Proc. Nati. Acad. Sci. USA Vol. 91, pp. 8195-8199, August 1994 Neurobiology A third , Syt3, in the mouse (calcium/central nervous system/exocytoss/synaptic vesicles/PC12 cells) BRIAN S. HILBUSH* AND JAMES I. MORGAN Roche Institute of Molecular Biology, Roche Research Center, 340 Kingsland Street, Nutley, NJ 07110 Communicated by John J. Burns, May 9, 1994

ABSTRACT The are integral membrane Microinjection of antibodies directed against the cytoplasmic ofsynaptic vesicles thought to serve as Ca2+ sensors in domains of synaptotagmins significantly reduces depolariza- the process ofvesicular trafficking and exocytosis. Results from tion-induced catecholamine release in PC12 cells (10). (ii) antibody microinjection and gene-disruption experiments have Injection of -derived peptides into squid giant led to a controversy over whether synaptotagmi are essential presynaptic nerve terminals inhibits evoked neurotransmitter for neurotransmission. However, the studies casting doubt on release (11). Although these studies established that synap- the role of synaptotgins have assumed that no further totagmins can play a role in neurosecretion, there has been a isoforms of these molecules exist. Here we report the isolation debate as to whether their function is indispensable for neu- of a third member of the synaptotagmin family (Syt3) from rotransmission. For example, clones of PC12 cells that are mouse brain. Although retaining the characteristic five-domain deficient for synaptotagmins still release catecholamines (12). structure of the other synaptotagmins, SYT3 is considerably Furthermore, mutations in Drosophila (13) and Caenorhabdi- more divergent at the level ofamino acid sequence. In the most tis elegans (14) that perturb the synaptotagmin (Syt) locus highly conserved C2 domain, the mammalian synaptotagmins, result in a compromise of neural function, although some SYTi and SYT2, share 88% sequence identity, whereas SYT3 neurotransmission still occurs. However, in other genetic has only -45% identity to either. Overall, SYT3 has the studies in Drosophila where the Syt locus was disrupted, greatest sequence identity with rat SYT2 and marine ray p65A synaptotagmin was essential for Ca2+-dependent release (15). (both 37%), although homology to all of the known synap- One explanation for these discrepancies may reside in the totagmins is >30%. However, SYT3 is most like p65C when distinction between spontaneous and evoked release of neu- comparing domain structure. Syt3 is expressed in many regions rotransmitter. However, another explanation would be the ofthe nervous system but is undetectable in extraneural tissues. existence of additional synaptotagmin family members. The three murine synaptotagmins have differential expression Molecular cloning has identified two synaptotagmin patterns in the brain. Furthermore, in PC12 cells, Syt3 is in mammals [(3, 5), referred to here as Syti and Syt2], coexpressed with Sytl and is more abundant than the latter. although a third gene is known in the marine ray (6). Syn- This result suggests that individual neurons may have specific aptotagmin homologs have been identified in species from C. combinations of synaptotagmins that could provide for diver- elegans (14) to human (16), and sequence comparisons show sity in vesicular release. that the domain structure of all synaptotagmins has been conserved, especially within the C2 region. For example, the The Ca2+-dependent release of neurotransmitters and neu- rat Syti and Syt2 genes encode proteins that share an 88% ropeptides from the presynaptic nerve terminal occupies a identity at the amino acid level across both C2 repeats (3), and central role in synaptic transmission (1). The basic events rat and Drosophila Syti homologs maintain a 78% identity in involved in neurotransmitter release include the mobiliza- this region (16). The high degree of conservation of the tion, docking, and fusion of synaptic vesicles with the pre- synaptotagmin structure throughout evolution indicates that synaptic membrane. Although these steps use many mole- members of this gene family perform