TY - JOUR
AU - Fukuda, Mitsunori
AB - Abstract Synaptotagmins (Syts) represent a large family of putative membrane trafficking proteins found in various species from different phyla. In this study, I identified a novel class of Syt (named Syt XIV) conserved from Drosophila to humans and its highly related molecule, Strep14 (Syt XIV-related protein). Although both Syt XIV and Strep14 belong to the C-terminal-type (C-type) tandem C2 protein family, only Syt XIV has a single transmembrane domain at the N-terminus and a putative fatty-acylation site just downstream of the transmembrane domain. Biochemical analyses have indicated that Syt XIV is a Ca2+-independent Syt (e.g., Syts VIII, XII, and XIII) and that, like other Syt family proteins, it is capable of forming a Ca2+-independent oligomer. Unlike other Syt isoforms, however, expression of Syt XIV and Strep14 mRNA is highly restricted to mouse heart and testis and absent in the brain, where most other Syts are abundantly expressed, suggesting that Syt XIV and Strep14 may be involved in membrane trafficking in specific tissues outside the brain. I also identified all of the C-type tandem C2 proteins in humans, the mouse, the fruit fly, a nematode, a plant, and a yeast and discuss the molecular evolution of the C-type tandem C2 protein families, including the Syt family, the Syt-like protein (Slp) family, and the Doc2 family. Key words: C2 domain, C-type tandem C2 protein, membrane trafficking, phospholipid binding, synaptotagmin. Abbreviations: C-type, C-terminal-type; En, embryonic day n; GST, glutathione S-transferase; HRP, horseradish peroxidase; Mid, Munc13-1 interacting domain; RACE, rapid amplification of cDNA ends; RT, reverse transcriptase; SHD, Slp homology domain; Slp(s), synaptotagmin-like protein(s); Strep, synaptotagmin-related protein; Syt(s), synaptotagmins. Received February 13, 2003; accepted March 3, 2003 Over the past five years it has become increasingly evident that several members of the synaptotagmin (Syt) family are involved in the regulation of Ca2+-triggered cellular events through Ca2+-regulated membrane trafficking, including communication between neurons (i.e., synaptic vesicle exocytosis) (reviewed in Refs. 1–5), peptide hormone secretion (i.e., dense-core vesicle exocytosis) (6–13), neurite outgrowth (i.e., growth cone vesicle exocytosis) (14, 15), fertilization (i.e., acrosome reaction) (16), and plasma membrane repair (e.g., lysosomal exocytosis) (17–19). All Syt family members consist of an N-terminal single transmembrane domain and C-terminal tandem C2 domains (known as the C2A domain and the C2B domain) that are homologous to the C2 regulatory region of Ca2+-dependent protein kinase C (1–5). Ca2+-binding to the two C2 domains of the Syt family is widely believed to be essential for Ca2+-regulated exocytosis, and the two C2 domains of Syt I, the best characterized Syt isoform, which is abundant on synaptic vesicles, have been shown to regulate neurotransmitter release (20–24). By contrast, however, the exact function of other Syt isoforms largely remains to be determined and is still a matter of controversy. The Syt family is the largest of the C-terminal-type (C-type) tandem C2 protein families (25–33). To date, 13 distinct syt genes in mice and humans and several corresponding syt genes have been found in invertebrates (2, 34–37). A recent genomic analysis by BLAST search indicated the presence of six additional putative syt genes that encode fragments of tandem C2 domains in the human genome (36). However, whether these putative syt genes are indeed transcribed and belong to the syt gene family has never been elucidated. In the present study, I identified a novel class of Syt (named Syt XIV) conserved from Drosophila to humans and its highly related molecule (named Strep14, Syt XIV-related protein), and demonstrated their Ca2+-independent phospholipid binding properties and their tissue distributions. I also searched for syt-related genes in humans, the mouse, the fruit fly (Drosophila), a nematode (Caenorhabditis elegans), a plant (Arabidopthis thaliana), and a yeast (Saccharomyces cerevisiae), classified them into several groups, and discuss the molecular evolution of the C-type tandem C2 protein family proteins (e.g., the Doc2 family, the Syt-like protein (Slp) family, and Rabphilin). MATERIALS AND METHODS Molecular Cloning of Mouse Syt XIV and Strep14 cDNAs encoding the N-terminal (or C-terminal) region of mouse Syt XIV and Strep14 were amplified from Marathon-Ready mouse spleen (or heart) cDNA by 5′-(or 3′-) rapid amplification of cDNA ends (RACE) (Clonetech Laboratories, Inc., Palo Alto, CA, USA), essentially as described previously (34). The first 5′-RACE reactions were carried out by using adapter primer 1 (5′-CCATCCTAATACGACTCACTATAGGGC-3′) and the following primers designed on the basis of the mouse genome sequence (similar to synaptotagmin; mouse chromosomal positions 1 H6 and 12 C3) (http://www.ncbi.nlm.nih.gov/genome/guide/mouse/): 5′-CTATGGATCAAAAGGTTTAG-3′ (Syt XIV-ΔC2 primer; antisense), 5′-CTACAAAAGCCTGTGTGGTA-3′ (Strep14-ΔC2 primer; antisense), or 5′-AAGCAATTCAAGATCTCA-3′ (Strep14-N1 primer; sense). 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 mouse genome resource: 5′-CCTCATTCTCTGAATGCA-3′ (Syt XIV-C1 primer; antisense), 5′-TGAGATCTTGAATTGCTT-3′ (Strep14-C1 primer; antisense), or 5′-CAGGTACCACACAGGCTTTT-3′ (Strep14-N2 primer; sense). Both PCR reactions were carried out in the presence of Perfect Match PCR Enhancers (Stratagene, La Jolla, CA, USA) for 40 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 purified second PCR products were directly inserted into the pGEM-T Easy vector (Promega, Madison, WI, USA), and both strands were completely sequenced as described previously (34). cDNAs encoding the open reading frame of the mouse Syt XIV and Strep14 were similarly amplified by reverse transcriptase (RT)-PCR from Marathon-Ready mouse spleen cDNA using the following pairs of primers with a BamHI site (underlined) or a stop codon (bold letters): 5′-CGGATCCATGGCGATCGAAGGTGGAGA-3′ (Syt XIV-Met primer; sense) and 5′-TCAGGACTCCAGCAGTGCGT-3′ (Syt XIV-stop primer; antisense), or 5′-CGGATCCATGGTGTTGACCATGGCATCTCAGGATGTCCAG-3′ (Strep14-Met primer; sense) and 5′-CTAGGATTCCAACAGCGTAT-3′ (Strep14-stop primer; antisense). The purified PCR products were subcloned into the pGEM-T Easy vector (named pGEM-T-Syt XIV or pGEM-T-Strep14) and verified by DNA sequencing. The human Syt XIV and Strep14 cDNAs were