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Synaptotagmin-like Protein 1-3: A Novel Family of C-Terminal-Type Tandem C2 Proteins
Biochemical and Biophysical Research Communications 281, 1226 –1233 (2001) doi:10.1006/bbrc.2001.4512, available online at http://www.idealibrary.com on
Synaptotagmin-like Protein 1-3: A Novel Family of C-Terminal-Type Tandem C2 Proteins Mitsunori Fukuda* ,1 and Katsuhiko Mikoshiba* ,† *Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; and †Division of Molecular Neurobiology, Department of Basic Medical Science, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received February 6, 2001
Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca 2ⴙdependent isoform, but Slp1 and Slp2 are Ca 2ⴙindependent isoforms, because only the Slp3 C2A domain showed Ca 2ⴙ-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin I␣ in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages. © 2001 Academic Press Key Words: C2 domain; granuphilin; Doc2; rabphilin; phospholipid binding.
The nucleotide sequence reported in this paper is deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases with Accession Nos. AB050741–AB050743. This work was supported in part by grants from the Science and Technology Agency to Japan (to K.M.) and Grants 11780571 and 12053274 from the Ministry of Education, Science, and Culture of Japan (to M.F.). Abbreviations used: GST, glutathione S-transferase; HRP, horseradish peroxidase; NxI␣, neurexin I␣; PC, phosphatidylcholine; PCR, polymerase chain reaction; PS, phosphatidylserine; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; Slp, synaptotagmin-like protein; Syt(s), synaptotagmin(s). 1 To whom correspondence should be addressed at Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: ⫹81-48-467-9744. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
The C2 domain, a conserved protein motif that consists of approximately 130 amino acids, was originally defined as a homologous domain to the C2 regulatory region of protein kinase C (reviewed in Refs. 1 and 2). The C2 domain is now known to be present in various molecules including kinases, GTPase-activating proteins, ubiquitination enzymes and proteins that are involved in vesicular trafficking. The number of C2 domains in one molecule varies from one to more than five (see http://smart.embl-heidelberg.de/). Although prototypical C2 domains bind Ca 2⫹ and allow proteins to translocate phospholipid membranes, some C2 domains (e.g., synaptotagmin family) function as protein interaction sites, and some fail to exhibit Ca 2⫹ binding due to amino acid substitutions (reviewed in Ref. 3). Among the various C2 domain-containing proteins, tandem C2 domains (named C2A and C2B domains), which are separated by a short linker (less than 50 amino acids), are often found in proteins that are involved in vesicular trafficking (reviewed in Ref. 4). To date, five different families of tandem C2 proteins have been reported (Fig. 1A, B), and they are structurally divided into two classes. The N-terminal type (named N-type; Fig. 1A), where the tandem C2 domains are located at the N-terminus, includes the GAP1 family (5–9) and copine family (10, 11). The C-terminal type (named C-type; Fig. 1B), where the tandem C2 domains are located at the C-terminus, includes the synaptotagmin (Syt) family (reviewed in Refs. 3, 4, 12, 13), rabphilin-3A/granuphilin-a (14, 15), and Doc2 family (16 –19), and they are suggested to be involved in vesicular trafficking. These protein families are distinguished from one another by unique N-terminal domains; a transmembrane domain (TM) in the Syt family, a rab3A-binding domain in rabphilin-3A, and a Munc13-1-interacting domain (Mid) in the Doc2 family. Three proteins, Syt I, rabphilin-3A and Doc2␣, have been identified on synaptic vesicles, and were shown to be involved in the regulation of synaptic vesicle cycles in neurons and secretory vesicle exocyto-
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sis in some endocrine cells (20 –34). Syt I, an integral membrane protein of synaptic vesicles, is essential for the fusion of the synaptic vesicle with the presynaptic plasma membrane, probably functioning as a Ca 2⫹sensor via the C2A domain (24, 25, 27). By contrast, rabphilin-3A and Doc2␣ play a regulatory role in a step before the final fusion of the synaptic vesicle with the presynaptic plasma membrane (31–34). In this paper, we report a novel family of C-type tandem C2 proteins which lack a transmembrane domain, designated Slp1-3 (synaptotagmin-like protein 1-3). Slp1-3 also has tandem C2 domains at the C-terminus, but the N-terminal domains of Slp1-3 have no apparent homology with one another. We compared the biochemical properties of the Slp C2 domains and C-terminus to those of the Syt family, and examined the tissue distribution of Slp1-3 mRNA expression. MATERIALS AND METHODS Materials. Ex Taq and recombinant Taq DNA polymerases were obtained from Takara Shuzo Co., Ltd. (Shiga, Japan) and Toyobo Biochemicals (Tokyo, Japan), respectively. Polyclonal antibody against FLAG peptide was obtained from Zymed Laboratories, Inc. (San Francisco, CA). Horseradish peroxidase (HRP)-conjugated anti-T7 tag antibody and anti-T7 tag antibody-conjugated agarose were from Novagen (Madison, WI). L-␣-Phosphatidylcholine, dipalmitoyl (PC), and L-␣-phosphatidylserine, dioleoyl (PS) were from Sigma (St. Louis, MO). All other chemicals were commercial products of reagent grade. Solutions were made up in deionized water prepared with an Elix10 Water Purification System and Milli-Q Biocel A10 System (Millipore Corp.; Bedford, MA). Molecular cloning of mouse Slp1-3. cDNAs encoding the open reading frame of mouse Slp1-3 were amplified from Marathon-Ready mouse adult brain cDNA by 5⬘ (or 3⬘)-rapid amplification of cDNA ends (RACE) (Clontech Laboratories, Inc., Palo Alto, CA) as described previously (19). The first 5⬘-RACE reactions were carried out using adapter primer 1 (5⬘-CCATCCTAATACGACTCACTATAGGGC-3⬘) and the following primers designed on the basis of the Slp1-3 sequences in the mouse EST sequence database (Accession Nos. AA763436, AA727330 and AW763978 for Slp1; AW580643 for Slp2; and AI019424 for Slp3): 5⬘-CTATGCCCTGGGGACCAGGTT-3⬘ (Slp1-C1 primer; antisense; amino acid residues 543–548), 5⬘-CATCTTCTCCCAGAGAGCAA-3⬘ (Slp2-C2 primer; antisense; amino acid residues 346 –352), 5⬘-CGGATCCAACCTAGAAGTGAAAGGTAGC-3⬘ (Slp2-C2A upper primer, sense; amino acid residues 67-73), or 5⬘-TCAGTGCAGGACGAGGGTCAT-3⬘ (Slp3-C1 primer; antisense; amino acid residues 388393). The second RACE reactions were carried out by using internal adapter primer 2 (5⬘-ACTCACTATAGGGCTCGAGCGGC-3⬘) and the following primers designed on the basis of the above mentioned mouse EST sequences: 5⬘-AGCCCATAACTGCTGCCTGTA-3⬘ (Slp1-C2 primer; antisense; amino acid residues 501–508), 5⬘AGGCTTCCATCAGATCTTCA-3⬘ (Slp2-C3 primer; antisense; amino acid residues 296 –303), 5⬘-CGGATCCGTTGCCCTCGAGACAGAAAAC-3⬘ (Slp2-C2B upper primer; sense; amino acid residues 204 –210), or 5⬘-GTCTGTCCACAGGTTTGGGCT-3⬘ (Slp3-C2 primer; antisense; amino acid residues 381–387). Both polymerase chain reactions (PCRs) were carried out in the presence of Perfect Match PCR Enhancer (Stratagene, La Jolla, CA) for 35 cycles, each consisting ofdenaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 3 min. The first and second PCR
products were purified from an agarose gel on a Micro-Spin column (Amersham Pharmacia Biotech; Buckinghamshire, UK) as described previously (35), and were directly inserted into the pGEM-T Easy vector (Promega; Madison, WI). Both strands of the cDNA inserts were randomly sequenced with a Hitachi SQ-5500 DNA sequencer until a cDNA clone including the putative initiation methionine or termination codons was obtained. Since two Met residues were present just downstream of the 3⬘-termination codon in Slp1 and Slp3, the translation initiation site was determined by expression of Slp1-T7 and Slp3-T7 in COS-7 cells. The translation of Slp1 and Slp3 proteins was confirmed to be initiated at first Met and second Met, respectively, in COS-7 cells (data not shown). In the course of screening Slp1-3 cDNAs, we found several alternative splice variants of Slp1-3 (data not shown). For example, the Slp2 cDNAs showed a 120-bp deletion which corresponded to 13–52 amino acids of the full-length Slp2. Expression constructs. Full-length Slp1-3 cDNAs including the open reading frame were constructed by using appropriate restriction enzyme sites on the pGEM-T Easy vector (named pGEM-T-Slp13). Addition of the T7 tag (or FLAG) to the N (or C) terminus of each Slp (T7-Slp1-3, FLAG-Slp1-3, and Slp1, 3-T7) was done by PCR using primers with a T7 tag sequence as described previously (35). In the case of Slp3-T7, for