Inositol 1,3,4,5-Tetrakisphosphate Binding Activities of Neuronal and Non-neuronal Synaptotagmins
IDENTIFICATION OF CONSERVED AMINO ACID SUBSTITUTIONS THAT ABOLISH INOSITOL 1,3,4,5-TETRAKISPHOSPHATE BINDING TO SYNAPTOTAGMINS III, V, AND X*

Keiji IbataDagger §, Mitsunori FukudaDagger §parallel , and Katsuhiko MikoshibaDagger §**

From the Dagger  Molecular Neurobiology Laboratory, Tsukuba Life Science Center, the Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan, the § Department of Molecular Neurobiology, the Institute of Medical Science, the University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-0071, Japan, and the ** Calciosignal Net Project, Exploratory Research for Advanced Technology, 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153-0064, Japan

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Synaptotagmins I and II are essential for Ca2+-regulated exocytosis of synaptic vesicles from neurons, probably serving as Ca2+ sensors. This Ca2+-sensing function is thought to be disrupted by binding of an inositol 1,3,4,5-tetrakisphosphate (IP4) to the C2B domain of synaptotagmin I or II (Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 10708-10712). Recently, several synaptotagmin isoforms, expressed outside the nervous system, have been identified in rats and proposed to be involved in constitutive vesicle traffic. To test whether the inositol high polyphosphates also regulate constitutive vesicle traffic by binding to the non-neuronal synaptotagmins, we examined the IP4 binding properties of the recombinant C2 domains of both neuronal (III, V, X, and XI) and non-neuronal (VI-VIII and IX) synaptotagmins. The C2B domains of synaptotagmins VII-IX and XI had strong IP4 binding activity, but the C2B domain of synaptotagmin VI showed very weak IP4 binding activity. In contrast, there was no significant IP4 binding activity of the C2B domains of synaptotagmins III, V, and X or any of the C2A domains. A phylogenetic tree of the C2 domains of 11 isoforms revealed that synaptotagmins III, V, VI, and X (IP4-insensitive or very weak IP4-binding isoforms) belong to the same branch. Based on the sequence comparison between the IP4-sensitive and -insensitive isoforms, we performed site-directed mutagenesis of synaptotagmin III and identified several amino acid substitutions that abolish IP4 binding activity. Our data suggest that the inositol high polyphosphates might also regulate constitutive vesicle traffic via binding to the IP4-sensitive non-neuronal synaptotagmins.

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Synaptotagmins are a family of vesicle membrane proteins characterized by a short intravesicular amino terminus, a single transmembrane region, and two copies of highly conserved repeats homologous to the C2 regulatory region of protein kinase C (named the C2A and C2B domains) in the cytoplasmic domain (reviewed in Ref. 1). To date, at least 11 isoforms (synaptotagmins I-XI) have been described in rats or mice (2-10). Based on their expression patterns in tissues, synaptotagmins I-V, X, and XI are classified as neuronal types (expressed abundantly in neurons), and others (synaptotagmins VI-IX) are expressed in a wide variety of tissues other than brain (so-called ubiquitous types). Most of the proteins involved in Ca2+-regulated exocytosis in neurons (e.g. synaptobrevin or syntaxin) have been reported to have homologues involved in constitutive membrane trafficking; and therefore, it has been suggested that the same protein family governs both constitutive and regulated vesicle traffic (11-13). Based on this idea, the ubiquitous isoforms of synaptotagmin are also thought to be involved in constitutive vesicle trafficking because synaptotagmin I (the best characterized neuronal type) is essential for Ca2+-regulated exocytosis in neurons and some endocrine cells (probably functioning as a Ca2+ sensor) (reviewed in Ref. 1). However, the exact localization and functions of the ubiquitous synaptotagmins remain unknown.

Recently, we demonstrated the distinct roles of two C2 domains of synaptotagmin I (or II) in Ca2+-regulated exocytosis in the squid giant presynapse (14, 15), superior cervical ganglion cells (16), chromaffin cells (17), and insulin-secreting cells (18) by using specific antibodies against each C2 domain. The C2A domain is crucial for Ca2+-regulated exocytosis and is directly involved in the fusion of synaptic vesicles with the presynaptic plasma membrane. This fusion step was strongly inhibited by binding of an inositol high polyphosphate (inositol 1,3,4,5-tetrakisphosphate (IP4),1 inositol 1,3,4,5,6-pentakisphosphate (IP5), and inositol hexakisphosphate (IP6)) to the C2B domain of synaptotagmin I (or II) (15-17, 19-23). In chromaffin cells particularly, IP5 is suggested to function as a fusion clamp for exocytosis because IP5 is rapidly accumulated after depolarizing stimulation (17). These observations raised the possibility that inositol high polyphosphates may also regulate other types of vesicle traffic (e.g. constitutive) via binding to ubiquitous members of the synaptotagmin family (24).

