©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Phospholipid Composition Dependence of Ca-dependent Phospholipid Binding to the C2A Domain of Synaptotagmin IV (*)

(Received for publication, December 5, 1995; and in revised form, January 18, 1996)

Mitsunori Fukuda (1) (2)(§) Toshio Kojima (1) (2) Katsuhiko Mikoshiba (1) (2) (3)

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, the (2)Department of Molecular Neurobiology, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108, and (3)Calciosignal Net Project, Exploratory Research for Advanced Technology (ERATO), 2-9-3 Shimo-meguro, Meguro-ku, Tokyo 153, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Synaptotagmins I and II are Ca- and phospholipid-binding proteins of synaptic vesicles that may function as Ca receptors for neurotransmitter release via their first C2 domains. Herein, we describe the phospholipid binding properties of C2A domains of multiple synaptotagmins (II-VI). We demonstrate that all synaptotagmins can bind negatively charged phospholipids (phosphatidylserine (PS) and phosphatidylinositol (PI)) in a Ca-dependent manner, although it was previously reported that synaptotagmins IV and VI do not bind phospholipids. The Ca-dependent interaction of the C2A domain of synaptotagmin IV with PS was found to have two components with EC values of approximately 5 and 120 µM free Ca and exhibited positive cooperativity (Hill coefficient of approximately 2 for both components). This value is lower than that of the C2A domain of synaptotagmin II (Hill coefficient of approximately 3). All other isoforms bound PS with high affinity (EC of 0.3-1 µM free Ca; Hill coefficient of 3-3.5). In addition, the C2A domain of synaptotagmin IV cannot bind liposomes consisting of PS (or PI) and phosphatidylcholine, PC (or phosphatidylethanolamine, PE) (1:1, w/w), indicating that the binding to negatively charged phospholipids is inhibited by the presence of PC or PE. In contrast, other isoforms bound all of the liposomes, which include either PS or PI, in a Ca-dependent manner. Mutational analysis indicated that this phospholipid composition-dependent Ca binding of synaptotagmin IV results in the substitution of Asp for Ser at position 244. The cytoplasmic domain of synaptotagmin IV also shows this unique phospholipid binding. However, it binds PS with a positive cooperativity and an affinity similar to those of the C2A domains of other isoforms. Our results suggest that synaptotagmin IV is also a potential Ca sensor for neurotransmitter release.


INTRODUCTION

Synaptotagmins constitute a family of vesicle membrane proteins that are characterized by a short intravesicular amino terminus, a single transmembrane region, and a larger cytoplasmic carboxyl terminus containing two copies of highly conserved repeats homologous to the C2 regulatory region of protein kinase C(1) . At least nine separate synaptotagmin isoforms have been identified in rodents(1, 2, 3, 4, 5, 6) and three (o-p65-A, -B, and -C) in the electric ray(7) . The role of synaptotagmin I (the best characterized form) in Ca-regulated exocytosis has been demonstrated in microinjection experiments utilizing neural cells (8, 9) and by studies of null mutants of Caenorhabditis elegans(10) , Drosophila(11, 12) , and mice(13) . However, whether all other isoforms can also mediate Ca-regulated exocytosis remains unknown.

Recently, we showed that two C2 domains of synaptotagmin have different functions in synaptic vesicle trafficking by injecting domain-selective antibodies against two C2 domains into the squid giant preterminal(14, 15) , superior cervical ganglion cells(27) , and chromaffin cells(28) . 1) The C2A domain functions as a Ca sensor in exocytosis because anti-C2A IgG, which inhibits Ca/phospholipid binding to the C2A domain, blocks synaptic vesicle fusion(14, 27, 28) . 2) The inositol high polyphosphate series (inositol 1,3,4,5-tetrakisphosphate, inositol 1,3,4,5,6-pentakisphosphate, and inositol 1,2,3,4,5,6-hexakisphosphate) blocks synaptic transmission by binding to the C2B domain(15, 16, 17, 18, 27, 28) . The C2B domain is also involved in endocytosis, probably by binding the clathrin assembly protein, AP2, to the C2B domain because anti-C2B IgG blocks synaptic vesicle uptake(15, 19, 27) .