determined by database searching (standard BLAST search) using the mouse Syt XIV sequence and Strep14 sequence, respectively, as a query. Molecular Cloning of Arabidopthis thaliana Syt A and Syt C cDNAs encoding the open reading frame of the A. thalianaSyt A and Syt C were similarly amplified by RT-PCR from the Superscript™ Arabidopthis cDNA library (Invitrogen; Carlsbad, CA, USA) using the following pairs of primers with a BamHI site (underlined) or a stop codon (bold letters) designed on the basis of the A. thaliana genome resource: 5′-CGGATCCATGGGCTTTTTCAGTACGAT-3′ (at-Syt A-Met primer, sense), 5′-TCAAGAGGCAGTTCGCCACT-3′ (at-Syt A-stop primer, antisense), 5′-CGGATCCATGGGTTTCTTCACCAGTGT-3′ (at-Syt C-Met primer, sense), and 5′-TTAACTAGTTGTCCAACGGA-3′ (at-Syt C-stop primer, antisense). The purified PCR products were subcloned into the pGEM-T Easy vector and were completely sequenced. Expression Constructs, Transfection, and Immunoprecipitation Addition of T7 tag (or FLAG tag) to the N terminus of Syt XIV or Strep14 and construction of mammalian expression vectors (named pEF-T7-Syt XIV, pEF-FLAG-Syt XIV, pEF-T7-Strep14, and pEF-FLAG-Strep14) were performed as described previously (38–40). Plasmid DNA was prepared using Qiagen (Chatsworth, CA, USA) Maxi prep kits. Cotransfection of pEF-T7-Syt XIV (or -Strep14) and pEF-FLAG-Syt XIV (or -Strep14) into COS-7 cells (7.5 × 105 cells, the day before transfection/10 cm dish) was performed as described previously (41). Proteins were solubilized at 4°C for 1 h with a buffer containing 1% Triton X-100, 250 mM NaCl, 1 mM MgCl2, 50 mM HEPES-KOH, pH 7.2, 0.1 mM phenylmethylsulfonyl fluoride, 10 µM leupeptin, and 10 µM pepstatin A. T7-tagged proteins were immunoprecipitated with anti-T7 tag antibody-conjugated agarose (Novagen; Madison, WI, USA) as described previously (34). SDS-PAGE and immunoblotting analyses with HRP (horseradish peroxidase)-conjugated anti-FLAG tag (Sigma Chemical, St. Louis, MO, USA) and anti-T7 tag antibodies (Novagen) were also performed as described previously (34). The blots shown in this paper are representative of two independent experiments. Phospholipid-Binding Assay Construction of pGEX-4T-3 vector (Amersham Biosciences, Tokyo) carrying each C2 domain of the mouse Syt XIV or Strep14 was essentially performed by PCR (39) using pGEM-T-Syt XIV or pGEM-T-Strep14, respectively, as a template. The following primers with a BamHI site (underlined) or a stop codon (bold letter) were used for amplification: Syt XIV-C2A upper primer (5′-CGGATCCGAGCCAGAAGCTAAATATGG-3′), Syt XIV-C2A lower primer (5′-CTAACAGCCAGAAGGATTGT-3′), Syt XIV-C2B upper primer (5′-CGGATCCGACAGTACATCCTCCTGTCA-3′), Syt XIV-stop primer, Strep14-C2A upper primer (5′-CGGATCCGAGCCTATCTCAAAATGCGG-3′), Strep14-C2A lower primer (5′-CTATCCACTGCTCAGATTAC-3′), Strep14-C2B upper primer (5′-CGGATCCGACAGTGCTTCATCCACGCA-3′), and Strep14-stop primer. Preparation of GST (glutathione S-transferase) fusion proteins containing the single C2 domain of Syt XIV (or Strep14) was performed as described previously (39). GST-Syt XIV-C2A contains amino acid residues 256–390 of mouse Syt XIV; GST-Syt XIV-C2B, amino acid residues 403–555 of mouse Syt XIV; GST-Strep14-C2A, amino acid residues 340–474 of mouse Strep14; and GST-Strep14-C2B, amino acid residues 487–639 of mouse Strep14. Liposome co-sedimentation assay was also performed as described previously (42, 43). The gels shown in this paper are representative of three independent experiments. Sequence Analyses Multiple sequence alignment was performed with the CLUSTALW program (http://hypernig.nig.ac.jp/homology/clustalw.shtml) set at the default parameters and then modified manually to optimize similarity. Depiction of the phylogenetic tree was also performed by use of the CLUSTALW program (25). RT-PCR Analysis Mouse first-strand cDNAs prepared from various tissues and developmental stages (whole embryo) were obtained from Clontech Laboratories, Inc. (mouse MTC Panel I) (25, 26). PCRs were carried out in the presence of Perfect Match PCR enhancer for 28 cycles (for G3PDH) or 40 cycles (for Syt XIV and Strep14), 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 Syt XIV-C2B upper primer and Syt XIV-stop primer, and the Strep14-C2B upper primer and Strep14-stop primer were used for amplification. The PCR products were analyzed by 1% agarose gel electrophoresis followed by ethidium bromide staining. The authenticity of the products was verified by subcloning into a pGEM-T Easy vector and DNA sequencing as described previously (34). RESULTS Identification of a Novel Class of Synaptotagmin (Syt XIV) Conserved from Drosophila to Humans To identify a novel gene that encodes a C-type tandem C2 protein, I searched for tandem C2 proteins homologous to mouse Syts I-XIII C2 domains in human and mouse genome databases by use of protein BLAST or SMART (simple modular architecture research tool; http://smart.embl-heidelberg.de/). Two distinct genes that potentially encode novel C-type tandem C2 proteins were found in the human and mouse genomes (human chromosomal positions 4q13.1 and 14q23.1 and mouse chromosomal positions 1 H6 and 12 C3), and their full open reading frame was cloned from the mouse cDNA library by 5′- and 3′-RACE (see Materials and Methods for details). The 1,731 nucleotide sequence and 3,058 nucleotide sequence contain a single open reading frame encoding 555 and 639 amino acids with a calculated molecular weight of 62,041 and 71,251, respectively (Fig. 1A). These two proteins contain tandem C2 proteins (i.e., C2A domain and C2B domain) at the C terminus, and therefore they should be classified as members of the C-type tandem C2 protein family (25). Interestingly, these two proteins show significant homology with each other, especially in the two C2 domains (66.7% similarity in the C2A domain, 83.8% similarity in the C2B domain, and 46.4% similarity in the entire proteins) (Fig. 2), but relatively weak similarity to other Syt isoforms reported thus far (34–37). According to the results of a hydrophobicity analysis (44), the 555 amino acid protein contains a single hydrophobic region sufficient for a transmembrane domain at the N terminus, but the 639 amino acid protein has no hydrophobic region (Fig. 1A and data not shown). I therefore named the former protein the 14th isoform of Syt (Syt XIV) and the latter protein Strep14 (Syt XIV-related protein). Interestingly, homology search analysis revealed that the orthologue of mammalian Syt XIV is found in