To address this question, we examined the inositol high polyphosphate binding properties of the C2 domains of all synaptotagmin isoforms identified to date (synaptotagmins I-XI) as an indicator of IP4 binding activity. In this study, we show that the C2B domains, but not the C2A domains, of non-neuronal synaptotagmin isoforms (VII-IX) also have strong IP4 binding activities. In addition, we newly identified a subclass of synaptotagmins deficient in IP4 binding activity, despite having a putative IP4-binding sequence as determined previously (20, 22). Interestingly, this class of synaptotagmins (III, V, VI, and X) is structurally related and distinguished from other isoforms by phylogenetic trees of the C2 domains. We further determined the conserved amino acid substitutions that abolish IP4 binding activity by site-directed mutagenesis. On the basis of these results, we discuss the functional difference between IP4-sensitive and -insensitive synaptotagmins in vesicular trafficking.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Molecular Cloning of Mouse Synaptotagmin Isoforms and Preparation of Recombinant Proteins of Their C2 Domains-- cDNAs encoding two C2 domains of synaptotagmin isoforms (V-VII, X, and XI) were amplified by reverse transcriptase-polymerase chain reaction (PCR) from mouse cerebellum cDNAs using the following primers designed on the basis of rat sequences (8-10) with the addition of appropriate restriction endonuclease sites: SytV, 5'-CGGGATCCGGGAGGAGAAGTAACAGCAA-3' (sense) and 5'-GGAATTCTCATCGTTTCTCCAGCAGAG-3' (antisense); SytVI, 5'-CGGGATCCAGAGGCAACATGGCGGATAA-3' (sense) and 5'-GGAATTCTCACAACCGGGGGGTTCCCT-3' (antisense); SytVII, 5'-CGTGATCAGAGGAGGATGAGGCCCATGA-3' (sense) and 5'-GGAATTCCACAGGCTGCCGGGGACGAG-3' (antisense); SytX, 5'-CGGGATCCGAGCCTGCAATAAAAATCAG-3' (sense) and 5'-GGAATTCTCACAAGGGGTGCCAGTGTGTGA (antisense); and SytXI, 5'- GAAGATCTATGGCTGAGATCACAAATAT-3' (sense) and 5'-CCAATTGTTAGTACTCGCTCAGACTGT-3' (antisense). Reactions were carried out for 30 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 2 min. After digestion with BamHI, BglII, or FbaI and EcoRI or MunI, the PCR products were purified on an agarose gel, extracted with a Geneclean II kit (BIO 101, Inc.), and then inserted into the BamHI-EcoRI site of the pGEX-2T vector (Amersham Pharmacia Biotech). Only the nucleotide sequences coding for two C2 domains were sequenced in both directions using a BcaBEST dideoxy sequencing kit (Takara Shuzo). As compared with rat sequences, several amino acid substitutions were found in mouse SytVI (Ser at position 471 was altered to Asn, S471N), SytVII (I218V), SytX (S263F and M362I), and SytXI (G188D and I357V). These changes are probably not due to PCR-induced errors because they were also found in two independent PCR products of each synaptotagmin. Using primers based on the mouse nucleotide sequences obtained above and the data base for mouse SytVIII, fragments encoding the C2A or C2B domains of synaptotagmin isoforms (SytV-, SytVII-, SytVIII-, SytX-, and SytXI-C2A or -C2B and SytVI- and SytIX-C2B) were amplified by PCR. After digestion with BamHI or BglII and EcoRI, the PCR products were inserted into the BamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. pGEX-2T vectors carrying mouse SytI-IV-C2A or -C2B and SytVI- and SytIX-C2A were constructed as described previously (20, 22, 25).