Previously, we demonstrated functional differences in the C2B domains of multiple synaptotagmins in terms of inositol high polyphosphate binding(20) . We then studied whether or not the C2A domains of multiple synaptotagmins are diversified. The Ca/phospholipid binding properties of the C2A domains of multiple synaptotagmins have been compared by Ullrich et al.(21) and Li et al.(6) , and synaptotagmins IV, VI, and VIII were described as Ca-insensitive isoforms. However, these were determined under very limited conditions (tested only using liposomes consisting of phosphatidylserine and phosphatidylcholine (1:2.5, w/w)). In this study, we examined the phospholipid selectivity of the C2A domains of synaptotagmins II-VI (three neuronal types and two non-neuronal types). Although all of the C2A domains can bind acidic phospholipids such as phosphatidylserine and phosphatidylinositol, synaptotagmin IV shows unique phospholipid composition-dependent Ca/phospholipid binding. We also determined the amino acid residues that account for this unique phospholipid binding by mutational analysis. On the basis of these results, we discuss the role of synaptotagmin IV in Ca-regulated exocytosis.


EXPERIMENTAL PROCEDURES

Chemicals

Phosphatidylserine (PS), (^1)phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylethanolamine (PE) were from Sigma. All other chemicals were commercial products of reagent grade. Solutions were prepared in deionized water.

Molecular Cloning of Mouse Synaptotagmins V and VI

The cDNAs encoding the C2A domains of synaptotagmins V (amino acids 105-234) and VI(227-357) from the mouse cerebellum were amplified by the reverse transcriptase polymerase chain reaction (PCR) (Ex Taq, Takara Shuzo) for 40 cycles, each consisting of denaturation at 94 °C for 1 min, annealing at 50 °C for 2 min, and extension at 72 °C for 3 min (sense 5`-GCGGATCCGACAAACACCAGCTAGGCCG-3` and antisense 5`-GCGAATTCTTGGGAGCCTCCTGCAGCTC-3`; sense 5`-CGGATCCGCCGCCAAGAGCTGTGGGAA-3` and antisense 5`-CGAATTCAGTAGCGTACTGGATGTCCT-3`, respectively, designed on the basis of the mouse (^2)and rat sequences(6) . After digestion with BamHI and EcoRI, the PCR product was purified on an agarose gel and extracted with a Geneclean Kit II (BIO 101, Inc.) and then inserted into the BamHI-EcoRI site of pGEX-2T vector (Pharmacia Biotech Inc.). Both strands were completely sequenced by using a BcaBEST dideoxy sequencing kit (Takara Shuzo). As compared with the rat sequences, Val at position 231 was altered to Glu in mouse synaptotagmin V(5) .

Preparation of GST Fusion Proteins

Construction of pGEX-2T vectors carrying fragments of several synaptotagmins proceeded as described by Fukuda et al.(17) . Glutathione S-transferase (GST) fusion proteins were expressed and purified on glutathione-Sepharose. GST fusion proteins of mouse synaptotagmins I-IV (GST-STI-IV-C2A) used in this study were also prepared as described(20) . GST-STV-C2A and -STVI-C2A encoded amino acids 105-234 and 227-357 of mouse synaptotagmin V and VI, respectively. GST-STIII encoded amino acids 76-587 of mouse synaptotagmin III, and GST-STIV encoded amino acids 39-425 of mouse synaptotagmin IV.