Drosophila (named dm-Syt XIV) (63.2% similarity in the transmembrane domain, 78.6% similarity in the Syt XIV homology domain (see below), 45.7% similarity in the C2A domain and 71.4% similarity in the C2B domain), but not in C. elegans, the plant, or the yeast (Figs. 2 and 3), suggesting that Syt XIV may have an important role in membrane trafficking conserved from Drosophila to humans. The mouse, human, and Drosophila Syt XIV proteins share several features characteristic of Syt family members. First, they contain several Cys residues at the interface between the transmembrane domain and the spacer domain (# in Fig. 2), and such Cys residues often undergo fatty-acylation, which is involved in SDS-insensitive oligomerization of Syt proteins (41). The putative extracellular domain of the Syt XIV protein was very short, as are those of Syts I-XII, and the mammalian Syt XIV protein contains two Cys residues in this region (small boxes in Fig. 2). Similar Cys residues were also found in the extracellular domain of Syts III, V, VI, and X (Fig. 1A), and they are involved in dimer formation through disulfide bonding (34, 40, 45). Unlike Syts I and II, the Syt XIV protein lacks a putative N-glycosylation site and di-Thr/Ser residues (i.e., putative O-glycosylation site) (46). Although the mouse and human Strep14 protein lacks a transmembrane domain as well as the known protein motifs of the C-type tandem C2 proteins, including Mid (Munc13-1 interacting domain) (47), Slp homology domain (SHD) (48, 49), and Rab3/8/27 binding domain (31, 48) (see Fig. 1B), it contains a short sequence (Thr/Ser- and Glu/Asp-rich region) highly conserved between Syt XIV and Strep14 (named Syt XIV homology domain; Fig. 2, solid bars). The physiological meaning of this sequence is currently unknown, but it should have an important role(s) in the expression of Syt XIV function, because it has been retained during evolution. In addition, both the Syt XIV and Strep14 proteins contain a WHXL motif at the C terminus, which may be crucial for docking to the plasma membrane and correct folding of the C2B domain (50–52). Ca2+-Independent Oligomerization Property of Mouse Syt XIV Since the Syt family proteins form Ca2+-independent and -dependent oligomers (34, 40, 41, 45, 53) and Syt I is thought to function as an oligomer during neurotransmitter release (1, 37, 54, 55), I investigated the oligomerization property of Syt XIV and Strep14 by dual-tag coexpression assay (40). In brief, T7- and FLAG-tagged Syt XIV were coexpressed in COS-7 cells, and their association was determined by immunoprecipitation assay. As shown in Fig. 4A (lanes 1 and 2), FLAG-Syt XIV protein was efficiently co-immunoprecipitated with T7-Syt XIV protein irrespective of the presence of Ca2+, in the same way as the group B Syts (e.g., Syts I, II, and VIII) (45). The Ca2+-independent oligomerization of Syt XIV was mainly mediated by the fatty-acylated Cys residues between the transmembrane domain and spacer domain (41), rather than by a disulfide-bond at the extracellular domain (34, 40, 45), because the SDS-insensitive dimer band of Syt XIV on SDS-PAGE was unaffected by the presence of β-mercaptoethanol, which cleaves disulfide bonds (data not shown). Interestingly, Strep14 also showed relatively weak homo-oligomerization and hetero-oligomerization with Syt XIV irrespective of the presence of Ca2+ (Fig. 4B and Fig. 4A, lane 3 and 4), suggesting that Syt XIV and Strep14 may function as a complex in membrane transport. Phospholipid-Binding Property of C2 Domains of Mouse Syt XIV and Strep14 Although the C2 domain was originally described as a domain responsible for Ca2+-dependent translocation of proteins to phospholipid membranes (56), some C2 domains from C-type tandem C2 proteins have been shown to lack such Ca2+/phospholipid-binding activity (29, 30, 33, 35, 57, 58). I recently found that the presence of five conserved acidic residues in loops 1 and 3 of the C2 structure is a good marker for the Ca2+-dependent phospholipid-binding ability of the C2A domain of C-type tandem C2 proteins, except for Slp3-a and Slp5 (27, 57). In contrast to the Syt I C2A domain, the Syt XIV and Strep14 C2 domains lack such acidic residues (i.e., only one Glu residue is found in loop 3 of the C2 structure) (Fig. 2, asterisks), suggesting that the Syt XIV and Strep14 proteins should be classified as Ca2+-independent tandem C2 proteins. As expected, both C2 domains of Syt XIV and Strep14 fused to GST bound liposomes (phosphatidylcholine and phosphatidylserine, 1 : 1, w/w) in a Ca2+-independent manner (Fig. 5). Under the same experimental conditions, GST-Syt I-C2A binds PS/PC liposomes in a Ca2+-dependent manner, whereas GST alone did not show liposome binding activity at all (data not shown) (27, 33, 42, 43). Tissue Distribution of Syt XIV and Strep14 RT-PCR with specific primers was performed to investigate the tissue distribution of mouse Syt XIV and Strep14 (Fig. 6, top and middle panels). Syt XIV mRNA and Strep14 mRNA were found in almost the same tissues and at similar developmental stages, suggesting that the Syt XIV·Strep14 complex described above may be formed under physiological conditions. Their expression levels were highest in the heart and testis and moderate in the kidney. In addition, expression of Syt XIV and Strep14 mRNA was very weak (or absent) on embryonic day 7 (E7) and remained constant from E11 to E17. It is of great interest that mRNA expression of Syt XIV and Strep14 is almost absent in brain, because the mRNAs of other Syt isoforms (I-XIII) are easily detected in brain by RT-PCR analysis (35, 58, 59) and most of the Syt proteins (even non-neuronal or ubiquitous type Syt proteins) are abundant in brain (11, 12, 19, 60–63). As far as I know, Syt XIV is the first Syt isoform that is only expressed in selected non-neuronal tissues outside the brain. DISCUSSION In the present study, I isolated and characterized a novel Syt isoform, Syt XIV, and its related molecule, Srep14. The mammalian and Drosophila Syt XIV and Strep14 form a small branch of the phylogenetic tree of C-type tandem C2 protein families, and they possess a unique Thr/Ser- and Glu/Asp-rich sequence (named Syt XIV homology domain) at the N-terminal domain (downstream of the transmembrane domain of Syt XIV) (Figs. 1–3). Biochemical analyses indicated that both Syt XIV and Strep14 associate with phospholipids irrespective of the presence of Ca2+ and that Syt XIV and Strep14 can form homo- and hetero-oligomers even in