Glutathione S-transferase (GST) fusion proteins of the C2A and C2B domains of mouse synaptotagmin isoforms (GST-STI-XI-C2A and -C2B, where STI-XI is synaptotagmins I-XI) were expressed in Escherichia coli JM109 and then purified by glutathione-Sepharose 4B (Amersham Pharmacia Biotech) chromatography according to the manufacturer's recommendations. GST-STI-C2A and -C2B encoded amino acids 138-266 and 268-392, respectively, of mouse synaptotagmin I. Similarly, the following fusion proteins were prepared: GST-STII-C2A, -C2B, and -C2BDelta C, corresponding to amino acids 139-267, 267-393, and 267-346 of mouse synaptotagmin II, respectively; GST-STIII-C2A, -C2B, and -C2BDelta C, corresponding to amino acids 290-421, 425-549, and 425-501 of mouse synaptotagmin III, respectively; GST-STIV-C2A and -C2B, corresponding to amino acids 151-281 and 281-408 of mouse synaptotagmin IV, respectively; GST-STV-C2A, -C2B, and -C2BDelta C, corresponding to amino acids 5-135, 135-261, and 135-213 of mouse synaptotagmin V, respectively; GST-STVI-C2A and -C2B, corresponding to amino acids 227-357 and 357-483 of mouse synaptotagmin VI, respectively; GST-STVII-C2A and -C2B, corresponding to amino acids 132-261 and 261-387 of mouse synaptotagmin VII, respectively; GST-STVIII-C2A and -C2B, corresponding to amino acids 70-195 and 195-318 of mouse synaptotagmin VIII, respectively; GST-STIX-C2A and -C2B, corresponding to amino acids 105-234 and 233-360 of mouse synaptotagmin IX, respectively; GST-STX-C2A, -C2B, and -C2BDelta C, corresponding to amino acids 228-358, 358-484, and 358-436 of mouse synaptotagmin X, respectively; and GST-STXI-C2A and -C2B, corresponding to amino acids 153-284 and 284-413 of mouse synaptotagmin XI, respectively. The amino acids of SytV-VIII, SytX, and SytXI were numbered according to previously described rat sequences (8-10). The protein concentrations of the purified recombinant proteins were initially determined using a Bio-Rad protein assay with bovine serum albumin used as a reference. Purified proteins were analyzed by 10% SDS-polyacrylamide gel electrophoresis followed by Coomassie Brilliant Blue R-250 staining. The purity of each recombinant protein was estimated by scanning the Coomassie Blue-stained SDS-polyacrylamide gel followed by densitometry with BioImage (Millipore Corp.), and the concentration of each GST fusion protein was then determined with bovine serum albumin as a standard.

Molecular Cloning of Mouse Synaptotagmin B/K and SrgI (Synaptotagmin-related Gene I)-- cDNAs encoding two C2 domains of SrgI and SytB/K were also amplified by reverse transcriptase-PCR from mouse cerebellum cDNAs using the following primers designed on the basis of rat sequences (26, 27) with the addition of appropriate restriction endonuclease sites: SrgI, 5'-GAAGATCTATGGCCGTGGACGTGACAGA (sense) and 3'-CCAATTGTTAGTTTCGCCGGACTGGAT-3' (antisense); and SytB/K, 5'-CGGGATCCATGGCGTACATCCAGTTGGA-3' (sense) and 5'-GGAATTCTCAGGTCACCTCCAGCGAGG-3' (antisense). Reaction conditions were the same as described above. After digestion with BamHI or BglII and EcoRI or MunI, the PCR products were purified on an agarose gel and extracted with a Geneclean II kit and then inserted into the BamHI-EcoRI site of the pGEX-2T vector. Only the nucleotide sequences coding for two C2 domains were sequenced in both directions using a BcaBEST dideoxy sequencing kit. As compared with rat sequences, three amino acid substitutions were found in mouse SrgI (D315E, T317S, and A393V). These changes are probably not due to PCR-induced errors because they were also found in two independent PCR products. Using primers based on the mouse nucleotide sequences obtained above, fragments encoding the C2A or C2B domains of SrgI and SytB/K were amplified by PCR. After digestion with BamHI or BglII and EcoRI, the PCR products were inserted into the BamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. GST-Srg-C2A, -C2B, and -C2BDelta C contained amino acids 149-278, 278-404, and 278-356, respectively, of mouse SrgI; and GST-STB/K-C2A, -C2B, and -C2BDelta C contained amino acids 180-315, 315-443, and 315-394, respectively, of mouse SytB/K.

Site-directed Mutagenesis of the Synaptotagmin III C2B Domain-- Site-directed mutagenesis of GST-STIII-C2Balpha -(P505F,E509Q,N510K) and GST-STIII-C2Bbeta 7-(H525K,V531K,C532I,R533F) was carried out by means of two-step PCR as follows (22). In GST-STIII-C2Balpha -(P505F,E509Q,N510K), for example, the right and left halves of the C2B domain were separately amplified with two pairs of oligonucleotides (primer A (5'-CGGGATCCGAAAAGGCAGATCTTGGGGA-3') and mutagenic primer B (5'-GCGACGTCGAACACCAGGGCCT-3') (left half); mutagenic primer C (5'-TCGACGTCGCTTTCGAGAGCGTGCAGAAAGTGGGTCTCAG-3') and primer D (5'-GGAATTCATCTCTGCCCAGTGTTCTC-3') (right half)). The two resulting PCR fragments were digested with AatII (underlined), ligated to each other, and reamplified with primers A and D. The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin III (P505F,E509Q,N510K) was subcloned into the BamHI-EcoRI site of pGEX-2T and verified by DNA sequencing. Site-directed mutagenesis of GST-STIII-C2Bloopbeta 7-8-(E537N,A539T,D540G,G543L) was achieved by PCR using primer A and a mutagenic primer (primer E, 5'-GGAATTCATCTCTGCCCAGTGTTCTCTGAGGTGTGGGCCGGTAGCGTTTGGGCCCACG-3'). The obtained PCR fragment encoding the mutant C2B domain of synaptotagmin III (E537N,A539T,D540G,G543L) was subcloned into the BamHI-EcoRI site of the pGEX-2T vector and verified by DNA sequencing. Other plasmids encoding the mutant C2B domain of synaptotagmin were similarly constructed by means of PCR using mutagenic primers.