Site-directed mutagenesis of GST-STII-C2A(D231N), (D231S), GST-STIV-C2A(S244D) and deletion of GST-STII-C2A(Delta180-183) proceeded by means of two-step PCR as follows(20) . In GST-STII-C2A (D231S) for example, the right and left halves of the C2A domain were separately amplified with two pairs of oligonucleotides (primer A, 5`-CGGGATCCGAGCCGGAGAACCTGGGCAA-3` and mutagenic primer C, 5`-CACTAGTGTCTTGCCTCCTAA-3` (right half); mutagenic primer D, 5`-CACTAGTGATGGCAATCTATAGCTTTG-3` and primer B, 5`-CGGAATTCTCTCCGCCTTGTAGGTC-3` (left half)). The two resulting PCR fragments were digested with SpeI (underlined), ligated to each other, and reamplified with primers A and B. The obtained PCR fragment encoding the mutant C2A domain of synaptotagmin II (D231S) was subcloned into the BamHI-EcoRI site of pGEX-2T and verified by DNA sequencing.

By means of the same two-step PCR that introduced an artificial SpeI site into the C2A domain of synaptotagmins, we produced chimera from synaptotagmins II and III. GST-STII/III-C2A contained amino acids 139-177 of mouse synaptotagmin II followed by amino acids 332-421 of mouse synaptotagmin III. GST-STIII/II-C2A contains amino acids 290-330 of mouse synaptotagmin III followed by amino acids 177-267 of mouse synaptotagmin II.

Phospholipid Binding Assay

Ten kinds of liposomes were prepared in 50 mM HEPES-KOH (pH 7.2) and 100 mM NaCl by sonication, collected by centrifugation, and equilibrated with 50 mM HEPES-KOH (pH 7.2) in the presence of 2 mM EGTA or various concentrations of Ca-EGTA buffers, which were made with 0.1 M EGTA and 0.1 M CaCl(2) standard solution (Nacalai Tesque, Kyoto). GST fusion proteins (5-10 µg) were incubated with liposomes corresponding to 160 µg of phospholipid for 15 min at room temperature(17) . After centrifugation at 12,000 times g for 10 min at room temperature, the phospholipid pellets were washed in 500 µl of the above equilibration buffer and then extracted with 300 µl of acetone at -20 °C for 30 min to remove excess lipid. The pellets obtained by centrifugation at 12,000 times g for 15 min at 4 °C were dissolved in SDS sample buffer. The proteins in the supernatants were precipitated by adding an equal volume of 20% trichloroacetic acid. After a 15-min incubation on ice, the samples were centrifuged at 12,000 times g for 15 min at 4 °C, and the precipitates were mixed with SDS sample buffer. Equal proportions of the supernatants and pellets were analyzed by 10% SDS-polyacrylamide gel electrophoresis, followed by Coomassie Brilliant Blue R-250 staining. The proportion of the phospholipid binding fractions was quantified using a Bio Image (Millipore). The protein concentrations were determined by the Bio-Rad protein assay kit using bovine serum albumin as a reference.


RESULTS

Phospholipid Dependence of Ca-dependent Liposome Binding to the C2A Domains of Multiple Synaptotagmins