the absence of Ca2+ (Figs. 4 and 5). Moreover, the mRNA expression profiles of Syt XIV and Strep14 in mouse tissues were almost identical, with both mRNAs being highly expressed in the heart, kidney, and testis, but not being expressed in the brain (Fig. 6). These results imply that Syt XIV, or possibly a Syt XIV·Strep14 complex, may be involved in non-neuronal-type membrane trafficking. Further work is necessary to elucidate the exact functions of Syt XIV and Strep14. A previous study by Craxton (36) identified six additional putative syt genes in the human genome (chr10 syt, chr11 syt, chr2 syt, chr3 syt, chr14 syt, and chr415 syt), and sequence comparison revealed that the human Syt XIV and Strep14 correspond to the products of chr415 syt and chr14 syt, respectively. Further database searching revealed the products of chr10 syt, chr11 syt, chr2 syt, and chr3 syt to be a single C2 domain-containing protein with a single N-terminal transmembrane domain (GenBank™ accession no., XM_089553), Slp2-a (26), KIAA1228, which contains three C2 domains with a single N-terminal transmembrane domain (64), and a protein highly homologous to the mouse MBC2 (membrane bound C2 domain containing protein) (GenBank™ accession no., XM_125941) or the rat GLUT4 vesicle protein (65), respectively. Therefore, the Syt XIV identified in this study is most likely to be the final member of the Syt family that is conserved from Drosophila to mammals. Phylogenetic relationships of the tandem C2 proteins from the mouse (black background), Drosophila (red background), C. elegans (blue background), A. thaliana (gray background), and yeast (boxed) are summarized in Fig. 3. The Syt family (magenta trees) is the largest family among the tandem C2 protein families, and 14 Syt proteins are found in the mouse (Syts I-XIV), 5 in C. elegans (Syts I, IV, VII, A, and B), 7 in Drosophila (Syts I, IV, VII, XII, XIV, A, and B), and 5 in the plant (at-Syts A-E). Mammalian Syts I, IV, and VII homologues are found in both Drosophila and C. elegans (66), but mammalian Syts XII and XIV homologues are found only in Drosophila. By contrast, no vertebrate homologues of the Syt III/V/VI/X subfamily (34, 45, 67) are found in invertebrates, and no invertebrate homologues of Syts A and B are found in vertebrates, suggesting that some Syt members may have a species-specific role(s). Interestingly, five members of the plant Syt family form a branch completely different from the animal Syt family (i.e., having a different origin from the animal syt genes), even though they have C-terminal tandem C2 domains and an N-terminal single transmembrane domain without an extracellular domain, the same structure as mouse Syt XIII (Fig. 1A). By careful database searching, I found that the plant Syt proteins are highly homologous to yeast Tricalbin proteins, which have three C2 domains and an N-terminal single transmembrane domain (Fig. 1A). As judged from the phylogenetic tree and sequence alignment (blue trees in Fig. 3), the plant Syt proteins probably evolved from the yeast Tricalbin proteins by losing a third C2 domain (i.e., C2C of Fig. 1A). Since most of the C2 domains of plant Syt and yeast Tricalbin contain four or five conserved Asp/Glu residues, which may be crucial for Ca2+-binding (56, 68, 69), they may function as Ca2+-dependent regulators of membrane trafficking, in the same way as the animal Syt proteins, although no research has been attempted on the plant Syt proteins and yeast Tricalbin proteins. Further work is necessary to determine whether they are indeed involved in membrane trafficking. Both Drosophila and C. elegans have one Rabphilin homologue, a putative Rab3/8/27 effector (31, 48, 70), but they do not have any Doc2 homologue (green trees in Fig. 3). Five Slp members (Slp1-5) are present in the mouse and humans, (26, 27) (yellow trees in Fig. 3), and they are thought to function as effectors for Rab27, one of the small GTP-binding proteins, by directly interacting with the N-terminal SHD (48, 49). Although one Drosophila Slp (named dm-Slp) has been found, it lacks an N-terminal SHD, as do the mouse Slp2-b and Slp3-b (26), and thus the dm-Slp is unlikely to function as a Rab27 effector. Since Rab27 is highly conserved from C. elegans to Drosophila and to humans (71), while other Rab27 effectors, Slps and Slac2s (48, 72, 73), are not found in invertebrates, what the Rab27 effector(s) are in invertebrates is an open question. In summary, I first identified a novel class of Syt proteins (Syt XIV), presumably the final Syt in mammals, and analyzed the phylogenetic relationships of the C-type tandem C2 proteins from five different species. The results indicate that the Syt family is the largest C-type tandem C2 protein family and that several Syt isoforms (e.g., Syts I, IV, VII, XII, and XIV) are evolutionarily conserved from Drosophila to humans, suggesting important roles of the Syt family proteins in membrane trafficking conserved across phylogeny. Future studies will clarify the function of individual Syt isoforms in membrane trafficking. Note Added in Proof: After the acceptance of this paper we found that chr10 syt also encoded a tandem C2 domain–containing protein with a single N-terminal transmembrane domain. We refer to the product of chr10 syt as synaptotagmin XV (Syt XV) [Biochem. Biophys. Res. Commun. (2003) 306, 64–71]. I thank Eiko Kanno and Yukie Ogata for expert technical assistance. This work was supported in part by Grants-in-Aid for Young Scientists (B) from the Ministry of Education, Culture, Sports, and Technology of Japan (13780624). The nucleotide sequence reported in this paper is deposited in the DDBJ, EMBL, and GenBank nucleotide sequence databases with accession numbers of AB102947-AB102952. + To whom correspondence should be addressed. Tel: +81-48-462-4994, Fax: +81-48-462-4995, E-mail: mnfukuda@brain.riken.go.jp. View largeDownload slide Fig. 1. Schematic representation of C-type tandem C2 protein families. (A) Syts from mouse (m), Drosophila (dm), and plant (at) and yeast (sc) Tricalbin. (B) Other C-type tandem C2 protein families from mice. The C-type tandem C2 protein is defined as a protein that contains tandem C2 domains (shaded boxes) at the C terminus. Mammalian Syt XIV, Drosophila Syt XIV, and Strep14 contain a Syt XIV homology domain (hatched boxes; see also Fig. 2, solid bars). Amino acid numbers are given on both sides. Y, glycosylation sites of Syt I; TM, transmembrane domain (black boxes); Cx, numbers of cysteine