fundamental and con- cules and processes that are ubiquitous components of gen- served functions in neurosecretion. eral membrane trafficking, the mechanism underlying the Here we report the cloning of a cDNA encoding a third Ca2+-evoked release characteristic of neurons is incom- synaptotagmin isoform, Syt3, in mouse.t The deduced amino pletely understood. However, at least some ofthe molecules acid sequence of this member shows that SYT3, while that provide for Ca2+-dependent vesicular release are thought retaining the domain structure ofall previously characterized to be located on the surface of the , where synaptotagmins, is the most divergent synaptotagmin known. they might mediate membrane fusion after stimulation (2). The identification of a more structurally divergent synap- Attractive candidates for the Ca2+ sensor are members of totagmin has a number of important implications for the the synaptotagmin (p65) family (3-6). The synaptotagmins possible mechanism ofaction ofthis class of synaptic are integral membrane proteins of synaptic vesicles and in neurotransmitter release. chromaffin granules and are found only in neural and neu- roendocrine tissues (3, 6). The hallmark of these proteins is MATERIALS AND METHODS a large cytoplasmic domain containing two internal repeats Northern Blot Analysis. Total RNA was prepared from that have homology to the C2 regulatory region found in frozen tissues or cells using the RNAzol B method (17). RNA several isoforms ofprotein kinase C (5, 7) and phospholipase samples (5 Mg) were electrophoresed in 0.8% agarose- A2 (8). The C2 domains of these proteins constitute a W+- formaldehyde gels, transferred to nitrocellulose, and hybrid- and phospholipid-binding motif that in synaptotagmin are ized with 32P-labeled cDNA probes, as described (18). thought to function both in Ca2+ sensing and as a component Probe Preparation. Probes for murine Sytl, -2, and -3 were of the fusion machinery (9). Several studies suggest that the generated by PCR and subcloned into Bluescript II KS+ C2 repeats ofsynaptotagmins are essential for its function. (i) Abbreviation: CNS, central nervous system. The publication costs ofthis article were defrayed in part by page charge *To whom reprint requests should be addressed. payment. This article must therefore be hereby marked "advertisement" tThe sequence reported in this paper has been deposited in the in accordance with 18 U.S.C. §1734 solely to indicate this fact. GenBank data base (accession no. U10355).

8195 Downloaded by guest on October 2, 2021 81% Neurobiology: Hilbush and Morgan Proc. Natl. Acad Sci. USA 91 (1994) (Stratagene). Adult mouse brain cDNA was obtained by domain, and a C-terminal tail. Kyte-Doolittle analysis of reverse transcription of RNA using oligo(dT) as primer em- SYT3 identified a single hydrophobic region of 25 amino ploying the Superscript system (GIBCO/BRL). Nondegener- acids near the N terminus (Fig. 2A). This potential trans- ate primer pairs were used to amplify a portion of each Syt membrane region in SYT3 is preceded by a short stretch of cDNA corresponding to the amino acid positions as follows: 13 amino acids with no significant homology to N-terminal Syti, 55-61 and 261-267; Syt2, 108-115 and 293-299, and sequences in other synaptotagmins. The hydrophobic do- Syt3, 212-218 and 362-368. Oligonucleotide primers (100 main is followed by a 109-amino acid stretch containing pmol) were used in a final reaction volume of 100 ,ul containing several serine/threonine-rich clusters. The C-terminal half of an aliquot of cDNA following the recommendations of the SYT3 contains two internal repeats that are highly homolo- manufacturer. Cycling conditions consisted of an initial dena- gous to the C2A and C2B repeats found in all synaptotagmins turation step at 94TC for 5 min, followed by 940C for 30 sec, and rabphilin 3A, a rab3A-binding protein (22) and to the 550C for 30 sec, and 720C for 30 sec (25 times) with a final 720C single C2 regulatory domain of several protein kinase C for 10 min using the Perkin-Elmer system 9600 thermal cycler. isoforms (7). Because SYT3 