example, the Slp3-T7 fragment was amplified by Slp3-Met primer (5⬘-GCGGATCCATGGACCCCAGGCAGTGTGT-3⬘) and Slp3-T7 primer (5⬘-CTAACCCATTTGCTGTCCACCAGTCATGCTAGCCATCAATTGGTGCAGGACGAGGGTCATGTC-3⬘; T7-tag, italics; restriction enzyme sites, underlines; and stop codons, bold letters) using pGEM-T-Slp3 as a template. Purified PCR products were inserted into the pGEM-T Easy vector and verified by DNA sequencing. The full length Slp3 inserts with T7 tag were then excised from the pGEM-T Easy vector by NotI digestion and subcloned into the NotI site of a modified pEF-BOS mammalian expression vector (36, 37) (pEF-T7Slp1-3, -FLAG-Slp1-3, and pEF-Slp1, 3-T7). Other expression constructs (pEF-T7-Syt II) were also prepared as described previously (35). Plasmid DNA was prepared using Wizard-mini preps (Promega) or QIAGEN Maxi prep kits. Preparation of GST fusion proteins. Construction of pGEX-4T-3 (or pGEX-2T) vector (Amersham Pharmacia Biotech) carrying fragments of the Slp1-3 C2A or C2B domain were essentially performed by PCR (36) using pGEM-T-Slp1-3 as a template. The following pairs of oligonucleotides with appropriate restriction enzyme sites (underlined) and/or termination codons (bold letters) were used for amplification: Slp1-C2A upper primer (sense) 5⬘-GCGGATCCACGGTGCAGGTGCGAGGCTC-3⬘ and Slp1-C2A lower primer (antisense) 5⬘-GCACTAGTCAAGATGGAGGAACCCGAGGCT-3⬘; Slp1C2B upper primer (sense) 5⬘-GCAGATCTTCTCCAGATGAGCTTCCCAG-3⬘ and SP6 primer; Slp2-C2A upper primer described above and Slp2-C2A lower primer (antisense) 5⬘-GCACTAGTCAAACTGGTGCTGTCTTCCT-3⬘; Slp2-C2B upper primer described above and SP6 primer; Slp3-C2A upper primer (sense) 5⬘GCGGATCCAATGCTAATGTCACTGGAGA-3⬘ and Slp3-C2A lower primer (antisense) 5⬘-GCACTAGTCAAGCCTTGGCCCGCAGAGGAT-3⬘; and Slp3-C2B upper primer (sense) 5⬘-GCGGATCCGCTGAGAAATACGAAGAGAA-3⬘ and SP6 primer. The resulting pGEX-Slp1-3-C2A and -C2B were confirmed by DNA sequencing and transformed into Escherichia coli, JM109. Glutathione S-transferase (GST) fusion proteins were expressed and purified on glutathione Sepharose (Amersham Pharmacia Biotech) by the standard method (38). GST-Slp1-C2A and -Slp1-C2B encoded amino acids 268 –397 and 397–567 of mouse Slp1, respectively. GST-Slp2-C2A and -Slp2C2B encoded amino acids 67–204 and 204 –376 of mouse Slp2, respectively. GST-Slp3-C2A and -Slp3-C2B encoded amino acids 88 – 222 and 222–393 of mouse Slp3, respectively. GST-Syt I-C2A was prepared as described previously (36). Reverse transcriptase (RT)-PCR analysis. Mouse first-strand cDNAs prepared from various tissues and developmental stages
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were obtained from Clontech Laboratories (Mouse MTC Panel I). PCRs were carried out in the presence of Perfect Match PCR Enhancer (Stratagene) for 30 cycles, each consisting of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min. The C2B upper and C1 primers described above (Slp2 C1 primer, 5⬘-GCACTAGTCACTTGGAAAGCTTGGCAAT-3⬘) were used for amplification. The PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The authenticity of the products was verified by subcloning into a pGEM-T Easy vector and DNA sequencing. Sequence analyses. Multiple sequence alignment and depiction of the phylogenetic tree of the mouse synaptotagmin family were performed by using the CLUSTALW program (http://watson. genes.nig.ac.jp/homology/clustalw.shtml) set at the default parameters (gapopen ⫽ 10, gapext ⫽ 0.05, gapdist ⫽ 8, and maxdiv ⫽ 40). Miscellaneous procedures. Subcellular fractionation of COS-7 cells and in vitro neurexin I␣ (NxI␣) binding assay was performed as described previously (39, 40). Preparation of liposomes consisting of PC and PS (1:1, w/w) and phospholipid binding assay was also performed as described previously (41, 42). The protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a reference. Association of T7- and FLAG-Slps in COS-7 cells were evaluated by immunoprecipitation as described previously (35, 43, 44).
RESULTS AND DISCUSSION Molecular Cloning of Novel Synaptotagmin-Like Proteins (Slp1-3) To identify novel C-type tandem C2 proteins that may be involved in vesicular trafficking, we searched for tandem C2 proteins homologous to the mouse Syts I–XII C2 domains in a mouse EST sequence database by tfasta using the default parameters. Fragments of five distinct genes that encoded three novel tandem C2 domains, Doc2␥ (19), and Syt XIII (40) were found, and their full open reading frame was cloned from a mouse adult brain cDNA library by 5⬘-RACE (see Materials and Methods for details). The 1824-, 1313-, and 1332-base sequences contained a single open reading frame encoding 567, 376, and 393 amino acids with a calculated molecular weight of 62,349, 42,759, and 43,781, respectively (Fig. 1C). These three proteins contain tandem C2 domains (Fig. 2), but they have no hydrophobic region sufficient to be a transmembrane region (data not shown). Homology search analysis revealed that the C2 domains of these proteins showed higher homology to those of granuphilin-a than to those of other C-type tandem C2 proteins (more than 32%; Fig. 2). In addition, the short C-terminus of Slp1-3 and granuphilin-a are highly conserved and different from that of Syt family, Doc2 family and rabphilin-3A proteins (Fig. 2, underline). The length of the N-terminal domain varies among the three Slp proteins and they do not show any apparent homology to one another or to any known protein motifs (PIR and SWISS-PROT protein databases). How-
FIG. 1. Schematic representation of mouse tandem C2 protein family. (A) Schematic representation of N-terminal type (N-type) tandem C2 proteins: GAP1 family (5–9) and copine family (10, 11). (B) Schematic representation of C-terminal type (C-type) tandem C2 proteins reported to date: synaptotagmin family (3), rabphilin3A/granuphilin-a (14, 15), and Doc2 family (16 –19). (C) Schematic representation of novel C-type tandem C2 proteins determined in this study (Slp1-3). Solid line indicates the deletion by alternative splicing in Slp2. Amino acid numbers are given on both sides. GRD, GTPase-activating protein-related domain; PH, pleckstrin homology; TM, transmembrane domain; rab3A, rab3A binding domain; Mid, Munc13-1 interacting domain. (D) Phylogenetic tree of mouse C-type tandem C2 protein family reported to date. The phylogenetic tree is depicted as described under Materials and Methods. Note that the Slp family (boxed) form a distinct branch from the synaptotagmin, rabphilin-3A and Doc2 family.
ever, we found that the Slp1 N-terminal domain showed very weak similarity to granuphilin-a (13.6% identity and about 22% similarity at the N-terminus), but it lacks the conserved Cys residues responsible for Zn 2⫹ binding (45) in rabphilin-3A and granuphilin-a (data not shown). The overall amino acid identity between Slp1 and granuphilin-a was about 31%, which was much higher than that be-
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FIG. 2. Sequence alignment of the mouse C2 domains of C-type tandem C2 proteins. Sequence alignment of the mouse C2 domains of Syt I (Accession No. D37792), Syt B/K (unpublished), Slp1-3 (AB050741-3), granuphilin-a (AB025258), and Doc2␣ (D50000). Residues conserved or similar between sequences are shown on a black background or shaded background, respectively. Asterisks indicate the five conserved aspartate or glutamate residues, which may be crucial for Ca 2⫹ binding by analogy with the Syt I-C2A domain (52, 53). The number signs (#) indicate the key amino acids responsible for high affinity neurexin I␣ binding in vitro (58). The dashed lines and solid line indicate two C2 domains and a short carboxyl terminus, respectively.
tween granuphilin-a and rabphilin-3A (about 22%). Consistent with this, Slp1-3 and granuphilin-a form a small branch in the phylogenetic tree (Fig. 1D). Therefore, granuphilin-a belongs to the Slp family rather than the rabphilin-3A homologues. Membrane Association of Slp1-3 in COS-7 Cells Since Slp1-3 did not contain a transmembrane domain, we first examined whether Slp1-3 are soluble or membrane-associated proteins. To address this, subcellular fractionation of COS-7 cells expressing Slp1-3
was performed. All three proteins were recovered in the membrane fraction like Syt II, an integral membrane protein of synaptic vesicles (46), although half of the Slp1 proteins were also recovered in the soluble fraction (Fig. 3). Since Slp1-3 lack any apparent transmembrane domain, we treated the membrane fraction with 0.1 M Na 2CO 3, which should release most peripheral membrane proteins from the membranes. Under these conditions Slp1-3, but not Syt II, were released from the membrane fraction. Therefore, we concluded that Slp1-3 are peripheral, but not integral, membrane proteins.