Measurements of [3H]IP4 Binding to GST Fusion Proteins-- Measurement of IP4 binding was performed as described previously (20) with slight modifications. Briefly, the buffer system was changed from 20 mM Tris-HCl (pH 8.0) to 50 mM HEPES-KOH (pH 7.2) because GST-STII-C2B showed stronger IP4 binding activity in the latter buffer. GST fusion proteins (1-2.5 µg) were incubated with 9.6 nM [3H]IP4 (specific radioactivity of 777 GBq/mmol; NEN Life Science Products) in 49 µl of 50 mM HEPES-KOH (pH 7.2) for 10 min at 4 °C. The sample was then mixed with 1 µl of 50 mg/ml gamma -globulins and 50 µl of a solution containing 30% (w/v) polyethylene glycol 6000 and 50 mM HEPES-KOH (pH 7.2) and placed on ice for 5 min. The precipitate obtained by centrifugation at 10,000 × g for 5 min was solubilized in 500 µl of Solvable (Packard Instrument Co.), and radioactivity was measured in Aquasol 2 (Packard Instrument Co.) with a liquid scintillation counter. Nonspecific binding was determined in the presence of 10 µM nonradioactive IP4

Sequence Analyses-- Multiple sequence alignment of the C2 domains of synaptotagmin isoforms was performed using the PILEUP program of the GCG program (Version 8.1). Calculation of genetic distance and suitable depiction of the phylogenetic tree using the neighbor joining method were performed with the SINCA program (Fujitsu). Putative IP4-binding sites were aligned referring to the multiple alignment results.

Data Processing-- Statistical analysis and curve fitting were done using the GraphPad PRISM computer program (Version 2.0).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

IP4 Binding Activity of Synaptotagmin Isoforms (V-XI)-- The C2 domain, originally identified as a sequence motif of protein kinase C, is a conserved protein module of ~120 amino acids found in many proteins (28). Among them, synaptotagmins are apparently distinguished from other proteins in that they have a single transmembrane region and tandem C2 domains (C2A and C2B domains) with a short spacer. In our previous studies, we showed that neuronal synaptotagmins I, II, and IV, but not synaptotagmin III, are IP4- or inositol high polyphosphate-binding proteins (20, 22, 23). To further examine whether other neuronal and non-neuronal isoforms of synaptotagmins are also regulated by inositol high polyphosphates like synaptotagmin I, we prepared GST fusion proteins of C2 domains of synaptotagmins V-XI and tested for their IP4 binding activity (Fig. 1 and Table I). GST-STVII, -STVIII, -STIX, and -STXI-C2B had strong IP4 binding activity like synaptotagmin II, but GST-STVI-C2B showed weak IP4 binding activity (<20% of that of GST-STII-C2B). In contrast, GST-STV and -STX and all the GST-ST-C2A fusion proteins showed no significant IP4 binding activity under our experimental conditions.


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Fig. 1.   IP4 binding activities of the C2 domains of multiple synaptotagmins. GST fusion proteins from the C2 domains of synaptotagmins II and V-XI (1-2.5 µg) were analyzed by [3H]IP4 binding assay as described under "Materials and Methods." Open and hatched bars indicate the IP4 binding activities of the GST-C2A and -C2B fusion proteins, respectively. Significant IP4 binding was observed in the C2B domains of synaptotagmins II, VI-IX, and XI under these conditions. The data are means ± S.D. of three or four measurements, normalized to 100% for binding to GST-STII-C2B (100% specific binding = 6.93 ± 0.25 pmol/nmol of protein).