residues; Mid, Munc13-1 interacting domain; Rab3/8/27, Rab3/8/27 binding domain; Zn2+, zinc finger motif; SHD, Slp homology domain; RBD27, Rab binding domain specific for Rab27 isoforms. View largeDownload slide Fig. 1. Schematic representation of C-type tandem C2 protein families. (A) Syts from mouse (m), Drosophila (dm), and plant (at) and yeast (sc) Tricalbin. (B) Other C-type tandem C2 protein families from mice. The C-type tandem C2 protein is defined as a protein that contains tandem C2 domains (shaded boxes) at the C terminus. Mammalian Syt XIV, Drosophila Syt XIV, and Strep14 contain a Syt XIV homology domain (hatched boxes; see also Fig. 2, solid bars). Amino acid numbers are given on both sides. Y, glycosylation sites of Syt I; TM, transmembrane domain (black boxes); Cx, numbers of cysteine residues; Mid, Munc13-1 interacting domain; Rab3/8/27, Rab3/8/27 binding domain; Zn2+, zinc finger motif; SHD, Slp homology domain; RBD27, Rab binding domain specific for Rab27 isoforms. View largeDownload slide Fig. 2. Sequence alignment of the mouse, human, and Drosophila Syt XIV and its related molecule, Strep14. Residues conserved and similar between sequences are shown on a black background and shaded background, respectively. Asterisks indicate the five conserved aspartate or glutamate residues in the C2 domains, which may be crucial for Ca2+ binding by analogy with the Syt I-C2A domain (68, 69). The symbol # indicates the Cys residues that may be fatty-acylated between the transmembrane domain (large box) and spacer domains (41). The cysteine residues located at the extracellular domain of mammalian Syt XIV are indicated by small boxes. The solid lines indicate the Syt XIV homology domain conserved across phylogeny. The protein IDs for newly named proteins in this paper (see http://smart.embl-heidelberg.de/) are indicated in parentheses: ce-Syt VII (Q9TZL3), ce-Syt A (Q22372), ce-Syt B (O16700), ce-Strep9 (Q20354), ce-Tricalbin (O76835), dm-Syt A (Q9I7M6), dm-Syt B (Q9VUJ3), dm-Syt XII (Q9VYE1), dm-Syt XIV (Q9VN04), dm-Slp (Q9VFF9), dm-Tricalbin (Q9VC62), at-Syt B (Q9LNT5), at-Syt D (Q9FY55), at-Syt E (Q8L706), sc-Tricalbin A (YNI7_YEAST), sc-Tricalbin B (Q12466), and sc-Tricalbin C (YMH2_YEAST). The GenBank™ accession numbers of mouse and human Syt XIV and Strep14 and of plant Syts A and C are AB102947- AB102952, respectively. View largeDownload slide Fig. 2. Sequence alignment of the mouse, human, and Drosophila Syt XIV and its related molecule, Strep14. Residues conserved and similar between sequences are shown on a black background and shaded background, respectively. Asterisks indicate the five conserved aspartate or glutamate residues in the C2 domains, which may be crucial for Ca2+ binding by analogy with the Syt I-C2A domain (68, 69). The symbol # indicates the Cys residues that may be fatty-acylated between the transmembrane domain (large box) and spacer domains (41). The cysteine residues located at the extracellular domain of mammalian Syt XIV are indicated by small boxes. The solid lines indicate the Syt XIV homology domain conserved across phylogeny. The protein IDs for newly named proteins in this paper (see http://smart.embl-heidelberg.de/) are indicated in parentheses: ce-Syt VII (Q9TZL3), ce-Syt A (Q22372), ce-Syt B (O16700), ce-Strep9 (Q20354), ce-Tricalbin (O76835), dm-Syt A (Q9I7M6), dm-Syt B (Q9VUJ3), dm-Syt XII (Q9VYE1), dm-Syt XIV (Q9VN04), dm-Slp (Q9VFF9), dm-Tricalbin (Q9VC62), at-Syt B (Q9LNT5), at-Syt D (Q9FY55), at-Syt E (Q8L706), sc-Tricalbin A (YNI7_YEAST), sc-Tricalbin B (Q12466), and sc-Tricalbin C (YMH2_YEAST). The GenBank™ accession numbers of mouse and human Syt XIV and Strep14 and of plant Syts A and C are AB102947- AB102952, respectively. View largeDownload slide Fig. 3. Phylogenetic tree of the C-type tandem C2 protein families from various species. The phylogenetic tree is depicted as described under “materials and methods.” Note that the plant Syt family and the Tricalbin family forms a branch distinct from the animal Syt family (blue letters, mouse Syts), the Doc2/Rabphilin family (green letters), and the Slp family (yellow letters). The animal Syt family is further classified into several subfamilies (i.e., Syt I/II/IX subfamily, Syt III/V/VI/X subfamily, and Syt IV/XI subfamily). Proteins from mouse (m), Drosophila (dm), C. elegans (ce), the plant (at), and the yeast (sc) are indicated by a black background, red background, blue background, gray background, and boxes, respectively. View largeDownload slide Fig. 3. Phylogenetic tree of the C-type tandem C2 protein families from various species. The phylogenetic tree is depicted as described under “materials and methods.” Note that the plant Syt family and the Tricalbin family forms a branch distinct from the animal Syt family (blue letters, mouse Syts), the Doc2/Rabphilin family (green letters), and the Slp family (yellow letters). The animal Syt family is further classified into several subfamilies (i.e., Syt I/II/IX subfamily, Syt III/V/VI/X subfamily, and Syt IV/XI subfamily). Proteins from mouse (m), Drosophila (dm), C. elegans (ce), the plant (at), and the yeast (sc) are indicated by a black background, red background, blue background, gray background, and boxes, respectively. View largeDownload slide Fig. 4. Oligomerization properties of mouse Syt XIV and Strep14. (A) Homo- and hetero-oligomerization property of Syt XIV in COS-7 cells as revealed by coexpression assay. (B) Oligomerization of Strep14 in COS-7 cells as revealed by coexpression assay. pEF-T7-Syt XIV (or pEF-T7-Strep14) and pEF-FLAG-Syt XIV (or pEF-FLAG-Strep14) were cotransfected into COS-7 cells. Expressed proteins were solubilized with 1% Triton X-100 and immunoprecipitated by anti-T7 tag antibody-conjugated agarose as described under “materials and methods.” Co-immunoprecipitated FLAG-tagged proteins were first detected with HRP-conjugated anti-FLAG tag antibody (1:10,000 dilution) (middle panels, Blot: anti-FLAG and IP: anti-T7). Then the same blots were stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure that equivalent amounts of T7-tagged proteins had been loaded (1:10,000 dilution) (bottom panels, Blot: anti-T7 and IP: anti-T7). The upper panels indicate the total expressed FLAG-tagged proteins (1/80 volume of the reaction mixture) used for immunoprecipitation. Note that Syt XIV and Strep14 formed a weak Ca2+-independent hetero-oligomer (lanes 3 and 4 in A). The positions of the molecular weight markers (×10–3) are shown on the