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(PS/PC liposomes) binding assay was performed using GST-fusion proteins (Fig. 4). Unexpectedly, however, the Slp3-C2A domain bound PS/PC liposomes only in the presence of calcium, indicating that the Ca 2⫹binding pocket of the Slp3-C2A domain is different from that of Syt I. By contrast, none of the other C2 domains showed any phospholipid binding activity. Since none of the C2 domains of Slp showed any Ca 2⫹independent phospholipid binding activity, the membrane association of Slp1-3 in Fig. 3 can not be attributable to the phospholipid binding activity of the C2 domains. Oligomerization Properties of Slp1-3 FIG. 3. Subcellular fractionation of COS-7 cells expressing T7Slp1-3. Subcellular fractionation of COS-7 cells expressing pEF-Syt II (top panel), -Slp1 (second panel), -Slp2 (third panel), or -Slp3 (fourth panel). Membrane (M) and soluble (S) fractions were separated as described previously (39, 40). The membrane fraction was further resuspended in a buffer containing 0.1 M Na 2CO 3, pH 11 and incubated for 1 h at 4°C. After centrifugation at 100,000 ⫻ g for 1 h, the supernatants (Su) were recovered. Equal proportions of total (T), membrane, soluble, and supernatants treated with 0.1 M Na 2CO 3, pH 11 were subjected to 10% SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp.), followed by immunoblotting with HRP-conjugated anti-T7 tag antibody. The positions of the molecular weight markers (⫻ 10 ⫺3) are shown on the right. The results shown are representative of three independent experiments.
Recent genetic and biochemical studies indicate that Syt I functions as an oligomer in synaptic vesicle exo-
Phospholipid Binding Properties of Slp1-3 C2 Domains In the next set of experiments, we compared the biochemical properties of the Slp family to those of Syt family. First, we focused on the tandem C2 domains, because the C2 domain was originally found in Ca 2⫹dependent protein kinase C, and is thought to function as a phospholipid binding site (reviewed in ref. 2). In Syt I, a well characterized C-type tandem C2 protein, the C2A domain binds negatively-charged phospholipids in a Ca 2⫹-dependent manner, whereas the C2B domain binds phospholipids irrespective of the presence of Ca 2⫹ (36, 41, 47). The Ca 2⫹/phospholipid binding site of the C2A domain has been shown to be important for secretory vesicle exocytosis (25, 27–29), neurite outgrowth of chick dorsal root ganglion neurons and PC12 cells (48, 49), and axonal repair (50, 51). Crystallographic and mutational analysis of the Syt I C2A domain indicated that five aspartate (or glutamate) residues are essential for Ca 2⫹ binding (52, 53) (see Fig. 2, asterisks). Interestingly, Slp1-3 lack such aspartate or glutamate residues both in the C2A and C2B domains (Slp1-C2A, Arg at amino acid positions 302 (R302), H359, and I367, and Slp1-C2B, R443, G505, and Q511; Slp2-C2A, H162, S170, and Slp2-C2B, R250, N255, Y312, and F318; and Slp3-C2A, C128, H183, G185, and V191 and Slp3-C2B, S279, N284, and A341), suggesting that Slp1-3 should be classified as Ca 2⫹-independent types. To address this, phospholipid
FIG. 4. Phospholipid binding properties of Slp1-3 C2 domains. Liposomes and GST fusion proteins were incubated in 50 mM HepesKOH, pH 7.2, in the presence of 2 mM EGTA or 1 mM Ca 2⫹ for 15 min at room temperature. After centrifugation at 12,000g for 10 min, the supernatants (S, non-binding fraction) and pellets (P, phospholipid binding fraction) were separated as described previously (41, 42). Equal proportions of the supernatants and pellets were subjected to 10% SDS–polyacrylamide gel electrophoresis and then stained with Coomassie brilliant blue R-250. Except for the C2A domain of Slp3, the C2 domains of Slp1-3 did not show phospholipid binding activity. By contrast, the C2A domains of Syt I and Slp3 only bound liposomes in a Ca 2⫹-dependent manner. The bottom panel indicates the negative control, GST. The results shown are representative of three independent experiments.
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ing to speculate that this weak interaction with NxI␣ may result in only half of the Slp1 proteins being present in the membrane fraction. Differential Tissue Distribution of Slp1-3
FIG. 5. Neurexin I␣ binding property of Slp1-3. GST pull-down assay was performed as described previously (40). After immunoblotting with HRP-conjugated anti-T7 tag antibody (upper panels), blots were stained with amido black to ensure that equivalent amounts of GST fusion proteins were loaded (lower panels). T7- Slp1-3 or Syt VI⌬TM proteins were incubated with GST (lanes 1– 4) or GST-NxI␣ (lanes 5– 8), respectively. The 1/80 volume of total proteins used for assay were loaded on lanes 9 –12. Note that Slp1-3 did bind GSTNx1␣, but not GST alone, the same as Syt VI⌬TM. However, the Slp1-NxI␣ interaction is apparently weaker than others (lane 5). The results shown are representative of two independent experiments.
cytosis (22, 54 –56). In addition, Syt isoforms have been shown to form Ca 2⫹-dependent and -independent hetero-oligomers (35, 43, 44, 56). By contrast, Slp1-3 did not show any apparent homo- and heterooligomerization activities (data not shown), indicating that Slp1-3 is present at a monomer state in living cells. The C-Terminus of Slp1-3 Binds Neurexin I␣ in Vitro In a previous study, we showed that the conserved C-terminus of the synaptotagmin family is essential for plasma membrane association in PC12 cells (39, 57) and that the Syt I-C terminus is indeed involved in synaptic vesicle docking to presynaptic plasma membranes in the squid giant synapse (57). Since the polymorphic plasma membrane protein neurexin binds the conserved C-terminus of Syt family in vitro (58, 59), neurexin (I-III) is suggested to be a common plasma membrane receptor for Syt family. To further examine whether the Slp1-3 C-terminus is involved in membrane association, a neurexin I␣ binding assay was performed. Briefly, T7-Slp1-3 proteins were expressed in COS-7 cells and solubilized proteins were incubated with glutathione Sepharose coupled with GST-NxI␣ or GST alone. After extensive washing of the beads with incubation buffer, GST-NxI␣-bound proteins were analyzed by immunoblotting with HRP-conjugated anti-T7 tag antibody. As shown in Fig. 5, Slp1-3 bound GST-NxI␣, but not GST alone, the same as Syt VI⌬TM (lanes 5– 8). However, the Slp1-NxI␣ interaction was apparently weaker than the Slp2, 3-NxI␣ interactions (Fig. 5, compare lanes 5 and 6). Therefore, it is tempt-
Finally, the tissue distribution of mouse Slp1-3 was examined by RT-PCR with specific primers (Fig. 6). The tissue distribution of Slp1-3 were apparently different and their transcripts were not enriched in the brain. For example, the expression level of Slp1 mRNA was highest in lung, moderate in spleen, liver and kidney, lowest in heart, brain and skeletal muscle, and absent in testis. By contrast, the expression of Slp2 mRNA was almost complementary to that of Slp1: highest expression was seen in skeletal muscle, kidney, and testis, and moderate expression in heart, brain, and lung. Slp3 mRNA expression was found in spleen, lung, kidney and testis. In addition, the expression pattern of Slp1-3 during development was also different. The expression of Slp1 and Slp3 transcripts seemed to be highest on embryonic day 7 and then decreased to adult, whereas expression of Slp2 seemed to be constant from embryonic day 7 to 17. CONCLUSION In summary, we have isolated and characterized three novel C-type tandem C2 protein family proteins, named Slp1-3, which are differentially distributed in different mouse tissues and at different developmental stages. The Slp1-3 family shows the highest homology with granuphilin-a C2 domains (more than 32% identity) and form a distinct branch from Syt, rabphilin-3A and Doc2 family proteins. Like synaptotagmin and
FIG. 6. Differential tissue distribution of mouse Slp1-3. RT-PCR analysis of Slp1-3 expression in various tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis) and on embryonic days 7 (E7), 11, 15, and 17 (upper three panels). RT-PCR analysis of G3PDH expression was also performed (bottom panel) to ensure that equivalent amounts of first strand cDNA were used for RT-PCR analysis. ⫺ means without templates as a negative control. The size of the molecular weight markers (/StyI) is shown at the left of the panel. The results shown are representative of two independent experiments.
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Doc2 family proteins, the Slp family contains both Ca 2⫹-dependent (Slp3) and -independent (Slp1 and Slp2) isoforms. In addition, the C-terminus of Slp1-3 also binds neurexin I␣ in vitro the same as the synaptotagmin family, suggesting that Slp1-3 may also regulate docking of transport vesicles to plasma membrane. Since the Slp1-3 mRNAs are expressed in various tissues outside the brain, we speculate that the Slp1-3 proteins are involved in constitutive vesicle traffic and/or regulated exocytosis in endocrine (or exocrine) cells. Further work is necessary to elucidate whether the Slp family is indeed involved in vesicular trafficking. ACKNOWLEDGMENTS We thank Dr. Shigekazu Nagata for the expression vector, and Eiko Kanno for technical assistance.
