From the 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
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ABSTRACT |
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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.
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INTRODUCTION |
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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.
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MATERIALS AND METHODS |
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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 -C2BMolecular 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 -C2BC contained
amino acids 149-278, 278-404, and 278-356, respectively, of mouse
SrgI; and GST-STB/K-C2A, -C2B, and -C2B
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-C2B-(P505F,E509Q,N510K) and
GST-STIII-C2B
7-(H525K,V531K,C532I,R533F) was carried out by means of
two-step PCR as follows (22). In GST-STIII-C2B
-(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-C2Bloop
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 -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).
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RESULTS |
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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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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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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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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 -helix between the
5 and
6
strands (Pro-505, Glu-509, and Asn-510), the
7 strand (His-525,
Val-531, Cys-532, and Arg-533), and the loop between the
7 and
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-C2B
-
7-loop
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-C2B
-(P505F,E509Q,N510K), GST-STIII-C2B
7-(H525K,V531K,C532I,R533F), and
GST-STIII-C2Bloop
7-8-(E537N,A539T,D540G,G543L) (Fig. 5). Among
these mutants, the
-helix and loop
7-8 mutants showed
IP4 binding activity (23 and 10% of that of GST-STII-C2B), whereas the
7 mutations were unaffected. These results indicate that
the combination of the
-helix and loop
7-8 mutations, without a
7 mutation, is required for adequate restoration of IP4
binding. This was confirmed with a double
-helix and loop
7-8
mutant, i.e. GST-STIII-C2B
-loop
7-8. This mutant
showed the same IP4 binding activity as
GST-STIII-C2B
-
7-loop
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
-helix and loop
7-8 region. In single loop
7-8 mutants, the IP4 binding activities of
GST-STIII-C2Bloop
7-8-(E537N) and GST-STIII-C2Bloop
7-8-(D540G)
recovered to the level of whole loop
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-C2B
8-(E537N) mutant. In contrast, the single or
double
-helix mutation apparently had a cumulative effect on the
restoration of IP4 binding (compare single and double
-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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DISCUSSION |
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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 Doc2, and Doc2
(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 sandwich, named C2 key. As shown in Fig. 4A, one-third of
the C terminus of the C2B domain corresponds to the
-helix and the
6,
7, and
8 strands; and conserved substitutions among
synaptotagmins III, V, VI, and X are found in the
-helix, the
7
strand, and the loop between the
7 and
8 strands. Among these,
the
-helix between the
5 and
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
-helix breaker,
i.e. Pro-505. Consistent with this finding, the
-helix
mutation (P505F,E509Q,N510K) more effectively restored IP4
binding than the mutation in the
7 strand
(H525K, V531K,C532I,R533F) or in the loop between the
7 and
8
strands (E537N,A539T,D540G,G543L). Although, at this stage, we do not
know how
7-8 loop substitutions affect the IP4-binding
site, the combination of
-helix and loop
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.
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ACKNOWLEDGEMENTS |
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We thank Hiroyuki Kabayama, Toshio Kojima, and Fumiaki Hamazato for advice.
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FOOTNOTES |
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* 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.
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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REFERENCES |
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