left. View largeDownload slide Fig. 4. Oligomerization properties of mouse Syt XIV and Strep14. (A) Homo- and hetero-oligomerization property of Syt XIV in COS-7 cells as revealed by coexpression assay. (B) Oligomerization of Strep14 in COS-7 cells as revealed by coexpression assay. pEF-T7-Syt XIV (or pEF-T7-Strep14) and pEF-FLAG-Syt XIV (or pEF-FLAG-Strep14) were cotransfected into COS-7 cells. Expressed proteins were solubilized with 1% Triton X-100 and immunoprecipitated by anti-T7 tag antibody-conjugated agarose as described under “materials and methods.” Co-immunoprecipitated FLAG-tagged proteins were first detected with HRP-conjugated anti-FLAG tag antibody (1:10,000 dilution) (middle panels, Blot: anti-FLAG and IP: anti-T7). Then the same blots were stripped and reprobed with HRP-conjugated anti-T7 tag antibody to ensure that equivalent amounts of T7-tagged proteins had been loaded (1:10,000 dilution) (bottom panels, Blot: anti-T7 and IP: anti-T7). The upper panels indicate the total expressed FLAG-tagged proteins (1/80 volume of the reaction mixture) used for immunoprecipitation. Note that Syt XIV and Strep14 formed a weak Ca2+-independent hetero-oligomer (lanes 3 and 4 in A). The positions of the molecular weight markers (×10–3) are shown on the left. View largeDownload slide Fig. 5. Phospholipid-binding properties of C2 domains of mouse Syt XIV and Strep14. Liposomes and GST fusion proteins were incubated in 50 mM HEPES-KOH, pH 7.2, in the presence of 2 mM EGTA or 1 mM Ca2+ for 15 min at room temperature. After centrifugation at 12,000 ×g for 10 min, the supernatants (non-binding fraction) and pellets (phospholipid-binding fraction) were separated as described previously (42, 43). The pelleted portion of the input samples was subjected to 10% SDS-PAGE and then stained with Coomassie Brilliant Blue R-250. Note that the C2 domains of Syt XIV (or Strep14) were recovered in the pelleted portion only in the presence of liposomes, but regardless of the presence of Ca2+. The results shown are representative of three independent experiments. View largeDownload slide Fig. 5. Phospholipid-binding properties of C2 domains of mouse Syt XIV and Strep14. Liposomes and GST fusion proteins were incubated in 50 mM HEPES-KOH, pH 7.2, in the presence of 2 mM EGTA or 1 mM Ca2+ for 15 min at room temperature. After centrifugation at 12,000 ×g for 10 min, the supernatants (non-binding fraction) and pellets (phospholipid-binding fraction) were separated as described previously (42, 43). The pelleted portion of the input samples was subjected to 10% SDS-PAGE and then stained with Coomassie Brilliant Blue R-250. Note that the C2 domains of Syt XIV (or Strep14) were recovered in the pelleted portion only in the presence of liposomes, but regardless of the presence of Ca2+. The results shown are representative of three independent experiments. View largeDownload slide Fig. 6. Tissue distribution of mouse Syt XIV and Strep14. RT-PCR analysis of Syt XIV (or Strep14) expression in various mouse tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis) and on embryonic days 7 (E7), 11, 15, and 17 (upper two 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. Note that tissue distributions of mouse Syt XIV and Strep14 mRNA were almost identical. Abundant expression was found in heart and testis, and moderate expression in kidney. “–” means without templates as a negative control. The size of the molecular weight markers (λ/StyI) is shown at the left of the panel. The results shown are representative of two independent experiments. View largeDownload slide Fig. 6. Tissue distribution of mouse Syt XIV and Strep14. RT-PCR analysis of Syt XIV (or Strep14) expression in various mouse tissues (heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis) and on embryonic days 7 (E7), 11, 15, and 17 (upper two 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. Note that tissue distributions of mouse Syt XIV and Strep14 mRNA were almost identical. Abundant expression was found in heart and testis, and moderate expression in kidney. “–” means without templates as a negative control. The size of the molecular weight markers (λ/StyI) is shown at the left of the panel. The results shown are representative of two independent experiments. References 1. Chapman, E.R. ( 2002) Synaptotagmin: a Ca2+ sensor that triggers exocytosis? Nat. Rev. Mol. Cell Biol.  3, 498–508 Google Scholar 2. Südhof, T.C. ( 2002) Synaptotagmins: why so many? J. Biol. Chem.  277, 7629–7632 Google Scholar 3. Marquèze, B., Berton, F., and Seagar, M. ( 2000) Synaptotagmins in membrane traffic: which vesicles do the tagmins tag? Biochimie  82, 409–420 Google Scholar 4. Schiavo, G., Osborne, S.L., and Sgouros, J.G. ( 1998) Synaptotagmins: more isoforms than functions? Biochem. Biophys. Res. Commun.  248, 1–8 Google Scholar 5. Fukuda, M. and Mikoshiba, K. ( 1997) The function of inositol high polyphosphate binding proteins. Bioessays  19, 593–603 Google Scholar 6. Thomas, D.M., Ferguson, G.D., Herschman, H.R., and Elferink, L.A. ( 1999) Functional and biochemical analysis of the C2 domains of synaptotagmin IV. Mol. Biol. Cell  10, 2285–2295 Google Scholar 7. Brown, H., Meister, B., Deeney, J., Corkey, B.E., Yang, S.N., Larsson, O., Rhodes, C.J., Seino, S., Berggren, P.O., and Fried, G. ( 2000) Synaptotagmin III isoform is compartmentalized in pancreatic β-cells and has a functional role in exocytosis. Diabetes  49, 383–391 Google Scholar 8. Gao, Z., Reavey-Cantwell, J., Young, R.A., Jegier, P., and Wolf, B.A. ( 2000) Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet β-cells. J. Biol. Chem.  275, 36079–36085 Google Scholar 9. Gut, A., Kiraly, C.E., Fukuda, M., Mikoshiba, K., Wollheim, C.B., and Lang, J. ( 2001) Expression and localisation of synaptotagmin isoforms in endocrine β-cells: their function in insulin exocytosis. J. Cell Sci.  114, 1709–1716 Google Scholar 10. Sugita, S., Shin, O.H., Han, W., Lao, Y., and Südhof, T.C. ( 2002) Synaptotagmins form a hierarchy of exocytotic Ca2+ sensors with distinct Ca2+ affinities. EMBO J.  21, 270–280 Google Scholar 11. Fukuda, M., Kowalchyk, J.A., Zhang, X., Martin, T.F.J., and Mikoshiba, K. ( 2002) Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells. J. Biol. Chem.  277, 4601–4604 Google Scholar 12. Saegusa, C., Fukuda, M., and Mikoshiba, K. ( 2002) Synaptotagmin V is targeted to dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells. J. Biol. Chem.  277, 24499–24505 Google Scholar 13. Fukuda, M., Kanno, E., Ogata, Y., Saegusa, C., Kim, T., Peng Loh, Y., and Yamamoto, A. ( 2003) Nerve growth factor-dependent sorting of synaptotagmin IV protein to mature dense-core vesicles that undergo calcium-dependent exocytosis in PC12 cells. J. Biol. Chem.  278, 3220–3226 Google Scholar 14. Kabayama, H., Takei, K., Fukuda, M., Ibata, K., and Mikoshiba, K. ( 1999) Functional involvement of synaptotagmin I/II C2A domain in neurite outgrowth of chick dorsal root ganglion neuron. Neuroscience  88, 999–1003 Google Scholar 15. Fukuda, M. and Mikoshiba, K. ( 2000) Expression of synaptotagmin I or II promotes neurite outgrowth in PC12 cells. Neurosci. Lett.  295, 33–36 Google Scholar 16. Michaut, M., De Blas, G., Tomes, C.N., Yunes, R., Fukuda, M., and Mayorga, L.S. ( 2001) Synaptotagmin VI participates in the acrosome reaction of human spermatozoa. Dev. Biol.  235, 521–529 Google Scholar 17. 