REFERENCES 1. Ponting, C. P., and Parker, P. J. (1996) Protein Sci. 5, 162–166. 2. Nalefski, E. A., and Falke, J. J. (1996) Protein Sci. 5, 2375–2390. 3. Marque`ze, B., Berton, F., and Seagar, M. (2000) Biochimie 82, 409 – 420. 4. Su¨dhof, T. C., and Rizo, J. (1996) Neuron 17, 379 –388. 5. Maekawa, M., Li, S., Iwamatsu, A., Morishita, T., Yokota, K., Imai, Y., Kohsaka, S., Nakamura, S., and Hattori, S. (1994) Mol. Cell Biol. 14, 6879 – 6885. 6. Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527– 530. 7. Yamamoto, T., Matsui, T., Nakafuku, M., Iwamatsu, A., and Kaibuchi, K. (1995) J. Biol. Chem. 270, 30557–30561. 8. Baba, H., Fuss, B., Urano, J., Poullet, P., Watson, J. B., Tamanoi, F., and Macklin, W. B. (1995) J. Neurosci. Res. 41, 846 – 858. 9. Fukuda, M., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 18838 –18842. 10. Creutz, C. E., Tomsig, J. L., Snyder, S. L., Gautier, M. C., Skouri, F., Beisson, J., and Cohen, J. (1998) J. Biol. Chem. 273, 1393– 1402. 11. Nakayama, T., Yaoi, T., Yasui, M., and Kuwajima, G. (1998) FEBS Lett. 428, 80 – 84. 12. Fukuda, M., and Mikoshiba, K. (1997) BioEssays 19, 593– 603. 13. Schiavo, G., Osborne, S. L., and Sgouros, J. G. (1998) Biochem. Biophys. Res. Commun. 248, 1– 8. 14. Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, S., Yamaguchi, T., Wada, K., Miyazaki, M., and Takai, Y. (1993) Mol. Cell. Biol. 13, 2061–2068. 15. Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542–28548. 16. Orita, S., Sasaki, T., Naito, A., Komuro, R., Ohtsuka, T., Maeda, M., Suzuki, H., Igarashi, H., and Takai, Y. (1995) Biochem. Biophys. Res. Commun. 206, 439 – 448. 17. Sakaguchi, G., Orita, S., Maeda, M., Igarashi, H., and Takai, Y. (1995) Biochem. Biophys. Res. Commun. 217, 1053–1061. 18. Kojima, T., Fukuda, M., Aruga, J., and Mikoshiba, K. (1996) J. Biochem. (Tokyo) 120, 671– 676. 19. Fukuda, M., and Mikoshiba, K. (2000) Biochem. Biophys. Res. Commun. 276, 626 – 632.
20. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Su¨dhof, T. C. (1994) Cell 79, 717–727. 21. Nonet, M. L., Grundahl, K., Meyer, B. J., and Rand, J. B. (1993) Cell 73, 1291–1305. 22. Littleton, J. T., and Bellen, H. J. (1995) Trends Neurosci. 18, 177–183. 23. Elferink, L. A., Peterson, M. R., and Scheller, R. H. (1993) Cell 72, 153–159. 24. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163–165. 25. Mikoshiba, K., Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., and Llina´s, R. (1995) Proc. Natl. Acad. Sci. USA 92, 10703–10707. 26. Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llina´s, R. (1995) Proc. Natl. Acad. Sci. USA 92, 10708 –10712. 27. Mochida, S., Fukuda, M., Niinobe, M., Kobayashi, H., and Mikoshiba, K. (1997) Neuroscience 77, 937–943. 28. Ohara-Imaizumi, M., Fukuda, M., Niinobe, M., Misonou, H., Ikeda, K., Murakami, T., Kawasaki, M., Mikoshiba, K., and Kumakura, K. (1997) Proc. Natl. Acad. Sci. USA 94, 287–291. 29. Lang, J., Fukuda, M., Zhang, H., Mikoshiba, K., and Wollheim, C. B. (1997) EMBO J. 16, 5837–5846. 30. Thomas, D. M., and Elferink, L. A. (1998) J. Neurosci. 18, 3511– 3520. 31. Mochida, S., Orita, S., Sakaguchi, G., Sasaki, T., and Takai, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 11418 –11422. 32. Burns, M. E., Sasaki, T., Takai, Y., and Augustine, G. J. (1998) J. Gen. Physiol. 111, 243–255. 33. Schluter, O. M., Schnell, E., Verhage, M., Tzonopoulos, T., Nicoll, R. A., Janz, R., Malenka, R. C., Geppert, M., and Su¨dhof, T. C. (1999) J. Neurosci. 19, 5834 –5846. 34. Sakaguchi, G., Manabe, T., Kobayashi, K., Orita, S., Sasaki, T., Naito, A., Maeda, M., Igarashi, H., Katsuura, G., Nishioka, H., Mizoguchi, A., Itohara, S., Takahashi, T., and Takai, Y. (1999) Eur. J. Neurosci. 11, 4262– 4268. 35. Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421–31427. 36. Fukuda, M., Aruga, J., Niinobe, M., Aimoto, S., and Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206 –29211. 37. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5332. 38. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31– 40. 39. Fukuda, M., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31428 –31434. 40. Fukuda, M., and Mikoshiba, K. (2001) Biochem. J. 354, 249 –257. 41. Fukuda, M., Kojima, T., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 8430- 8434. 42. Fukuda, M., Kojima, T., and Mikoshiba, K. (1997) Biochem. J. 323, 421– 425. 43. Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180 –28185. 44. Fukuda, M., and Mikoshiba, K. (2000) J. Biochem. (Tokyo) 128, 637– 645. 45. Misra, S., and Hurley, J. H. (1999) Cell 97, 657– 666. 46. Niinobe, M., Yamaguchi, Y., Fukuda, M., and Mikoshiba, K. (1994) Biochem. Biophys. Res. Commun. 205, 1036 –1042. 47. Damer, C. K., and Creutz, C. E. (1994) J. Biol. Chem. 269, 31115–31123. 48. Fukuda, M., and Mikoshiba, K. (2000) Neurosci. Lett. 295, 33– 36.
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49. Kabayama, H., Takei, K., Fukuda, M., Ibata, K., and Mikoshiba, K. (1999) Neuroscience 88, 999 –1003. 50. Detrait, E., Eddleman, C. S., Yoo, S., Fukuda, M., Nguyen, M. P., Bittner, G. D., and Fishman, H. M. (2000) J. Neurobiol. 44, 382–391. 51. Detrait, E. R., Yoo, S., Eddleman, C. S., Fukuda, M., Bittner, G. D., and Fishman, H. M. (2000) J. Neurosci. Res. 62, 566 – 573. 52. Shao, X., Davletov, B. A., Sutton, R. B., Su¨dhof, T. C., and Rizo, J. (1996) Science 273, 248 –251. 53. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Su¨dhof, T. C., and Sprang, S. R. (1995) Cell 80, 929 –938.
54. Sugita, S., Hata, Y., and Su¨dhof, T. C. (1996) J. Biol. Chem. 271, 1262–1265. 55. Davis, A. F., Bai, J., Fasshauer, D., Wolowick, M. J., Lewis, J. L., and Chapman, E. R. (1999) Neuron 24, 363–376. 56. Fukuda, M., Kabayama, H., and Mikoshiba, K. (2000) FEBS Lett. 482, 269 –272. 57. Fukuda, M., Moreira, J. E., Liu, V., Sugimori, M., Mikoshiba, K., and Llina´s, R. R. (2000) Proc. Natl. Acad. Sci. USA 97, 14715– 14719. 58. Perin, M. S. (1996) Biochemistry 35, 13808 –13816. 59. Hata, Y., Davletov, B., Petrenko, A. G., Jahn, R., and Su¨dhof, T. C. (1993) Neuron 10, 307–315.
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Synaptotagmin-like Protein 1-3: A Novel Family of C-Terminal-Type Tandem C2 Proteins Mitsunori Fukuda* ,1 and Katsuhiko Mikoshiba* ,† *Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan; and †Division of Molecular Neurobiology, Department of Basic Medical Science, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
Received February 6, 2001
Synaptotagmins (Syt), rabphilin-3A, and Doc2 belong to a family of carboxyl terminal type (C-type) tandem C2 proteins and are thought to be involved in vesicular trafficking. We have cloned and characterized a novel family of C-type tandem C2 proteins, designated Slp1-3 (synaptotagmin-like protein 1-3). The Slp1-3 C2 domains show high homology to granuphilin-a C2 domains, but the amino-terminal domain of Slp1-3 does not contain any known protein motifs or a transmembrane domain. A subcellular fractionation study indicated that Slp1-3 proteins are peripheral membrane proteins. Phospholipid binding experiments indicated that Slp3 is a Ca 2ⴙdependent isoform, but Slp1 and Slp2 are Ca 2ⴙindependent isoforms, because only the Slp3 C2A domain showed Ca 2ⴙ-dependent phospholipid binding activity. The C-terminus of Slp1-3 also bound neurexin I␣ in vitro, in the same manner as Syt family proteins, which may be important for the membrane association of Slp1-3. In addition, Slp family proteins are differentially distributed in different mouse tissues and at different developmental stages. © 2001 Academic Press Key Words: C2 domain; granuphilin; Doc2; rabphilin; phospholipid binding.