possesses a hydrophobic do- PCR Amplification of Syt. Degenerate oligonucleotide main, it is unlikely to be an additional rabphilin member primer pairs were designed to amplify cDNAs encoding because rabphilin 3A lacks a transmembrane domain. There- potential C2 domains present in synaptotagmins by PCR fore, the overall domain organization characteristic of the using 100 pmol in a reaction volume of 100 Al: C2.1, 5'- synaptotagmins is conserved in SYT3. ATACGGATCCA(T/C)(A/C)CNTA(T/C)(T/C)T; C2.2, 5'- Data base searches using the TFASTA algorithm (19) and ATAGGAATICCA(A/T)(A/G)GGACC(A/C/G)AAC(A/ subsequent protein alignments show that SYT3 is most G)TC. Restriction sites for BamHI and EcoRI were included closely related to synaptotagmins. The overall identity of the to facilitate subcloning and are underlined; degenerate posi- amino acid sequences composing the C2 repeats of SYT3 was tions are in parentheses. Cycling conditions used were 94°C highest when compared with other synaptotagmins (43-47%) for 5 min, 42°C for 1.5 min, and 72°C for 1.5 min (once); 94°C than to either rabphilin 3A (37%) or protein kinase C isoforms for 1 min, 45°C for 1.5 min, and 72°C for 1.5 min (three times); (27-29%). The homology of SYT3 to the synaptotagmin and 94°C for 1 min, 50°C for 1 min, and 72°C for 1 min (25 family extends outside of the C2 domain, as significant times). identity was found in the conserved C-terminal tail region. Library Screening. cDNA was prepared from 5 pg of Comparison of the amino acid sequence for the C2 domain poly(A)+ mRNA isolated from adult mouse brain and intro- and C-terminal tail of murine SYT3 to those of synaptotag- duced into the Uni-ZAP XR vector (Stratagene). Approxi- mins from rat (SYT1 and -2) and marine ray (p65A, -B, and mately 5 x 104 recombinants were screened using the Syt3 -C) is shown in Fig. 2B. Only limited was PCR probe and two related clones, p122 (1.5 kb) and p171 (4.0 detected outside of the C-terminal half of SYT3 when com- kb), were isolated and sequenced on both strands. Compar- pared with other synaptotagmins. The overall sequence iden- ison of the nucleotide sequences of these clones revealed an tity of SYT3 to these synaptotagmins is as follows: SYT1, exact match in the overlapping region. DNA sequencing 31%; SYT2, 37%; p65A, 37%; p65B, 34%; p65C, 34%. By reactions were done with the Prism cycle sequencing kit comparison, SYT3 shares a 28% identity overall to rabphilin (Applied Biosystems) and electrophoresed and analyzed on 3A. Sequence comparisons were also done between SYT3 an Applied Biosystems 373 DNA sequencer. The p171 clone and murine SYT1 and -2. cDNAs corresponding to portions contained a single open reading frame and coupled transcrip- of SYT1 and -2 were subcloned and sequenced from the tion/translation reactions with this clone resulted in the mouse (see Materials and Methods). Murine SYT1 and -2 are synthesis of a 47.5-kDa protein, as predicted from the pro- highly homologous to their rat counterparts (>95% overall posed initiator methionine (data not shown). amino acid identity) and are clearly distinct from SYT3. Data Base Searches and Protein Alignments. Data base Thus, in the absence of functional data, these results suggest searches were done by using the TFASTA algorithm (19), and that SYT3 represents a third, highly divergent member of the analysis ofsequence relatedness and protein alignments were synaptotagmin family in rodents. done with the GAP and PILEUP programs (version 7.3, Ge- Expression of Sytl, -2, and -3 mRNA in Mouse Tissue and netics Computer Group, Madison, WI). The symbol compar- PC12 Cells. The synaptotagmins are vesicle proteins that are ison table used for assigning amino acid relatedness was as expressed only in neural and neuroendocrine tissues (3, 4, 6). described in ref. 20. To determine whether Syt3 is expressed in a similar manner, the distribution of Syt transcripts in various tissues was analyzed by Northern blot (Fig. 3). A cDNA probe specific RESULTS for Syt3 recognized a single transcript of -4.5 kb in brain Cloning and Structure of Murine Synaptotagmin 3. Degener- tissue. Syt3 