                              
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Table I
Comparison of IP4 binding properties of tandem C2 domains of synaptotagmins, SrgI, synaptotagmin B/K, rabphilin 3A, and Doc2

Analysis of the Phylogenetic Tree of Synaptotagmin C2 Domains-- To understand the relationship between the molecular evolution of the C2 domains of synaptotagmins and IP4 binding capacity, a phylogenetic tree of the C2 domains of synaptotagmin isoforms was constructed using the neighbor joining method (Fig. 2). In this phylogenetic tree, the C2B domain of synaptotagmin I is expressed as the most primitive or original form of the C2B domain because this domain of invertebrate synaptotagmins has been identified in Drosophila (31), Caenorhabditis elegans (32), Aplysia (33), and squid (Loligo pealei) (14) as having a less distant genetic relationship to mouse synaptotagmin I than the other isoforms (data not shown). According to this phylogenetic tree, the C2 domains of synaptotagmin isoforms are classified into two distinct groups, C2A and C2B, and the C2 domain from the same isoform is located at very similar positions in the two groups, suggesting that mammalian synaptotagmin isoforms were separated after the tandem C2 domains had been produced. When synaptotagmins that bind IP4 strongly or weakly are solid-boxed or broken-boxed, respectively, it is apparent that the C2B domains of synaptotagmins III, V, VI, and X (IP4-insensitive or weak binding isoforms) form a small but distinct branch (Fig. 2).


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Fig. 2.   Phylogenetic tree of the C2 domains of the mouse synaptotagmin family. The phylogenetic tree depiction was derived by the neighbor joining method using the results of multiple alignments of the C2A and C2B domains of synaptotagmin isoforms. To achieve a suitable depiction, the C2B domain of synaptotagmin I was taken as the root of the C2B domain among all synaptotagmin isoforms. The scale above the tree represents genetic distance. Boxes show the IP4-sensitive isoforms of synaptotagmin, but the broken box indicates weak IP4 binding ability (synaptotagmin VI). The sequences of mouse synaptotagmins I and II are from Ref. 20; mouse synaptotagmin III is from Ref. 22; mouse synaptotagmin IV is from Ref. 5; and mouse synaptotagmins V-XI are from this study.

Sequence Comparison of Putative IP4-binding Domains-- To further examine whether these synaptotagmins (III, V, VI, and X) have a common sequence responsible for the lack of IP4 binding at the amino acid level, we compared the putative IP4-binding sites of all synaptotagmin isoforms as determined previously (20, 22) (Fig. 3). However, in this region, no apparent differences were observed between IP4-sensitive and -insensitive synaptotagmin isoforms. Within the putative IP4-binding domain, three positively charged amino acids responsible for high affinity IP4 binding activity (Lys at positions 327, 328, and 332 of synaptotagmin II (22); asterisks in Fig. 3) were highly conserved among isoforms, whereas the corresponding positions in the C2A domains of virtually none of these molecules are occupied by positively charged amino acids (data not shown) (24). Since SytVIII-C2B lacks one of the important Lys residues (Ser at position 252, Ser-252), its IP4 binding activity was weaker than that of synaptotagmin II (Fig. 3), which is consistent with our previous mutational analysis (22).


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Fig. 3.   Alignment of the putative IP4-binding domains of the synaptotagmin family (I-XI). Thick white letters indicate residues that are identical in all 11 sequences. Residues that were conserved in half of the sequences are shown on a black background. Asterisks indicate the most important Lys residues for IP4 binding to synaptotagmin II as determined in Ref. 22. Note that these positions are occupied by Lys (or Arg) in all the synaptotagmin C2B domains, except for that of synaptotagmin VIII (Ser at position 252).

Although no substitutions corresponding to the three important positively charged amino acids were found in the C2B domains of synaptotagmins III, V, and X, they showed no IP4 binding activity whatsoever. The results indicate that these three residues are important for IP4 binding activity, but are not solely responsible for this binding. Furthermore, chimeric and deletion analyses of synaptotagmin III revealed the loss of IP4 binding activity to be due mainly to one-third of the C terminus of the C2B domain of synaptotagmin III (22). Since synaptotagmins III, V, VI, and X belong to the same small branch of the phylogenetic tree (Fig. 2), it seems likely that loss of the IP4 binding activity of synaptotagmins V and X or the weak IP4 binding ability of synaptotagmin VI also results from alterations in the sequence of the C termini of their C2B domains. To address this question, C-terminal deletion mutants of synaptotagmins V and X (GST-STV-C2BDelta C and -STX-C2BDelta C) were produced (Fig. 4B). As expected, both GST-STV-C2BDelta C and -STX-C2BDelta C showed IP4 binding activity (70% of that of GST-STII-C2BDelta C) (Fig. 4C), similar to the observations in our previous study using GST-STIII-C2BDelta C. Such recovery of IP4 binding by deletion of one-third of the C terminus seems to be a unique event occurring only in synaptotagmins III, V, and X because other tandem C2 proteins closely related to synaptotagmin, SrgI (26) and SytB/K (27), did not bind IP4 even when their C termini were deleted (Fig. 4C).