The nucleotide sequence reported in this paper is deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases with Accession Nos. AB050741–AB050743. This work was supported in part by grants from the Science and Technology Agency to Japan (to K.M.) and Grants 11780571 and 12053274 from the Ministry of Education, Science, and Culture of Japan (to M.F.). Abbreviations used: GST, glutathione S-transferase; HRP, horseradish peroxidase; NxI␣, neurexin I␣; PC, phosphatidylcholine; PCR, polymerase chain reaction; PS, phosphatidylserine; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; Slp, synaptotagmin-like protein; Syt(s), synaptotagmin(s). 1 To whom correspondence should be addressed at Laboratory for Developmental Neurobiology, Brain Science Institute, RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. Fax: ⫹81-48-467-9744. E-mail: [email protected]. 0006-291X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
The C2 domain, a conserved protein motif that consists of approximately 130 amino acids, was originally defined as a homologous domain to the C2 regulatory region of protein kinase C (reviewed in Refs. 1 and 2). The C2 domain is now known to be present in various molecules including kinases, GTPase-activating proteins, ubiquitination enzymes and proteins that are involved in vesicular trafficking. The number of C2 domains in one molecule varies from one to more than five (see http://smart.embl-heidelberg.de/). Although prototypical C2 domains bind Ca 2⫹ and allow proteins to translocate phospholipid membranes, some C2 domains (e.g., synaptotagmin family) function as protein interaction sites, and some fail to exhibit Ca 2⫹ binding due to amino acid substitutions (reviewed in Ref. 3). Among the various C2 domain-containing proteins, tandem C2 domains (named C2A and C2B domains), which are separated by a short linker (less than 50 amino acids), are often found in proteins that are involved in vesicular trafficking (reviewed in Ref. 4). To date, five different families of tandem C2 proteins have been reported (Fig. 1A, B), and they are structurally divided into two classes. The N-terminal type (named N-type; Fig. 1A), where the tandem C2 domains are located at the N-terminus, includes the GAP1 family (5–9) and copine family (10, 11). The C-terminal type (named C-type; Fig. 1B), where the tandem C2 domains are located at the C-terminus, includes the synaptotagmin (Syt) family (reviewed in Refs. 3, 4, 12, 13), rabphilin-3A/granuphilin-a (14, 15), and Doc2 family (16 –19), and they are suggested to be involved in vesicular trafficking. These protein families are distinguished from one another by unique N-terminal domains; a transmembrane domain (TM) in the Syt family, a rab3A-binding domain in rabphilin-3A, and a Munc13-1-interacting domain (Mid) in the Doc2 family. Three proteins, Syt I, rabphilin-3A and Doc2␣, have been identified on synaptic vesicles, and were shown to be involved in the regulation of synaptic vesicle cycles in neurons and secretory vesicle exocyto-
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sis in some endocrine cells (20 –34). Syt I, an integral membrane protein of synaptic vesicles, is essential for the fusion of the synaptic vesicle with the presynaptic plasma membrane, probably functioning as a Ca 2⫹sensor via the C2A domain (24, 25, 27). By contrast, rabphilin-3A and Doc2␣ play a regulatory role in a step before the final fusion of the synaptic vesicle with the presynaptic plasma membrane (31–34). In this paper, we report a novel family of C-type tandem C2 proteins which lack a transmembrane domain, designated Slp1-3 (synaptotagmin-like protein 1-3). Slp1-3 also has tandem C2 domains at the C-terminus, but the N-terminal domains of Slp1-3 have no apparent homology with one another. We compared the biochemical properties of the Slp C2 domains and C-terminus to those of the Syt family, and examined the tissue distribution of Slp1-3 mRNA expression. MATERIALS AND METHODS Materials. Ex Taq and recombinant Taq DNA polymerases were obtained from Takara Shuzo Co., Ltd. (Shiga, Japan) and Toyobo Biochemicals (Tokyo, Japan), respectively. Polyclonal antibody against FLAG peptide was obtained from Zymed Laboratories, Inc. (San Francisco, CA). Horseradish peroxidase (HRP)-conjugated anti-T7 tag antibody and anti-T7 tag antibody-conjugated agarose were from Novagen (Madison, WI). L-␣-Phosphatidylcholine, dipalmitoyl (PC), and L-␣-phosphatidylserine, dioleoyl (PS) were from Sigma (St. Louis, MO). All other chemicals were commercial products of reagent grade. Solutions were made up in deionized water prepared with an Elix10 Water Purification System and Milli-Q Biocel A10 System (Millipore Corp.; Bedford, MA). Molecular cloning of mouse Slp1-3. cDNAs encoding the open reading frame of mouse Slp1-3 were amplified from Marathon-Ready mouse adult brain cDNA by 5⬘ (or 3⬘)-rapid amplification of cDNA ends (RACE) (Clontech Laboratories, Inc., Palo Alto, CA) as described previously (19). The first 5⬘-RACE reactions were carried out using adapter primer 1 (5⬘-CCATCCTAATACGACTCACTATAGGGC-3⬘) and the following primers designed on the basis of the Slp1-3 sequences in the mouse EST sequence database (Accession Nos. AA763436, AA727330 and AW763978 for Slp1; AW580643 for Slp2; and AI019424 for Slp3): 5⬘-CTATGCCCTGGGGACCAGGTT-3⬘ (Slp1-C1 primer; antisense; amino acid residues 543–548), 5⬘-CATCTTCTCCCAGAGAGCAA-3⬘ (Slp2-C2 primer; antisense; amino acid residues 346 –352), 5⬘-CGGATCCAACCTAGAAGTGAAAGGTAGC-3⬘ (Slp2-C2A upper primer, sense; amino acid residues 67-73), or 5⬘-TCAGTGCAGGACGAGGGTCAT-3⬘ (Slp3-C1 primer; antisense; amino acid residues 388393). The second RACE reactions were carried out by using internal adapter primer 2 (5⬘-ACTCACTATAGGGCTCGAGCGGC-3⬘) and the following primers designed on the basis of the above mentioned mouse EST sequences: 5⬘-AGCCCATAACTGCTGCCTGTA-3⬘ (Slp1-C2 primer; antisense; amino acid residues 501–508), 5⬘AGGCTTCCATCAGATCTTCA-3⬘ (Slp2-C3 primer; antisense; amino acid residues 296 –303), 5⬘-CGGATCCGTTGCCCTCGAGACAGAAAAC-3⬘ (Slp2-C2B upper primer; sense; amino acid residues 204 –210), or 5⬘-GTCTGTCCACAGGTTTGGGCT-3⬘ (Slp3-C2 primer; antisense; amino acid residues 381–387). Both polymerase chain reactions (PCRs) were carried out in the presence of Perfect Match PCR Enhancer (Stratagene, La Jolla, CA) for 35 cycles, each consisting ofdenaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 3 min. The first and second PCR
products were purified from an agarose gel on a Micro-Spin column (Amersham Pharmacia Biotech; Buckinghamshire, UK) as described previously (35), and were directly inserted into the pGEM-T Easy vector (Promega; Madison, WI). Both strands of the cDNA inserts were randomly sequenced with a Hitachi SQ-5500 DNA sequencer until a cDNA clone including the putative initiation methionine or termination codons was obtained. Since two Met residues were present just downstream of the 3⬘-termination codon in Slp1 and Slp3, the translation initiation site was determined by expression of Slp1-T7 and Slp3-T7 in COS-7 cells. The translation of Slp1 and Slp3 proteins was confirmed to be initiated at first Met and second Met, respectively, in COS-7 cells (data not shown). In the course of screening Slp1-3 cDNAs, we found several alternative splice variants of Slp1-3 (data not shown). For example, the Slp2 cDNAs showed a 120-bp deletion which corresponded to 13–52 amino acids of the full-length Slp2. Expression constructs. Full-length Slp1-3 cDNAs including the open reading frame were constructed by using appropriate restriction enzyme sites on the pGEM-T Easy vector (named pGEM-T-Slp13). Addition of the T7 tag (or FLAG) to the N (or C) terminus of each Slp (T7-Slp1-3, FLAG-Slp1-3, and Slp1, 3-T7) was done by PCR using primers with a T7 tag sequence as described previously (35). In the case of Slp3-T7, for example, the Slp3-T7 fragment was amplified by Slp3-Met primer (5⬘-GCGGATCCATGGACCCCAGGCAGTGTGT-3⬘) and Slp3-T7 primer (5⬘-CTAACCCATTTGCTGTCCACCAGTCATGCTAGCCATCAATTGGTGCAGGACGAGGGTCATGTC-3⬘; T7-tag, italics; restriction enzyme sites, underlines; and stop codons, bold letters) using pGEM-T-Slp3 as a template. Purified PCR products were inserted into the pGEM-T Easy vector and verified by DNA sequencing. The full length Slp3 inserts with T7 tag were then excised from the pGEM-T Easy vector by NotI digestion and subcloned into