mRNA was absent from all other tissues exam- ate oligonucleotide primer pairs were designed to C2 domains ined, including adrenal gland, but was present as an abundant to amplify cDNAs encoding unusual synaptotagmins by PCR. transcript in PC12 cells, a cell type that harbors both cate- One primer pair (C2.1 and C2.2) amplified a 450-bp fiagment cholamine-containing dense-core vesicles and small synaptic that appeared to encode a C2 domain. This 450-bp cDNA was vesicles. The brain-specific restriction of Syt3 is similar to used as a probe to screen an adult mouse brain cDNA library that found for Syt2 (Fig. 3 and ref. 3), although no expression (see Materials and Methods). Two clones were identified that of Syt2 could be detected in PC12 cells as reported (12). contained significant homology to sequences encoding the C2 Analysis of Sytl expression revealed an abundant transcript repeats present in synaptotagmin (5). The longest clone, p171 in brain and a low level of expression in adrenal tissue (Fig. (4.0 kb), contained a single open reading frame predicted to 3). Surprisingly, in comparison with Syt3, Sytl was expressed encode a protein of 425 amino acids (Fig. 1). at very low levels in PC12 cells (Fig. 3). These data indicate The predicted amino acid sequence of the p171 clone and that Syt3 is expressed in a neural-specific manner in mouse its overall structure identified the protein as another member tissue and represents the most abundant synaptotagmin of the synaptotagmin family, which we designate SYT3. mRNA in PC12 cells. Synaptotagmins are defined by a characteristic primary Syt Gene Expression in Mouse Central Nervous System structure consisting of five domains (21). These include an (CNS). The distribution of Syt transcripts in both the rat and N-terminal domain with low sequence conservation among marine ray nervous systems indicates that members of this members, a single hydrophobic stretch of25-27 amino acids, gene family are expressed in a complementary and restricted a spacer region of variable length, the highly conserved C2 manner (3, 6). To examine whether Syt3 is expressed coor- Downloaded by guest on October 2, 2021 Neurobiology: Hilbush and Morgan Proc. Natl. Acad. Sci. USA 91 (1994) 8197

1 !AAU 85 86 :AC 170 171 255 256 340 341 425 426 510 511 595 596 680 681 ITG 765 766 TG 850 851 CA 935 936 CA 1020 1021 AG 1105 1106 1190 1191. V:G 1275 1276 "'TCrcx 1360 1361 TI 1445 1446 Au 1530 1531 .Tc 1615 1616 1700 1701 1785 1 M A P I TT S R V E F D E I P T V V G I F S A F 24 1786 ILGGCCICGIICtTCTGAGCTAGATCAGA CAAATCCAACAAG&CTCCTCCATACAA 1870 25 G L V F T V S L F A W I C C Q R R S A K S N R T P P Y K 52 1871 GIrrGT~CACGTGCTrAAAGGAGTTGATATCTACCCAGAAAACCTAAGTAGCCAAAAGAAGTTTrGAGGAGATGACAAGAGTGAA 1955 53 F V H V L R G. V D I Y P E N L S S Q K R F G G DD K S E 80 1956 GTGAAGGGTAAAACCGCTCGCCCAACC CCCTGCATGACCTA GAAGCGAGACCTCAATGGCAACTTCCCCAAAGCCA 2040 81 V K G K T A L P N L S L H L D L E K R D L N G N F P K A N 109 2041 ACCCCAC CGCTCTATCTGGAATGTCACCCCAAGCTCrACGGAGACAGAAGGAGGCCAATTCCCCTGA 2125 110 P K A G S S S D L E N V T P R L F T E T E X E A N S P E 137 2126 GAGCTGAAGCAGCACTCCC GTCAGAGGAG AGAGA ACA AGAGTACAACTTC 2210 138 S L K S S T S L T S E E K Q E KL G T L F L S LE Y N F 165 2211 GAGAAGAAAGCATT'GTGGTGAACATCAAGGAAGCCCAGGGCTTACCAGCCATBGACGAGCAATCCATGACCTCTGACCCGTACA 2295 166 E KK A F V V N I K E A Q G L P A M D E Q S M T S D P Y I 194 2296 TCAAAATrACAATC¶rACCAGAGAAGAAGCACAGAGG.AAGACCAGAGTGCTCAGGAAGACGCTGGACCCTG'1'r?1'GGTGAAAC 2380 195 K M T I L P E K K H R V K T R V LR K T L D P V F G E T 222 2381 CTTCACATTCTATGGAATCCCTTATCCC CACACCTTTCTAGA 2465 223 F T F Y G I P Y P H I Q E L S L HF T V L S F D R F S R 250 2466 GATGATGTCATTGGAGAAGTCCTGATTCCCTCAGGGAT1'GAATTATCAGATGGAAAAATGTTAATGATAAGAGATCATCA 2550 251 D D V I G E V L I P L S G I EL S D G K M L M T R E I K 279 2551 AGAGAAATGCTAAGAAGTCTTCTGGCGCGGTGAACT-T1TCTGG FATCAGTCCACTACAAACACGCTCACTGTGGT 2635 280 R N A KK S S G R G E L L V S L C Y Q S T T N T L T V V 307 2636 GGTCTTAAAAGCGCGGCACCTACCGAAATGTCTGGATCTGCTTCAGATCCCTACGTCAAAGTGAACCTGTACCATGCCAAG 2720 308 V L K A R H L P K S D V S G L S D P Y V K