To determine whether the C2A domains of multiple synaptotagmins are functionally similar, we examined the Ca-dependent phospholipid binding properties of the C2A domains of synaptotagmins II-VI (Fig. 1). All of the C2A domains fused to GST bound to acidic phospholipids (PS or PI) in a Ca-dependent manner, as exemplified by GST-STI-C2A(22) . In contrast, there was no interaction with PC or PE (synaptotagmins II, III, and IV) and only a weak interaction with PE (synaptotagmin V) or PC (synaptotagmin VI). Our results were completely different from those of Ullrich et al.(21) ; the C2A domain of synaptotagmin IV did not show Ca/phospholipid binding. Since 1 mM Mg did not activate phospholipid binding to GST-STIV-C2A (data not shown), our results cannot be attributed to a nonspecific ionic interaction. To clarify this discrepancy, we performed a phospholipid binding assay under the conditions described by Ullrich et al.(21) using ^3H-labeled liposomes (PS/PC; 1:2.5, w/w) and confirmed their results (data not shown). The observed differences seemed to be derived from the composition of the liposomes used in the assays. GST-STII-C2A bound PS liposomes (Fig. 2, open circles) with high affinity (EC of approximately 0.4 µM free Ca) and exhibited strong positive cooperativity (Hill coefficient of approximately 3), although in a high Ca concentration range (0.2-1 mM) phospholipid binding was suppressed as compared with the low Ca concentration range. In contrast, GST-STIV-C2A bound PS with lower affinity and exhibited biphasic curves (EC of approximately 5 and 120 µM free Ca and a Hill coefficient of approximately 2 for both components; Fig. 2, open triangles). The phospholipid binding capacity of GST-STIV-C2A to PS/PC liposomes (PS/PC; 1:2.5, w/w) was completely lost, whereas GST-STII-C2A continued to show Ca-dependent phospholipid binding with a slightly lower affinity than that for PS liposomes alone (EC of approximately 1.5 µM free Ca and a Hill coefficient of approximately 3; Fig. 2, closed circles).


Figure 1: Ca-dependent phospholipid binding to the C2A domains of synaptotagmins II-VI fused to GST. Liposomes and GST fusion proteins were incubated for 15 min at room temperature in 50 mM HEPES-KOH (pH 7.2) in the presence of 2 mM EGTA or 1 mM Ca. After centrifugation at 12,000 times g for 10 min, the pellets (P; phospholipid binding fraction) and supernatants (S; nonbinding fraction) were separated as described under ``Experimental Procedures.'' Equal proportions of the supernatants and pellets were resolved by 10% SDS-polyacrylamide gel electrophoresis. Note that all of the C2A domains from synaptotagmins can bind PS and PI, which are negatively charged phospholipids, but essentially not PE and PC. GST-STV-C2A and GST-STVI-C2A weakly interacted with PE and PC, respectively.




Figure 2: Ca concentration dependence of phospholipid binding to the C2A domains of synaptotagmins II and IV. Binding of PS or PS/PC (1:2.5, w/w) liposomes to GST-STII-C2A (open and closed circles, respectively) or to GST-STIV-C2A (open and closed triangles, respectively) was examined as a function of the free Ca concentration using Ca/EGTA buffers. Phospholipid binding was measured as described in the legend to Fig. 1. The proportion of the phospholipid binding fractions was quantified using a Bio Image (Millipore). GST-STII-C2A binds both PS and PS/PC (1:2.5) liposomes with high affinity (EC of approximately 0.4 and 1.5 µM free Ca, respectively) and with strong positive cooperativity (Hill coefficient of approximately 3). GST-STIV-C2A also binds PS liposomes alone with two binding constants (EC of approximately 5 and 120 µM free Ca) but cannot bind PS/PC (1:2.5) liposomes. Note that within a high Ca concentration range (0.2-1 mM), the phospholipid binding properties of GST-STII-C2A were suppressed. The data are means ± S.E. of three measurements.



To examine further the effects of the phospholipid composition, six kinds of liposomes (PS/PE, PS/PC, PS/PI, PI/PE, PI/PC, PE/PC; 1:1, w/w) were tested. GST-STII, V, VI-C2A bound liposomes containing negatively charged phospholipids (PS or PI) in a Ca-dependent manner but did not bind PE/PC liposomes (Fig. 3, left and data not shown). In addition, GST-STIII-C2A showed Ca-independent binding to PS/PE or PI/PE liposomes (combination of negatively charged phospholipids with PE, Fig. 3, middle). This unique binding may be due to one-third of the amino terminus of the C2A domain of synaptotagmin III, a region that is different from those of other isoforms (Fig. 4). Chimeric analysis between synaptotagmins II and III confirmed these predictions. GST-STIII/II-C2A, which carries one-third of the amino terminus of the C2A domain of synaptotagmin III fused to two-thirds of the carboxyl terminus of the C2A domain of synaptotagmin II, showed Ca-independent binding to PS/PE liposomes, whereas GST-STIII/II-C2A, which is a reverse chimera of GST-STII/III-C2A, showed Ca-dependent binding to PS/PE liposomes (data not shown). However, since the entire cytoplasmic domain of synaptotagmin III (GST-STIII) lacks this Ca-independent binding to PS/PE liposomes (data not shown), these unique binding properties of GST-STIII-C2A may result from deletion of the C2A domain from the entire protein. In contrast, GST-STIV-C2A binds only PS/PI liposomes (Fig. 3, right), indicating that the binding of GST-STIV-C2A to PS or PI is restricted by the presence of PC or PE.