the NotI site of a modified pEF-BOS mammalian expression vector (36, 37) (pEF-T7Slp1-3, -FLAG-Slp1-3, and pEF-Slp1, 3-T7). Other expression constructs (pEF-T7-Syt II) were also prepared as described previously (35). Plasmid DNA was prepared using Wizard-mini preps (Promega) or QIAGEN Maxi prep kits. Preparation of GST fusion proteins. Construction of pGEX-4T-3 (or pGEX-2T) vector (Amersham Pharmacia Biotech) carrying fragments of the Slp1-3 C2A or C2B domain were essentially performed by PCR (36) using pGEM-T-Slp1-3 as a template. The following pairs of oligonucleotides with appropriate restriction enzyme sites (underlined) and/or termination codons (bold letters) were used for amplification: Slp1-C2A upper primer (sense) 5⬘-GCGGATCCACGGTGCAGGTGCGAGGCTC-3⬘ and Slp1-C2A lower primer (antisense) 5⬘-GCACTAGTCAAGATGGAGGAACCCGAGGCT-3⬘; Slp1C2B upper primer (sense) 5⬘-GCAGATCTTCTCCAGATGAGCTTCCCAG-3⬘ and SP6 primer; Slp2-C2A upper primer described above and Slp2-C2A lower primer (antisense) 5⬘-GCACTAGTCAAACTGGTGCTGTCTTCCT-3⬘; Slp2-C2B upper primer described above and SP6 primer; Slp3-C2A upper primer (sense) 5⬘GCGGATCCAATGCTAATGTCACTGGAGA-3⬘ and Slp3-C2A lower primer (antisense) 5⬘-GCACTAGTCAAGCCTTGGCCCGCAGAGGAT-3⬘; and Slp3-C2B upper primer (sense) 5⬘-GCGGATCCGCTGAGAAATACGAAGAGAA-3⬘ and SP6 primer. The resulting pGEX-Slp1-3-C2A and -C2B were confirmed by DNA sequencing and transformed into Escherichia coli, JM109. Glutathione S-transferase (GST) fusion proteins were expressed and purified on glutathione Sepharose (Amersham Pharmacia Biotech) by the standard method (38). GST-Slp1-C2A and -Slp1-C2B encoded amino acids 268 –397 and 397–567 of mouse Slp1, respectively. GST-Slp2-C2A and -Slp2C2B encoded amino acids 67–204 and 204 –376 of mouse Slp2, respectively. GST-Slp3-C2A and -Slp3-C2B encoded amino acids 88 – 222 and 222–393 of mouse Slp3, respectively. GST-Syt I-C2A was prepared as described previously (36). Reverse transcriptase (RT)-PCR analysis. Mouse first-strand cDNAs prepared from various tissues and developmental stages
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were obtained from Clontech Laboratories (Mouse MTC Panel I). PCRs were carried out in the presence of Perfect Match PCR Enhancer (Stratagene) for 30 cycles, each consisting of denaturation at 94°C for 1 min, annealing at 55°C for 2 min, and extension at 72°C for 2 min. The C2B upper and C1 primers described above (Slp2 C1 primer, 5⬘-GCACTAGTCACTTGGAAAGCTTGGCAAT-3⬘) were used for amplification. The PCR products were analyzed by 1% agarose gel electrophoresis and ethidium bromide staining. The authenticity of the products was verified by subcloning into a pGEM-T Easy vector and DNA sequencing. Sequence analyses. Multiple sequence alignment and depiction of the phylogenetic tree of the mouse synaptotagmin family were performed by using the CLUSTALW program (http://watson. genes.nig.ac.jp/homology/clustalw.shtml) set at the default parameters (gapopen ⫽ 10, gapext ⫽ 0.05, gapdist ⫽ 8, and maxdiv ⫽ 40). Miscellaneous procedures. Subcellular fractionation of COS-7 cells and in vitro neurexin I␣ (NxI␣) binding assay was performed as described previously (39, 40). Preparation of liposomes consisting of PC and PS (1:1, w/w) and phospholipid binding assay was also performed as described previously (41, 42). The protein concentrations were determined with a Bio-Rad protein assay kit (Bio-Rad Laboratories, Hercules, CA) using bovine serum albumin as a reference. Association of T7- and FLAG-Slps in COS-7 cells were evaluated by immunoprecipitation as described previously (35, 43, 44).
RESULTS AND DISCUSSION Molecular Cloning of Novel Synaptotagmin-Like Proteins (Slp1-3) To identify novel C-type tandem C2 proteins that may be involved in vesicular trafficking, we searched for tandem C2 proteins homologous to the mouse Syts I–XII C2 domains in a mouse EST sequence database by tfasta using the default parameters. Fragments of five distinct genes that encoded three novel tandem C2 domains, Doc2␥ (19), and Syt XIII (40) were found, and their full open reading frame was cloned from a mouse adult brain cDNA library by 5⬘-RACE (see Materials and Methods for details). The 1824-, 1313-, and 1332-base sequences contained a single open reading frame encoding 567, 376, and 393 amino acids with a calculated molecular weight of 62,349, 42,759, and 43,781, respectively (Fig. 1C). These three proteins contain tandem C2 domains (Fig. 2), but they have no hydrophobic region sufficient to be a transmembrane region (data not shown). Homology search analysis revealed that the C2 domains of these proteins showed higher homology to those of granuphilin-a than to those of other C-type tandem C2 proteins (more than 32%; Fig. 2). In addition, the short C-terminus of Slp1-3 and granuphilin-a are highly conserved and different from that of Syt family, Doc2 family and rabphilin-3A proteins (Fig. 2, underline). The length of the N-terminal domain varies among the three Slp proteins and they do not show any apparent homology to one another or to any known protein motifs (PIR and SWISS-PROT protein databases). How-
FIG. 1. Schematic representation of mouse tandem C2 protein family. (A) Schematic representation of N-terminal type (N-type) tandem C2 proteins: GAP1 family (5–9) and copine family (10, 11). (B) Schematic representation of C-terminal type (C-type) tandem C2 proteins reported to date: synaptotagmin family (3), rabphilin3A/granuphilin-a (14, 15), and Doc2 family (16 –19). (C) Schematic representation of novel C-type tandem C2 proteins determined in this study (Slp1-3). Solid line indicates the deletion by alternative splicing in Slp2. Amino acid numbers are given on both sides. GRD, GTPase-activating protein-related domain; PH, pleckstrin homology; TM, transmembrane domain; rab3A, rab3A binding domain; Mid, Munc13-1 interacting domain. (D) Phylogenetic tree of mouse C-type tandem C2 protein family reported to date. The phylogenetic tree is depicted as described under Materials and Methods. Note that the Slp family (boxed) form a distinct branch from the synaptotagmin, rabphilin-3A and Doc2 family.
ever, we found that the Slp1 N-terminal domain showed very weak similarity to granuphilin-a (13.6% identity and about 22% similarity at the N-terminus), but it lacks the conserved Cys residues responsible for Zn 2⫹ binding (45) in rabphilin-3A and granuphilin-a (data not shown). The overall amino acid identity between Slp1 and granuphilin-a was about 31%, which was much higher than that be-
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FIG. 2. Sequence alignment of the mouse C2 domains of C-type tandem C2 proteins. Sequence alignment of the mouse C2 domains of Syt I (Accession No. D37792), Syt B/K (unpublished), Slp1-3 (AB050741-3), granuphilin-a (AB025258), and Doc2␣ (D50000). Residues conserved or similar between sequences are shown on a black background or shaded background, respectively. Asterisks indicate the five conserved aspartate or glutamate residues, which may be crucial for Ca 2⫹ binding by analogy with the Syt I-C2A domain (52, 53). The number signs (#) indicate the key amino acids responsible for high affinity neurexin I␣ binding in vitro (58). The dashed lines and solid line indicate two C2 domains and a short carboxyl terminus, respectively.
tween granuphilin-a and rabphilin-3A (about 22%). Consistent with this, Slp1-3 and granuphilin-a form a small branch in the phylogenetic tree (Fig. 1D). Therefore, granuphilin-a belongs to the Slp family rather than the rabphilin-3A homologues. Membrane Association of Slp1-3 in COS-7 Cells Since Slp1-3 did not contain a transmembrane domain, we first examined whether Slp1-3 are soluble or membrane-associated proteins. To address this, subcellular fractionation of COS-7 cells expressing Slp1-3
was performed. All three proteins were recovered in the membrane fraction like Syt II, an integral membrane protein of synaptic vesicles (46), although half of the Slp1 proteins were also recovered in the soluble fraction (Fig. 3). Since Slp1-3 lack any apparent transmembrane domain, we treated the membrane fraction with 0.1 M Na 2CO 3, which should release most peripheral membrane proteins from the membranes. Under these conditions Slp1-3, but not Syt II, were released from the membrane fraction. Therefore, we concluded that Slp1-3 are peripheral, but not integral, membrane proteins.