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Fig. 4.   Inhibitory effect of the C termini of the C2B domains of synaptotagmins III, V, and X on IP4 binding activity. A, sequence comparison of the C termini of the C2B domains between synaptotagmins I, II, and IX and synaptotagmins III, V, VI, and X. Residues that are identical in the five sequences are shown on a black background. Residues that are highly conserved only among synaptotagmins III, V, VI, and X are shaded, and their positions are boxed. Bars above the sequence indicate the corresponding positions of secondary structures of the C2A domain of synaptotagmin I (29). Among these subdomains, the alpha -helix between the beta 5 and beta 6 strands, the beta 7 strand, and the loop between the beta 7 and beta 8 strands contain conserved amino acids only among IP4-insensitive isoforms (see below the sequence and Fig. 5A). The amino acid (Asn-471) probably involved in the weak IP4 binding activity of synaptotagmin VI (see "Results") is indicated (#). B, schematic representation of synaptotagmin III and its C-terminal truncated mutant and the wild-type GST fusion protein. Other C-terminal truncated mutants (GST-STII-C2BDelta C, -STV-C2BDelta C, -STX-C2BDelta C, -Srg-C2BDelta C, and -STB/K-C2BDelta C) lack a corresponding C-terminal region like GST-STIII-C2BDelta C. The transmembrane region (TM), the two C2 domains, and GST are designated by open, hatched, and cross-hatched boxes, respectively. The putative IP4-binding sites (amino acid residues 470-501; see Fig. 3) are indicated below. C, effect of C-terminal deletion of the C2B domain of SytII, SytIII, SytV, SytX, SrgI, and SytB/K on IP4 binding activity. GST fusion proteins (1-2.5 µg) were analyzed by [3H]IP4 binding assay as described under "Materials and Methods." Black, open, and hatched bars indicate the IP4 binding activities of GST-C2A, -C2B, and -C2BDelta C fusion proteins, respectively. Note that GST-STIII-C2BDelta C, -STV-C2BDelta C, and -STX-C2BDelta C showed IP4 binding activity similar to that of GST-STII-C2BDelta C, but GST-Srg-C2A, -C2B, or -C2BDelta C, and -STB/K-C2A, -C2B, or C2BDelta C did not show any significant IP4 binding activity. The data are means ± S.D. of three or four measurements, normalized to 100% for binding to GST-STII-C2B (100% specific binding = 6.93 ± 0.25 pmol/nmol of protein).

Mutational Analysis of the C Terminus of the C2B Domain of Synaptotagmin III-- To identify the amino acids that reduce the IP4 binding activity of synaptotagmins III, V, VI, and X, we compared C-terminal sequences between IP4-sensitive (I, II, and IX) and -insensitive (or weak IP4-binding) synaptotagmins (III, V, VI, and X) (Fig. 4A). We initially focused on highly conserved amino acids among synaptotagmins III, V, VI, and X, yet these residues were apparently different from those of synaptotagmins I, II, and IX in their side chain character. Following these criteria, we selected 11 amino acids (Fig. 4A). Then, based on the three-dimensional structure of the C2A domain of synaptotagmin I (29), we divided these 11 amino acids into three groups based on secondary structures: the alpha -helix between the beta 5 and beta 6 strands (Pro-505, Glu-509, and Asn-510), the beta 7 strand (His-525, Val-531, Cys-532, and Arg-533), and the loop between the beta 7 and beta 8 strands (Glu-537, Ala-539, Asp-540, and Gly-543) (Fig. 4A). If one of these were involved in the inhibition of IP4 binding to synaptotagmin III, substitution with a synaptotagmin II amino acid at the corresponding position would restore IP4 binding activity. Therefore, in the first set of mutational experiments, we produced GST-STIII-C2Balpha -beta 7-loopbeta 7-8 containing P505F, E509Q, N510K, H525K, V531K, C532I, R533F, E537N, A539T, D540G, and G543L substitutions (Fig. 5A). As illustrated in Fig. 5B, this mutant showed restoration of IP4 binding activity (~70% of that of GST-STII-C2B). To further examine which region (or amino acid) is most effective for IP4 binding, we produced a series of three mutants, GST-STIII-C2Balpha -(P505F,E509Q,N510K), GST-STIII-C2Bbeta 7-(H525K,V531K,C532I,R533F), and GST-STIII-C2Bloopbeta 7-8-(E537N,A539T,D540G,G543L) (Fig. 5). Among these mutants, the alpha -helix and loop beta 7-8 mutants showed IP4 binding activity (23 and 10% of that of GST-STII-C2B), whereas the beta 7 mutations were unaffected. These results indicate that the combination of the alpha -helix and loop beta 7-8 mutations, without a beta 7 mutation, is required for adequate restoration of IP4 binding. This was confirmed with a double alpha -helix and loop beta 7-8 mutant, i.e. GST-STIII-C2Balpha -loopbeta 7-8. This mutant showed the same IP4 binding activity as GST-STIII-C2Balpha -beta 7-loopbeta 7-8, with a Kd value of 287 nM, which is comparable to that of GST-STII-C2B (Kd = 162 nM) (Fig. 6). We also performed a single or double mutation within the alpha -helix and loop beta 7-8 region. In single loop beta 7-8 mutants, the IP4 binding activities of GST-STIII-C2Bloopbeta 7-8-(E537N) and GST-STIII-C2Bloopbeta 7-8-(D540G) recovered to the level of whole loop beta 7-8 mutants (E537N,A539T,D540G,G543L), indicating that both acidic residues are required for an inhibitory effect on IP4 binding. Since synaptotagmin VI lacks one of these acidic residues (Asn-471; see Fig. 4A, #), GST-STVI-C2B bound IP4 at the same level as the GST-STIII-C2Bbeta 8-(E537N) mutant. In contrast, the single or double alpha -helix mutation apparently had a cumulative effect on the restoration of IP4 binding (compare single and double alpha -helix mutants in Fig. 5B). All of these mutational studies indicated that more than five simultaneous amino acid substitutions (i.e. Pro-505, Glu-509, Asn-510, Glu-537, and Asp-540 of synaptotagmin III) are involved in the inhibition of the IP4 binding activity of synaptotagmins III, V, and X. 