Figure 3: Phospholipid composition dependence of liposome binding to the C2A domains of synaptotagmins II, III, and IV. GST fusion proteins were incubated in the presence of 2 mM EGTA or 1 mM Ca with liposomes composed of PS mixed with PE (referred to as PS/PE; 1:1, w/w), PS/PC, PS/PI, PI/PE, PI/PC, and PE/PC. GST-STII, V, VI-C2A bound liposomes containing either PS or PI (left and data not shown). GST-STIII-C2A shows Ca-independent binding to PS/PE or PI/PE liposomes (middle). GST-STIV-C2A only can bind PS/PI liposomes (right), indicating that the binding of GST-STIV-C2A to PS or PI is inhibited by the coexistence of PC or PE. Abbreviations are the same as those for Fig. 1.




Figure 4: Sequence comparison of the C2A domain of synaptotagmins I-IV. Alignment of the first C2 domains of murine synaptotagmins (Syt) I-VI. Residues that are identical in the four sequences are shaded, and conserved residues in all sequences are shown by bold letters. Residue numbers are given on both sides. # and * indicate the essential aspartate residues for Ca binding to the C2A domain of synaptotagmin I as shown by crystallographic analysis(23) . Note that the C2A domain of synaptotagmin IV exhibits an aspartate to serine substitution (arrowhead). The sequence of mouse synaptotagmins I and II are from (17) ; mouse synaptotagmin III was from (20) ; mouse synaptotagmin IV was from (4) ; rat synaptotagmin V was from (5) ; and rat synaptotagmin VI was from (6) .



Mutational Analysis of Phospholipid-binding Domains of Synaptotagmins

Recently, Sutton et al.(23) reported the three-dimensional structure of the C2A domain of synaptotagmin I and identified 4 aspartate residues (amino acid positions at 172, 178, 230, and 232) that are important for Ca binding. These 4 residues are highly conserved among synaptotagmin isoforms (Fig. 4), although in synaptotagmin IV only 1 aspartate residue at position 244 was found to be replaced by serine. Thus, this substitution must be considered a prime candidate as the cause of the unique phospholipid binding properties of synaptotagmin IV. In the first set of experiments, we produced a mutant synaptotagmin II to determine whether it showed phospholipid composition-dependent Ca/phospholipid binding. As shown in Fig. 5, GST-STII-C2A(Delta180-183), which has a four-amino acid deletion at position 180-183 (Pro-Tyr-Val-Lys) just behind the first Ca binding loop(23) , completely lacked phospholipid binding capacity. GST-STII-C2A(D231S) and (D231N) showed phospholipid composition-dependent binding to liposomes, as was the case with GST-STIV-C2A. Their Ca/phospholipid (PS) binding curves were similar to that of GST-STIV-C2A (EC of 3-6 and 120-150 µM free Ca, data not shown). These observations indicate that the substitution of Ser for Asp is sufficient for phospholipid composition-dependent and low affinity Ca/phospholipid binding. In the second set of experiments, we attempted to restore the limited phospholipid binding capacity of the C2A domain of synaptotagmin IV by mutational analysis. GST-STIV-C2A(S244D) bound both PS and PS/PC liposomes Ca dependently with a positive cooperativity and an affinity similar to those of GST-STII-C2A (Fig. 5, bottom). These results indicate that the Ser at position 244 is involved in the unique phospholipid binding properties of synaptotagmin IV.