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(PS/PC liposomes) binding assay was performed using GST-fusion proteins (Fig. 4). Unexpectedly, however, the Slp3-C2A domain bound PS/PC liposomes only in the presence of calcium, indicating that the Ca 2⫹binding pocket of the Slp3-C2A domain is different from that of Syt I. By contrast, none of the other C2 domains showed any phospholipid binding activity. Since none of the C2 domains of Slp showed any Ca 2⫹independent phospholipid binding activity, the membrane association of Slp1-3 in Fig. 3 can not be attributable to the phospholipid binding activity of the C2 domains. Oligomerization Properties of Slp1-3 FIG. 3. Subcellular fractionation of COS-7 cells expressing T7Slp1-3. Subcellular fractionation of COS-7 cells expressing pEF-Syt II (top panel), -Slp1 (second panel), -Slp2 (third panel), or -Slp3 (fourth panel). Membrane (M) and soluble (S) fractions were separated as described previously (39, 40). The membrane fraction was further resuspended in a buffer containing 0.1 M Na 2CO 3, pH 11 and incubated for 1 h at 4°C. After centrifugation at 100,000 ⫻ g for 1 h, the supernatants (Su) were recovered. Equal proportions of total (T), membrane, soluble, and supernatants treated with 0.1 M Na 2CO 3, pH 11 were subjected to 10% SDS–PAGE and transferred to a polyvinylidene difluoride membrane (Millipore Corp.), followed by immunoblotting with HRP-conjugated anti-T7 tag antibody. The positions of the molecular weight markers (⫻ 10 ⫺3) are shown on the right. The results shown are representative of three independent experiments.
Recent genetic and biochemical studies indicate that Syt I functions as an oligomer in synaptic vesicle exo-
Phospholipid Binding Properties of Slp1-3 C2 Domains In the next set of experiments, we compared the biochemical properties of the Slp family to those of Syt family. First, we focused on the tandem C2 domains, because the C2 domain was originally found in Ca 2⫹dependent protein kinase C, and is thought to function as a phospholipid binding site (reviewed in ref. 2). In Syt I, a well characterized C-type tandem C2 protein, the C2A domain binds negatively-charged phospholipids in a Ca 2⫹-dependent manner, whereas the C2B domain binds phospholipids irrespective of the presence of Ca 2⫹ (36, 41, 47). The Ca 2⫹/phospholipid binding site of the C2A domain has been shown to be important for secretory vesicle exocytosis (25, 27–29), neurite outgrowth of chick dorsal root ganglion neurons and PC12 cells (48, 49), and axonal repair (50, 51). Crystallographic and mutational analysis of the Syt I C2A domain indicated that five aspartate (or glutamate) residues are essential for Ca 2⫹ binding (52, 53) (see Fig. 2, asterisks). Interestingly, Slp1-3 lack such aspartate or glutamate residues both in the C2A and C2B domains (Slp1-C2A, Arg at amino acid positions 302 (R302), H359, and I367, and Slp1-C2B, R443, G505, and Q511; Slp2-C2A, H162, S170, and Slp2-C2B, R250, N255, Y312, and F318; and Slp3-C2A, C128, H183, G185, and V191 and Slp3-C2B, S279, N284, and A341), suggesting that Slp1-3 should be classified as Ca 2⫹-independent types. To address this, phospholipid
FIG. 4. Phospholipid binding properties of Slp1-3 C2 domains. Liposomes and GST fusion proteins were incubated in 50 mM HepesKOH, pH 7.2, in the presence of 2 mM EGTA or 1 mM Ca 2⫹ for 15 min at room temperature. After centrifugation at 12,000g for 10 min, the supernatants (S, non-binding fraction) and pellets (P, phospholipid binding fraction) were separated as described previously (41, 42). Equal proportions of the supernatants and pellets were subjected to 10% SDS–polyacrylamide gel electrophoresis and then stained with Coomassie brilliant blue R-250. Except for the C2A domain of Slp3, the C2 domains of Slp1-3 did not show phospholipid binding activity. By contrast, the C2A domains of Syt I and Slp3 only bound liposomes in a Ca 2⫹-dependent manner. The bottom panel indicates the negative control, GST. The results shown are representative of three independent experiments.
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ing to speculate that this weak interaction with NxI␣ may result in only half of the Slp1 proteins being present in the membrane fraction. Differential Tissue Distribution of Slp1-3
FIG. 5. Neurexin I␣ binding property of Slp1-3. GST pull-down assay was performed as described previously (40). After immunoblotting with HRP-conjugated anti-T7 tag antibody (upper panels), blots were stained with amido black to ensure that equivalent amounts of GST fusion proteins were loaded (lower panels). T7- Slp1-3 or Syt VI⌬TM proteins were incubated with GST (lanes 1– 4) or GST-NxI␣ (lanes 5– 8), respectively. The 1/80 volume of total proteins used for assay were loaded on lanes 9 –12. Note that Slp1-3 did bind GSTNx1␣, but not GST alone, the same as Syt VI⌬TM. However, the Slp1-NxI␣ interaction is apparently weaker than others (lane 5). The results shown are representative of two independent experiments.
cytosis (22, 54 –56). In addition, Syt isoforms have been shown to form Ca 2⫹-dependent and -independent hetero-oligomers (35, 43, 44, 56). By contrast, Slp1-3 did not show any apparent homo- and heterooligomerization activities (data not shown), indicating that Slp1-3 is present at a monomer state in living cells. The C-Terminus of Slp1-3 Binds Neurexin I␣ in Vitro In a previous study, we showed that the conserved C-terminus of the synaptotagmin family is essential for plasma membrane association in PC12 cells (39, 57) and that the Syt I-C terminus is indeed involved in synaptic vesicle docking to presynaptic plasma membranes in the squid giant synapse (57). Since the polymorphic plasma membrane protein neurexin binds the conserved C-terminus of Syt family in vitro (58, 59), neurexin (I-III) is suggested to be a common plasma membrane receptor for Syt family. To further examine whether the Slp1-3 C-terminus is involved in membrane association, a neurexin I␣ binding assay was performed. Briefly, T7-Slp1-3 proteins were expressed in COS-7 cells and solubilized proteins were incubated with glutathione Sepharose coupled with GST-NxI␣ or GST alone. After extensive washing of the beads with incubation buffer, GST-NxI␣-bound proteins were analyzed by immunoblotting with HRP-conjugated anti-T7 tag antibody. As shown in Fig. 5, Slp1-3 bound GST-NxI␣, but not GST alone, the same as Syt VI⌬TM (lanes 5– 8). However, the Slp1-NxI␣ interaction was apparently weaker than the Slp2, 3-NxI␣ interactions (Fig. 5, compare lanes 5 and 6). Therefore, it is tempt-
Finally, the tissue distribution of mouse Slp1-3 was examined by RT-PCR with specific primers (Fig. 6). The tissue distribution of Slp1-3 were apparently different and their transcripts were not enriched in the brain. For example, the expression level of Slp1 mRNA was highest in lung, moderate in spleen, liver and kidney, lowest in heart, brain and skeletal muscle, and absent in testis. By contrast, the expression of Slp2 mRNA was almost complementary to that of Slp1: highest expression was seen in skeletal muscle, kidney, and testis, and moderate expression in heart, brain, and lung. Slp3 mRNA expression was found in spleen, lung, kidney and testis. In addition, the expression pattern of Slp1-3 during development was also different. The expression of Slp1 and Slp3 transcripts seemed to be highest on embryonic day 7 and then decreased to adult, whereas expression of Slp2 seemed to be constant from embryonic day 7 to 17. CONCLUSION In summary, we have isolated and characterized three novel C-type tandem C2 protein family proteins, named Slp1-3, which are differentially distributed in different mouse tissues and at different developmental stages. The Slp1-3 family shows the highest homology with granuphilin-a C2 domains (more than 32% identity) and form a distinct branch from Syt, rabphilin-3A and Doc2 family proteins. Like synaptotagmin and
FIG. 6. Differential tissue distribution of mouse Slp1-3. RT-PCR analysis of Slp1-3 expression in various tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis) and on embryonic days 7 (E7), 11, 15, and 17 (upper three panels). RT-PCR analysis of G3PDH expression was also performed (bottom panel) to ensure that equivalent amounts of first strand cDNA were used for RT-PCR analysis. ⫺ means without templates as a negative control. The size of the molecular weight markers (/StyI) is shown at the left of the panel. The results shown are representative of two independent experiments.
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Doc2 family proteins, the Slp family contains both Ca 2⫹-dependent (Slp3) and -independent (Slp1 and Slp2) isoforms. In addition, the C-terminus of Slp1-3 also binds neurexin I␣ in vitro the same as the synaptotagmin family, suggesting that Slp1-3 may also regulate docking of transport vesicles to plasma membrane. Since the Slp1-3 mRNAs are expressed in various tissues outside the brain, we speculate that the Slp1-3 proteins are involved in constitutive vesicle traffic and/or regulated exocytosis in endocrine (or exocrine) cells. Further work is necessary to elucidate whether the Slp family is indeed involved in vesicular trafficking. ACKNOWLEDGMENTS We thank Dr. Shigekazu Nagata for the expression vector, and Eiko Kanno for technical assistance.