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 Google Scholar 18. Detrait, E.R., Yoo, S., Eddleman, C.S., Fukuda, M., Bittner, G.D., and Fishman, H.M. ( 2000) Plasmalemmal repair of severed neurites of PC12 cells requires Ca2+ and synaptotagmin. J. Neurosci. Res.  62, 566–573 Google Scholar 19. Reddy, A., Caler, E.V., and Andrews, N.W. ( 2001) Plasma membrane repair is mediated by Ca2+-regulated exocytosis of lysosomes. Cell  106, 157–169 Google Scholar 20. Bommert, K., Charlton, M.P., DeBello, W.M., Chin, G.J., Betz, H., and Augustine, G.J. ( 1993) Inhibition of neurotransmitter release by C2-domain peptides implicates synaptotagmin in exocytosis. Nature  363, 163–165 Google Scholar 21. Mikoshiba, K., Fukuda, M., Moreira, J.E., Lewis, F.M.T., Sugimori, M., Niinobe, M., and Llinás, R. ( 1995) Role of the C2A domain of synaptotagmin in transmitter release as determined by specific antibody injection into the squid giant synapse preterminal. Proc. Natl. Acad. Sci. USA  92, 10703–10707 Google Scholar 22. Fukuda, M., Moreira, J.E., Lewis, F.M.T., Sugimori, M., Niinobe, M., Mikoshiba, K., and Llinás, R. ( 1995) Role of the C2B domain of synaptotagmin in vesicular release and recycling as determined by specific antibody injection into the squid giant synapse preterminal. Proc. Natl. Acad. Sci. USA  92, 10708–10712 Google Scholar 23. Fernandez-Chacon, R., Konigstorfer, A., Gerber, S.H., Garcia, J., Matos, M.F., Stevens, C.F., Brose, N., Rizo, J., Rosenmund, C., and Südhof, T.C. ( 2001) Synaptotagmin I functions as a calcium regulator of release probability. Nature  410, 41–49 Google Scholar 24. Mackler, J.M., Drummond, J.A., Loewen, C.A., Robinson, I.M., and Reist, N.E. ( 2002) The C2B Ca2+-binding motif of synaptotagmin is required for synaptic transmission in vivo. Nature  418, 340–344 Google Scholar 25. Fukuda, M. and Mikoshiba, K. ( 2001) Synaptotagmin-like protein 1–3: a novel family of C-terminal-type tandem C2 proteins. Biochem. Biophys. Res. Commun.  281, 1226–1233 Google Scholar 26. Fukuda, M., Saegusa, C., and Mikoshiba, K. ( 2001) Novel splicing isoforms of synaptotagmin-like proteins 2 and 3: identification of the Slp homology domain. Biochem. Biophys. Res. Commun.  283, 513–519 Google Scholar 27. Kuroda, T.S., Fukuda, M., Ariga, H., and Mikoshiba, K. ( 2002) Synaptotagmin-like protein 5: a novel Rab27A effector with C-terminal tandem C2 domains. Biochem. Biophys. Res. Commun.  293, 899–906 Google Scholar 28. Kwon, O.J., Gainer, H., Wray, S., and Chin, H. ( 1996) Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney. FEBS Lett.  378, 135–139 Google Scholar 29. Fukuda, M. and Mikoshiba, K. ( 2001) The N-terminal cysteine cluster is essential for membrane targeting of B/K protein. Biochem. J.  360, 441–448 Google Scholar 30. Fukuda, M. and Mikoshiba, K. ( 2001) Tac2-N, an atypical C-type tandem C2 protein localized in the nucleus. FEBS Lett.  503, 217–218 Google Scholar 31. Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, S., Yamaguchi, T., Wada, K., Miyazaki, M., and Takai, Y. ( 1993) Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol. Cell. Biol.  13, 2061–2068 Google Scholar 32. Duncan, R.R., Shipston, M.J., and Chow, R.H. ( 2000) Double C2 protein: a review. Biochimie  82, 421–426 Google Scholar 33. Fukuda, M., Saegusa, C., Kanno, E., and Mikoshiba, K. ( 2001) The C2A domain of double C2 protein γ contains a functional nuclear localization signal. J. Biol. Chem.  276, 24441–24444 Google Scholar 34. Fukuda, M., Kanno, E., and Mikoshiba, K. ( 1999) Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X. J. Biol. Chem.  274, 31421–31427 Google Scholar 35. Fukuda, M. and Mikoshiba, K. ( 2001) Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII). Biochem. J.  354, 249–257 Google Scholar 36. Craxton, M. ( 2001) Genomic analysis of synaptotagmin genes. Genomics  77, 43–49 Google Scholar 37. Adolfsen, B. and Littleton, J.T. ( 2001) Genetic and molecular analysis of the synaptotagmin family . Cell. Mol. Life Sci.  58, 393–402 Google Scholar 38. Mizushima, S. and Nagata, S. ( 1990) pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res.  18, 5332 Google Scholar 39. Fukuda, M., Aruga, J., Niinobe, M., Aimoto, S., and Mikoshiba, K. ( 1994) Inositol-1, 3, 4, 5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J. Biol. Chem.  269, 29206–29211 Google Scholar 40. Fukuda, M. and Mikoshiba, K. ( 2000) Distinct self-oligomerization activities of synaptotagmin family: unique calcium-dependent oligomerization properties of synaptotagmin VII. J. Biol. Chem.  275, 28180–28185 Google Scholar 41. Fukuda, M., Kanno, E., Ogata, Y., and Mikoshiba, K. ( 2001) Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains. J. Biol. Chem.  276, 40319–40325 Google Scholar 42. Fukuda, M., Kojima, T., and Mikoshiba, K. ( 1996) Phospholipid composition dependence of Ca2+-dependent phospholipid binding to the C2A domain of synaptotagmin IV. J. Biol. Chem.  271, 8430–8434 Google Scholar 43. Fukuda, M., Kojima, T., and Mikoshiba, K. ( 1997) Regulation by bivalent cations of phospholipid binding to the C2A domain of synaptotagmin III. Biochem. J.  323, 421–425 Google Scholar 44. Kyte, J. and Doolittle, R.F. ( 1982) A simple method for displaying the hydropathic character of a protein. J. Mol. Biol.  157, 105–132 Google Scholar 45. Fukuda, M. and Mikoshiba, K. ( 2000) Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family. J. Biochem. (Tokyo)  128, 637–645 Google Scholar 46. Fukuda, M. ( 2002) Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I. J. Biol. Chem.  277, 30351–30358 Google Scholar 47. Orita, S., Naito, A., Sakaguchi, G., Maeda, M., Igarashi, H., Sasaki, T., and