

In summary, we first identified the inositol polyphosphate binding site by mutational analysis of the C2B domain of synaptotagmin II and found that the C2B domains of multiple synaptotagmins are functionally different with respect to IHPS binding. Our findings suggest that the multiple synaptotagmins may have different roles in presynaptic function.


FOOTNOTES

*
This work was supported by grants from the Japanese Ministry of Education, Science, and Culture (to J. A. and K. M.), the Japan Society for the Promotion of Science (to M. F.), the Intractable Diseases Research Foundation (to J. A.), and the Human Frontier Science Program (to K. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 81-298-36-9170; Fax: 81-298-36-9040; fukuda@rtc.riken.go.jp.

(^1)
The abbreviations used are: IP(4), inositol 1,3,4,5-tetrakisphosphate; PCR, polymerase chain reaction; GST, glutathione S-transferase; STI-IV, synaptotagmin I-IV.

(^2)
M. Fukuda, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. Akihiro Mizutani for critical reading of the manuscript. We are grateful to members of the Mikoshiba Laboratory for valuable discussions.


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