Figure 5: Mutational analysis of Ca/phospholipid-binding domain of synaptotagmins. Phospholipid binding properties of mutant synaptotagmin in the presence of 2 mM EGTA or 1 mM Ca except for GST-STIV-C2A binding to PS/PC, which was measured at a free Ca concentration of 100 µM. GST-STII-C2A(Delta180-183) completely lacked the phospholipid binding capacity, whereas GST-STII-C2A(D231N) and (D231S) showed composition-dependent phospholipid binding properties similar to those of GST-STIV-C2A as shown in Fig. 2(open triangles). GST-STIV-C2A(S244D) bound both PS and PS/PC liposomes with high affinity (EC of 0.3-0.6 and 2-4 µM free Ca, respectively). The EC values were estimated by plotting the same graphs as shown in Fig. 2. dash, not determined because of a lack of phospholipid binding activity. Abbreviations are the same as those for Fig. 1.



Phospholipid Composition-dependent Ca/Phospholipid Binding of the Cytoplasmic Domain of Synaptotagmin IV

We further examined the phospholipid binding properties of the cytoplasmic domain of synaptotagmin IV fused to GST (referred to as GST-STIV). GST-STIV showed phospholipid composition-dependent binding properties similar to those of GST-STIV-C2A (Fig. 6A) but bound PS with high affinity and strong positive cooperativity (EC of 0.06-0.1 µM free Ca; Hill coefficient of 3-3.5, data not shown). These discrepancies seemed to be attributable to the C2A domain not folding properly when expressed only as the C2A domain or to the C2B domain of synaptotagmin IV being involved in Ca-dependent phospholipid binding and positive cooperativity. As shown in Fig. 6A, GST-STIV can bind the PS liposomes but not the PS/PC liposomes, in which PS and PC appear to be uniformly distributed. However, GST-STIV bound the ``PS+PC'' liposomes, in which PS and PC appear to be distributed in patches. These results indicate that, if a cluster of positively charged phospholipids is present in the membranes, as shown in Fig. 6B(b), synaptotagmin IV can bind phospholipid membranes in a Ca-dependent manner.


Figure 6: Ca-dependent phospholipid binding to the cytoplasmic domain of synaptotagmin IV. A, GST-STIV, containing the entire cytoplasmic domain of synaptotagmin IV, shows phospholipid composition-dependent binding properties similar to those of GST-STIV-C2A as shown in Fig. 3. Phospholipid binding to GST-STIV was also assayed as described in the legend to Fig. 1in the presence of 2 mM EGTA or 1 mM Ca. P, pellet; S, supernatant. PS+PC indicates that PS and PC liposomes were mixed well after separate sonication. PS/PC means that PS and PC dissolved in chloroform were mixed before sonication. Note that GST-STIV can bind PS+PC liposomes Ca dependently but not PS/PC liposomes. B, schematic representation of the limited phospholipid binding of synaptotagmin IV. Synaptotagmin IV cannot bind the membranes in which PS and PC are uniformly distributed (a) but can bind those in which PS is distributed in patches (b). closed circles, PS or PI; open circles, PC or PE; TM, transmembrane.