REFERENCES 1. Ponting, C. P., and Parker, P. J. (1996) Protein Sci. 5, 162–166. 2. Nalefski, E. A., and Falke, J. J. (1996) Protein Sci. 5, 2375–2390. 3. Marque`ze, B., Berton, F., and Seagar, M. (2000) Biochimie 82, 409 – 420. 4. Su¨dhof, T. C., and Rizo, J. (1996) Neuron 17, 379 –388. 5. Maekawa, M., Li, S., Iwamatsu, A., Morishita, T., Yokota, K., Imai, Y., Kohsaka, S., Nakamura, S., and Hattori, S. (1994) Mol. Cell Biol. 14, 6879 – 6885. 6. Cullen, P. J., Hsuan, J. J., Truong, O., Letcher, A. J., Jackson, T. R., Dawson, A. P., and Irvine, R. F. (1995) Nature 376, 527– 530. 7. Yamamoto, T., Matsui, T., Nakafuku, M., Iwamatsu, A., and Kaibuchi, K. (1995) J. Biol. Chem. 270, 30557–30561. 8. Baba, H., Fuss, B., Urano, J., Poullet, P., Watson, J. B., Tamanoi, F., and Macklin, W. B. (1995) J. Neurosci. Res. 41, 846 – 858. 9. Fukuda, M., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 18838 –18842. 10. Creutz, C. E., Tomsig, J. L., Snyder, S. L., Gautier, M. C., Skouri, F., Beisson, J., and Cohen, J. (1998) J. Biol. Chem. 273, 1393– 1402. 11. Nakayama, T., Yaoi, T., Yasui, M., and Kuwajima, G. (1998) FEBS Lett. 428, 80 – 84. 12. Fukuda, M., and Mikoshiba, K. (1997) BioEssays 19, 593– 603. 13. Schiavo, G., Osborne, S. L., and Sgouros, J. G. (1998) Biochem. Biophys. Res. Commun. 248, 1– 8. 14. Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, S., Yamaguchi, T., Wada, K., Miyazaki, M., and Takai, Y. (1993) Mol. Cell. Biol. 13, 2061–2068. 15. Wang, J., Takeuchi, T., Yokota, H., and Izumi, T. (1999) J. Biol. Chem. 274, 28542–28548. 16. Orita, S., Sasaki, T., Naito, A., Komuro, R., Ohtsuka, T., Maeda, M., Suzuki, H., Igarashi, H., and Takai, Y. (1995) Biochem. Biophys. Res. Commun. 206, 439 – 448. 17. Sakaguchi, G., Orita, S., Maeda, M., Igarashi, H., and Takai, Y. (1995) Biochem. Biophys. Res. Commun. 217, 1053–1061. 18. Kojima, T., Fukuda, M., Aruga, J., and Mikoshiba, K. (1996) J. Biochem. (Tokyo) 120, 671– 676. 19. Fukuda, M., and Mikoshiba, K. (2000) Biochem. Biophys. Res. Commun. 276, 626 – 632.
20. Geppert, M., Goda, Y., Hammer, R. E., Li, C., Rosahl, T. W., Stevens, C. F., and Su¨dhof, T. C. (1994) Cell 79, 717–727. 21. Nonet, M. L., Grundahl, K., Meyer, B. J., and Rand, J. B. (1993) Cell 73, 1291–1305. 22. Littleton, J. T., and Bellen, H. J. (1995) Trends Neurosci. 18, 177–183. 23. Elferink, L. A., Peterson, M. R., and Scheller, R. H. (1993) Cell 72, 153–159. 24. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, H., and Augustine, G. J. (1993) Nature 363, 163–165. 25. Mikoshiba, K., Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., and Llina´s, R. (1995) Proc. Natl. Acad. Sci. USA 92, 10703–10707. 26. Fukuda, M., Moreira, J. E., Lewis, F. M. T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llina´s, R. (1995) Proc. Natl. Acad. Sci. USA 92, 10708 –10712. 27. Mochida, S., Fukuda, M., Niinobe, M., Kobayashi, H., and Mikoshiba, K. (1997) Neuroscience 77, 937–943. 28. Ohara-Imaizumi, M., Fukuda, M., Niinobe, M., Misonou, H., Ikeda, K., Murakami, T., Kawasaki, M., Mikoshiba, K., and Kumakura, K. (1997) Proc. Natl. Acad. Sci. USA 94, 287–291. 29. Lang, J., Fukuda, M., Zhang, H., Mikoshiba, K., and Wollheim, C. B. (1997) EMBO J. 16, 5837–5846. 30. Thomas, D. M., and Elferink, L. A. (1998) J. Neurosci. 18, 3511– 3520. 31. Mochida, S., Orita, S., Sakaguchi, G., Sasaki, T., and Takai, Y. (1998) Proc. Natl. Acad. Sci. USA 95, 11418 –11422. 32. Burns, M. E., Sasaki, T., Takai, Y., and Augustine, G. J. (1998) J. Gen. Physiol. 111, 243–255. 33. Schluter, O. M., Schnell, E., Verhage, M., Tzonopoulos, T., Nicoll, R. A., Janz, R., Malenka, R. C., Geppert, M., and Su¨dhof, T. C. (1999) J. Neurosci. 19, 5834 –5846. 34. Sakaguchi, G., Manabe, T., Kobayashi, K., Orita, S., Sasaki, T., Naito, A., Maeda, M., Igarashi, H., Katsuura, G., Nishioka, H., Mizoguchi, A., Itohara, S., Takahashi, T., and Takai, Y. (1999) Eur. J. Neurosci. 11, 4262– 4268. 35. Fukuda, M., Kanno, E., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31421–31427. 36. Fukuda, M., Aruga, J., Niinobe, M., Aimoto, S., and Mikoshiba, K. (1994) J. Biol. Chem. 269, 29206 –29211. 37. Mizushima, S., and Nagata, S. (1990) Nucleic Acids Res. 18, 5332. 38. Smith, D. B., and Johnson, K. S. (1988) Gene 67, 31– 40. 39. Fukuda, M., and Mikoshiba, K. (1999) J. Biol. Chem. 274, 31428 –31434. 40. Fukuda, M., and Mikoshiba, K. (2001) Biochem. J. 354, 249 –257. 41. Fukuda, M., Kojima, T., and Mikoshiba, K. (1996) J. Biol. Chem. 271, 8430- 8434. 42. Fukuda, M., Kojima, T., and Mikoshiba, K. (1997) Biochem. J. 323, 421– 425. 43. Fukuda, M., and Mikoshiba, K. (2000) J. Biol. Chem. 275, 28180 –28185. 44. Fukuda, M., and Mikoshiba, K. (2000) J. Biochem. (Tokyo) 128, 637– 645. 45. Misra, S., and Hurley, J. H. (1999) Cell 97, 657– 666. 46. Niinobe, M., Yamaguchi, Y., Fukuda, M., and Mikoshiba, K. (1994) Biochem. Biophys. Res. Commun. 205, 1036 –1042. 47. Damer, C. K., and Creutz, C. E. (1994) J. Biol. Chem. 269, 31115–31123. 48. Fukuda, M., and Mikoshiba, K. (2000) Neurosci. Lett. 295, 33– 36.
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49. Kabayama, H., Takei, K., Fukuda, M., Ibata, K., and Mikoshiba, K. (1999) Neuroscience 88, 999 –1003. 50. Detrait, E., Eddleman, C. S., Yoo, S., Fukuda, M., Nguyen, M. P., Bittner, G. D., and Fishman, H. M. (2000) J. Neurobiol. 44, 382–391. 51. Detrait, E. R., Yoo, S., Eddleman, C. S., Fukuda, M., Bittner, G. D., and Fishman, H. M. (2000) J. Neurosci. Res. 62, 566 – 573. 52. Shao, X., Davletov, B. A., Sutton, R. B., Su¨dhof, T. C., and Rizo, J. (1996) Science 273, 248 –251. 53. Sutton, R. B., Davletov, B. A., Berghuis, A. M., Su¨dhof, T. C., and Sprang, S. R. (1995) Cell 80, 929 –938.
54. Sugita, S., Hata, Y., and Su¨dhof, T. C. (1996) J. Biol. Chem. 271, 1262–1265. 55. Davis, A. F., Bai, J., Fasshauer, D., Wolowick, M. J., Lewis, J. L., and Chapman, E. R. (1999) Neuron 24, 363–376. 56. Fukuda, M., Kabayama, H., and Mikoshiba, K. (2000) FEBS Lett. 482, 269 –272. 57. Fukuda, M., Moreira, J. E., Liu, V., Sugimori, M., Mikoshiba, K., and Llina´s, R. R. (2000) Proc. Natl. Acad. Sci. USA 97, 14715– 14719. 58. Perin, M. S. (1996) Biochemistry 35, 13808 –13816. 59. Hata, Y., Davletov, B., Petrenko, A. G., Jahn, R., and Su¨dhof, T. C. (1993) Neuron 10, 307–315.
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