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Fig. 5.   Mutational analysis of the C terminus of the C2B domain of synaptotagmin III. A, 15 synaptotagmin III mutants and the wild-type synaptotagmin III and II proteins are illustrated. Identical residues are in boldface, and the positions of mutation are indicated by boxes. Amino acid substitutions are indicated by single letter amino acid code below the wild-type sequence. Bars above the sequence indicate the corresponding positions of secondary structures of the C2A domain of synaptotagmin I (29). B, mutant and wild-type GST fusion proteins (1-2.5 µg) were analyzed by [3H]IP4 binding assay as described under "Materials and Methods." Shaded, hatched, black, cross-hatched and open bars indicate the IP4 binding activities of the GST-STIII-C2B alpha -helix, beta 7, loop beta 7-8, and combined alpha -helix/loop beta 7-8 mutants and the wild-type GST-ST-C2B fusion proteins, respectively. The data are means ± S.D. of three or four measurements, normalized to 100% for binding to GST-STII-C2B (100% specific binding = 6.93 ± 0.25 pmol/nmol of protein).


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Fig. 6.   Characterization of IP4 binding to GST-STII-C2B and GST-STIII-C2Balpha -loopbeta 7-8. The competition experiment was performed using 1-2 µg of GST fusion proteins in the presence of various concentrations of unlabeled IP4. The Kd and Bmax values were calculated to be 287 nM and 12.9 pmol/µg of protein for GST-STIII-C2Balpha -loopbeta 7-8 and 162 nM and 12.1 pmol/µg of protein for GST-STII-C2B, respectively, by GraphPad PRISM (Version 2.0). The data are means ± S.D. of three or four measurements. open circle , GST-STII-C2B; bullet , GST-STIII-C2Balpha -loopbeta 7-8.

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

In this study, we have demonstrated that IP4 binding to the C2 domains is unique to the C2B domains of certain synaptotagmin isoforms, but not to other tandem C2 domains of SrgI, SytB/K, rabphilin 3A (20) and Doc2alpha , and Doc2beta (30) (summarized in Table I). Based on a sequence comparison between IP4-sensitive and -insensitive isoforms (Figs. 3 and 4A) and deletion or mutational analysis of synaptotagmin III (Figs. 4 and 5), we propose that the primitive form of synaptotagmin originally bound IP4 and that, during the diversification of synaptotagmin in vertebrates, some isoforms (III, V, and X) lost their IP4 binding activity as a consequence of several amino acid substitutions mainly in the C-terminal region of the C2B domain, but not in the putative IP4-binding site itself. This hypothesis was supported by the following evidence. (i) In invertebrates (Drosophila, C. elegans, Aplysia, and squid), only one synaptotagmin, closely related to mouse synaptotagmin I, has been reported (14, 31-33). Since the putative IP4-binding site of synaptotagmin I is highly conserved between vertebrates and invertebrates and squid synaptotagmin also bound IP4 (15), the IP4 binding properties are likely to have been retained during evolution. (ii) IP4-insensitive or weak IP4-binding synaptotagmins (III, V, VI, and X) also have a putative IP4-binding site containing a KK(K/R)TXXK(K/R) basic sequence (Fig. 3) (22). (iii) The C-terminal truncated form of synaptotagmins III, V, and X showed IP4 binding activities comparable to that of synaptotagmin II (Fig. 4C). (iv) Substitutions of seven amino acids of synaptotagmin II for those of synaptotagmin III (P505F, E509Q, N510K, E537N, A539T, D540G, and G543L) restored IP4 binding activity with an affinity similar to that of synaptotagmin II (Fig. 6).