Takai, Y. ( 1997) Physical and functional interactions of Doc2 and Munc13 in Ca2+-dependent exocytotic machinery. J. Biol. Chem.  272, 16081–16084 Google Scholar 48. Kuroda, T.S., Fukuda, M., Ariga, H., and Mikoshiba, K. ( 2002) The Slp homology domain of synaptotagmin-like proteins 1–4 and Slac2 functions as a novel Rab27A binding domain. J. Biol. Chem.  277, 9212–9218 Google Scholar 49. Fukuda, M. ( 2002) Synaptotagmin-like protein (Slp) homology domain 1 of Slac2-a/melanophilin is a critical determinant of GTP-dependent, specific binding to Rab27A. J. Biol. Chem.  277, 40118–40124 Google Scholar 50. Perin, M.S. ( 1994) The COOH terminus of synaptotagmin mediates interaction with the neurexins. J. Biol. Chem.  269, 8576–8581 Google Scholar 51. Fukuda, M., Moreira, J.E., Liu, V., Sugimori, M., Mikoshiba, K., and Llinás, R.R. ( 2000) Role of the conserved WHXL motif in the C terminus of synaptotagmin in synaptic vesicle docking. Proc. Natl. Acad. Sci. USA  97, 14715–14719 Google Scholar 52. Fukuda, M., Yamamoto, A., and Mikoshiba, K. ( 2001) Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus: structural importance of the WHXL motif in the C2B domain. J. Biol. Chem.  276, 41112–41119 Google Scholar 53. Fukuda, M., Katayama, E., and Mikoshiba, K. ( 2002) The calcium-binding loops of the tandem C2 domains of synaptotagmin VII cooperatively mediate calcium-dependent oligomerization. J. Biol. Chem.  277, 29315–29320 Google Scholar 54. Fukuda, M., Kabayama, H., and Mikoshiba, K. ( 2000) Drosophila AD3 mutation of synaptotagmin impairs calcium-dependent self-oligomerization activity. FEBS Lett.  482, 269–272 Google Scholar 55. Littleton, J.T., Bai, J., Vyas, B., Desai, R., Baltus, A.E., Garment, M.B., Carlson, S.D., Ganetzky, B., and Chapman, E.R. ( 2001) synaptotagmin mutants reveal essential functions for the C2B domain in Ca2+-triggered fusion and recycling of synaptic vesicles in vivo. J. Neurosci.  21, 1421–1433 Google Scholar 56. Nalefski, E.A. and Falke, J.J. ( 1996) The C2 domain calcium-binding motif: structural and functional diversity. Protein Sci.  5, 2375–2390 Google Scholar 57. Fukuda, M. ( 2002) The C2A domain of synaptotagmin-like protein 3 (Slp3) is an atypical calcium-dependent phospholipid-binding machine: comparison with the C2A domain of synaptotagmin I. Biochem. J.  366, 681–687 Google Scholar 58. Li, C., Ullrich, B., Zhang, J.Z., Anderson, R.G.W., Brose, N., and Südhof, T.C. ( 1995) Ca2+-dependent and -independent activities of neural and non-neural synaptotagmins. Nature  375, 594–599 Google Scholar 59. Fukuda, M., Ogata, Y., Saegusa, C., Kanno, E., and Mikoshiba, K. ( 2002) Alternative splicing isoforms of synaptotagmin VII in the mouse, rat, and human. Biochem. J.  365, 173–180 Google Scholar 60. Butz, S., Fernandez-Chacon, R., Schmitz, F., Jahn, R., and Südhof, T.C. ( 1999) The subcellular localizations of atypical synaptotagmins III and VI: synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles. J. Biol. Chem.  274, 18290–18296 Google Scholar 61. Fukuda, M. and Mikoshiba, K. ( 1999) A novel alternatively spliced variant of synaptotagmin VI lacking a transmembrane domain: implications for distinct functions of the two isoforms. J. Biol. Chem.  274, 31428–31434 Google Scholar 62. Ibata, K., Fukuda, M., Hamada, T., Kabayama, H., and Mikoshiba, K. ( 2000) Synaptotagmin IV is present at the Golgi and distal parts of neurites. J. Neurochem.  74, 518–526 Google Scholar 63. Potter, G.B., Facchinetti, F., Beaudoin, G.M., III., and Thompson, C.C. ( 2001) Neuronal expression of synaptotagmin-related gene 1 is regulated by thyroid hormone during cerebellar development. J. Neurosci.  21, 4373–4380 Google Scholar 64. Nagase, T., Ishikawa, K., Kikuno, R., Hirosawa, M., Nomura, N., and Ohara, O. ( 1999) Prediction of the coding sequences of unidentified human genes. XV. The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro. DNA Res.  6, 337–345 Google Scholar 65. Morris, N.J., Ross, S.A., Neveu, J.M., Lane, W.S., and Lienhard, G.E. ( 1999) Cloning and preliminary characterization of a 121 kDa protein with multiple predicted C2 domains. Biochim. Biophys Acta  1431, 525–530 Google Scholar 66. Littleton, J.T., Serano, T.L., Rubin, G.M., Ganetzky, B., and Chapman, E.R. ( 1999) Synaptic function modulated by changes in the ratio of synaptotagmin I and IV. Nature  400, 757–760 Google Scholar 67. Ibata, K., Fukuda, M., and Mikoshiba, K. ( 1998) Inositol 1, 3, 4, 5-tetrakisphosphate binding activities of neuronal and nonneuronal synaptotagmins: identification of conserved amino acid substitutions that abolish inositol 1, 3, 4, 5-tetrakisphosphate binding to synaptotagmins III, V, and X. J. Biol. Chem.  273, 12267–12273 Google Scholar 68. Sutton, R.B., Davletov, B.A., Berghuis, A.M., Südhof, T.C., and Sprang, S.R. ( 1995) Structure of the first C2 domain of synaptotagmin I: a novel Ca2+/phospholipid-binding fold. Cell  80, 929–938 Google Scholar 69. Shao, X., Davletov, B.A., Sutton, R.B., Südhof, T.C., and Rizo, J. ( 1996) Bipartite Ca2+-binding motif in C2 domains of synaptotagmin and protein kinase C. Science  273, 248–251 Google Scholar 70. Fukuda, M. ( 2003) Distinct Rab binding specificity of Rim1, Rim2, Rabphilin, and Noc2: identification of a critical determinant of Rab3A/Rab27A recognition by Rim2. J. Biol. Chem.  278, 15373–15380 Google Scholar 71. Pereira-Leal, J.B. and Seabra, M.C. ( 2000) The mammalian Rab family of small GTPases: idefinition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. J. Mol. Biol.  301, 1077–1087 Google Scholar 72. Fukuda, M., Kuroda, T.S., and Mikoshiba, K. ( 2002) Slac2-a/melanophilin, the missing link between Rab27 and myosin Va: implications of a tripartite protein complex for melanosome transport. J. Biol. Chem.  277, 12432–12436 Google Scholar 73. Fukuda, M. and Kuroda, T.S. ( 2002) Slac2-c (synaptotagmin-like protein homologue lacking C2 domains-c), a novel linker protein that interacts with Rab27, myosin Va/VIIa, and actin. J. Biol. Chem.  277, 43096–43103 Google Scholar Author notes Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198
TI - Molecular Cloning, Expression, and Characterization of a Novel Class of Synaptotagmin (Syt XIV) Conserved from Drosophila to Humans
JF - The Journal of Biochemistry
DO - 10.1093/jb/mvg082
DA - 2003-05-01
UR - https://www.deepdyve.com/lp/oxford-university-press/molecular-cloning-expression-and-characterization-of-a-novel-class-of-g3VZtkD0Io
SP - 641
EP - 649
VL - 133
IS - 5
DP - DeepDyve
ER -