DISCUSSION

Herein, we have demonstrated that the C2A domains of multiple synaptotagmins (II-VI) can selectively bind negatively charged phospholipids (PS and PI). Synaptotagmin IV was found to have the following unique phospholipid binding capacity. (i) The cytoplasmic domain of synaptotagmin IV, including both C2A and C2B domains, bound PS with a positive cooperativity and an affinity similar to those of the C2A domain of synaptotagmin II. However, when deleted from the full-length protein, the C2A domain of synaptotagmin IV weakly interacted with negatively charged phospholipid (EC of approximately 5 and 120 µM free Ca) and lost the high cooperativity. (ii) Synaptotagmin IV showed phospholipid composition-dependent Ca/phospholipid binding, indicating that the Ca/phospholipid binding capacities of synaptotagmin IV are restricted by the composition of the phospholipid in the membrane. A sequence comparison of the C2A domains of synaptotagmins I-VI (Fig. 4) and mutational analysis (Fig. 5) indicated that these unique binding properties of synaptotagmin IV are attributable to the Ser residue at position 244, which is located in the putative second Ca binding loop(23) . In addition, the first putative Ca binding loop of the C2A domain of synaptotagmin IV is one amino acid longer and has sequences that are different from those of the other isoforms (between # in Fig. 4). Synaptotagmin VI, which is reportedly a Ca-insensitive isoform(6) , showed the same Ca/phospholipid binding properties as that of synaptotagmin II, at least under our binding conditions. These results are also supported by the observation that synaptotagmin VI has 4 conserved Asp residues responsible for Ca binding (# and * in Fig. 4).

Rather than Ca/phospholipid binding, considerable research has been focused on the Ca-dependent interaction of synaptotagmin I with syntaxin I(6, 24) , because its effective concentration (about 200 µM) corresponds to the Ca concentration required for neurotransmitter release (25) . Therefore, it is likely that within a low Ca concentration range (10-10M), synaptotagmin binds phospholipid membranes, whereas in a high Ca concentration range (10-10M), it interacts with syntaxin more efficiently than with phospholipid because the phospholipid binding capacity is suppressed at a high Ca concentration (Fig. 2). At present, we do not know whether these two Ca-dependent interactions are essential for neurotransmitter release. This is the first report, to our knowledge, showing the unique phospholipid composition-dependent binding of synaptotagmin IV. Furthermore, although the C2A domain of synaptotagmin IV fused to GST did not bind syntaxin I(6) , we believe that its Ca-dependent interaction with the syntaxin family remains to be fully elucidated because GST-STIV-C2A (amino acids 151-281) and GST-STIV (amino acids 39-425) have markedly different affinities for Ca in terms of phospholipid binding.

Synaptotagmin IV is distributed at low although uniform levels throughout the brain(21) , and little attention has been focused on this isoform as compared with synaptotagmin I. However, Vician and co-workers (26) reported that synaptotagmin IV is an immediate early gene induced by depolarization in the brain. After kainic acid-induced seizures in rats, synaptotagmin IV mRNA levels are elevated in the hippocampus and piriform cortex, whereas synaptotagmin I mRNA is only slightly decreased. On the basis of these results, together with our own observations, we propose that synaptotagmin IV is involved in Ca-dependent presynaptic events.


FOOTNOTES

*
This work was supported in part by grants from the Japanese Ministry of Education, Science and Culture (to K. M.) and the Japan Society for the Promotion of Science (to M. F.). 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: Molecular Neurobiology Laboratory, Tsukuba Life Science Center, The Institute of Physical and Chemical Research (RIKEN), 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-36-9170; Fax: 81-298-36-9040; fukuda{at}rtc.riken.go.jp.

(^1)
The abbreviations used are: PS, phosphatidylserine; PI, phosphatidylinositol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PCR, polymerase chain reaction; GST, glutathione S-transferase; STI-VI, synaptotagmin I-VI.

(^2)
M. Fukuda and K. Mikoshiba, unpublished data.


ACKNOWLEDGEMENTS

We thank Drs. Tohru Yoshioka and Jun Aruga for advice, Drs. Akihiro Mizutani and Michio Niinobe for critical reading of the manuscript, and Kuniko Takahashi and Masako Suenaga for technical assistance. We are grateful to members of the Mikoshiba Laboratory for valuable discussions.


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