Based on the three-dimensional structure of the C2A domain of synaptotagmin I (29), Lys-rich basic residues in the C2B domain are thought to be located in the fourth strand of the eight-stranded beta  sandwich, named C2 key. As shown in Fig. 4A, one-third of the C terminus of the C2B domain corresponds to the alpha -helix and the beta 6, beta 7, and beta 8 strands; and conserved substitutions among synaptotagmins III, V, VI, and X are found in the alpha -helix, the beta 7 strand, and the loop between the beta 7 and beta 8 strands. Among these, the alpha -helix between the beta 5 and beta 6 strands is predicted to be structurally closer to the IP4-binding site than the others (29) and may shield the basic region of synaptotagmin III from IP4 because of the presence of negatively charged rather than positively charged amino acids (e.g. Glu-509 of synaptotagmin III) or the presence of an alpha -helix breaker, i.e. Pro-505. Consistent with this finding, the alpha -helix mutation (P505F,E509Q,N510K) more effectively restored IP4 binding than the mutation in the beta 7 strand (H525K, V531K,C532I,R533F) or in the loop between the beta 7 and beta 8 strands (E537N,A539T,D540G,G543L). Although, at this stage, we do not know how beta 7-8 loop substitutions affect the IP4-binding site, the combination of alpha -helix and loop beta 7-8 mutations of synaptotagmin III greatly enhances IP4 binding activity (Fig. 5). Taken together, these results indicate that abolishing the IP4 binding capacity of the C2B domain of synaptotagmin requires a combination of amino acid substitutions. In contrast, the phospholipid binding properties of the C2A domain of synaptotagmins IV and XI changed dramatically with only one mutation (e.g. D244S of synaptotagmin IV) (10, 25).

In neurons, synaptotagmins I and II are thought to function as low affinity Ca2+ sensors for neurotransmitter release (1) and to be directly involved in the vesicular fusion step (14). In endoplasmic recticulum-Golgi transport, Ca2+ is now known to be required at a stage between vesicle docking and the actual membrane fusion event because EGTA inhibits this stage (34-36). Thus, it is possible that non-neuronal synaptotagmins VII-IX participate in this Ca2+-requiring event and that their function may be regulated by binding of IP4 to the C2B domain.

What is the functional difference between IP4-sensitive and -insensitive (SytIII, SytV, or SytX) isoforms of synaptotagmins? If synaptotagmin III and X function as Ca2+ sensors for vesicular exocytosis, it is most likely that their functions are unaffected by inositol high polyphosphates. The effects of inositol high polyphosphates on the in vitro biochemical nature of synaptotagmin III (e.g. Ca2+-dependent phospholipid and syntaxin binding) were not examined in this study. However, very recently, we demonstrated binding of the clathrin assembly protein AP-2 to the C2B domain of synaptotagmin II to be inhibited by IP6, whereas the SytIII-C2B/AP-2 interaction was not affected by IP6 (37). Thus, if IP6 functions as an inhibitor of endocytosis as proposed previously (24, 38), synaptotagmin III is a target of AP-2 even in the presence of IP6.

In summary, we investigated the IP4 binding properties of the C2 domains of neuronal and non-neuronal isoforms of synaptotagmin and showed that most of the C2B domains can bind IP4, suggesting that the inositol high polyphosphates may function as negative modulators of both regulated and constitutive vesicle trafficking, although the exact functions of isoforms other than synaptotagmins I and II remain to be clarified.

    ACKNOWLEDGEMENTS

We thank Hiroyuki Kabayama, Toshio Kojima, and Fumiaki Hamazato for advice.

    FOOTNOTES

* This work was supported in part by grants from the Japanese Ministry of Education, Science, Sports, and Culture (to K. M. and M. F.) and from the Science and Technology Agency of Japan (to K. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Present address: Developmental Neurobiology Laboratory, Brain Science Institute, the Institute of Physical and Chemical Research (RIKEN), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.

parallel To whom correspondence should be addressed: Developmental Neurobiology Laboratory, Brain Science Institute, RIKEN 2-1 Hirosawa, Wako, Saitama 351-0198 Japan. Tel.: 81-48-467-9745; Fax: 81-48-467-9744; E-mail: mnfukuda{at}brain.riken.go.jp.

1 The abbreviations used are: IP4, inositol 1,3,4,5-tetrakisphosphate; IP5, inositol 1,3,4,5,6-pentakisphosphate; IP6, inositol hexakisphosphate; PCR, polymerase chain reaction; Syt, synaptotagmin; GST, glutathione S-transferase.

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Abstract
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Materials & Methods
Results
Discussion
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