V N L Y H A K 335 2721 AAGAGAATCTCTAAAAAGAAGACTCACGTIrAC^AGAAA=GCACCAACGCAGTGTTCAATGAACTGITTGTCTTTGATATTCCTT 2805 336 K R I S R K K T H V K K C T P N A V F N E L F V F D I P C 364 2806 GTGAGAGTCTTGAAGAAATCAGTGTGAATTCTT PGlTCGmAA GGGG TCCGAAATGAGGTGAT GGACGGTTGGT 2890 365 E S L E E I S V E F L V L D S E. R G S R N E V I G RL V 392 2891 CCTGGGTGCCACAGCAGAAGGAA=W=GTGGGGGGCACTGGAAGGAGATCOGTGACT1~CCCCAGGAGACAAATTGCTAAGTGGCAC 2975 393 L G A T A E G S G G G H W K E I C D F P R R Q I A K W H 420 2976 ATGCTCTGTGATGGTTAGCTTCCTGGCCATGAGCTGCAACTTAAAGATC1FACTAGGCAAGAAGCAAPrFT1CTI ATACAC 3060 421 L C D G* 425 3061 3145 3146 AGACCAGAAATAG TAAATGTTTTTATGTAATTCCTC TGGT AATAAGATATCAG 3230 3231 TrTCCT¶TGTGGTATTAAGTIGGcCAACCCAGCCTGTTATGAGCACCCTAGCTTAACCTTGTCACAGATGTGTGTTGTAGGT 3315 3316 GTGrATCmGATGACGGAr¶rAGATAAATGGACAAC¶1TFTGTATGAAAAAT1AA 3400 3401 TCCATGTIrTCTrCGTrGGGGTGAATrAAATCCCAAGAT~McC AAATGTCCTCCAGTCmTT¶N'T' 3485 3486 TAGTTAAATACGTCATGTACAAATGATCG_ 3570 3571 ATCATAAAGGAGCTGuCTAAAAA_&AAG&AA ATIATAAGGFGAPAG 3655 3656 ACACTG TCATGTAA AATA¶ACACCTAAGAACCATGGTCT'G 3740 3741 AA¶'rA¶~AcmCCTGTCAGATTATICCA Am~rGGCCTAAGAACCCPArAIICMATGTGAAGTTGTCAGACTAGTGAAAAAAA 3825 3826 ATAAACCTAAGCTAGCAATTCAGTAAATACM¶ FAAATATAAAAATCTATCA0TGTACAAGTGTATFA _ GATTGT 3910 3911 CTCCCC C __AAAGAG 3992 FIG. 1. Nucleotide and predicted amino acid sequence ofmurine Syt3. Nucleotides for the Syt3 cDNA are numbered beginning with position 1, and the predicted amino acids are numbered beginning with the proposed initiator methionine codon. The first in-frame stop codon preceding the open reading frame in the 5' untranslated region is underlined, and the 3' terminator is indicated by a star. dinately with either Sytl or Syt2, the distribution of Syt DISCUSSION transcripts in the mouse CNS was determined by Northern Our results establish that the murine synaptotagmins are analysis. As shown in Fig. 4A, the Syti probe hybridized to encoded by a multigene family consisting of at least three a major transcript of 5.0 kb and, to a lesser extent, to a members, Sytl, -2, and -3. The identification of a third gene transcript of =7.0 kb, which is most likely due to cross- in rodents demonstrates that the level of complexity of this hybridization to Syt2 (compare Fig. 4 A and B). Syti expres- gene family has been maintained during the evolution of sion was highest in forebrain and diencephalon and was marine vertebrates to mammals because three synaptotagmin detectable in all brain regions examined, as reported previ- homologs have been described in the marine ray Discopyge ously in the rat CNS (3). The Syt2 probe hybridized to a ommata (6). The degree of sequence similarity between 7.0-kb transcript that was expressed predominantly in caudal synaptotagmins at the amino acid level, especially in the brain regions, with highest levels being found in brain stem region encompassing the Ca2+ and phospholipid-binding C2 (Fig. 4B). An additional transcript of -3.5 kb was detected in domain, suggests that this family consists of two closely cerebellum, which did not correspond to either Sytl or Syt3 related members (SYT1/SYT2 in mammals and p65A/p65B and may represent either a cerebellar-specific alternatively in the marine ray) and a third, more distantly related member spliced form ofSyt2 or a closely related transcript. In contrast (SYT3 in mammals and p65C in the ray). Based on overall to the differential pattern ofexpression observed for Sytl and amino acid sequence conservation, SYT3 is more related to Syt2, Syt3 transcripts were detected in most brain regions and p65A than it is to p65C. However, the domain structure of at rather uniform levels (Fig. 4C). The global distribution of SYT3 is more like p65C than any other known synaptotag- SYT3 mRNA demonstrates that the expression of this diver- min. For example, compared with other synaptotagmins, gent synaptotagmin homolog overlaps with that of SYT1 and both SYT3 and p65C contain a long cytoplasmic loop sepa- SYT2 and implies a more general utilization of the Syt3 gene rating the C2 domain from the hydrophobic region (109 and 149 product in the CNS. amino acids for SYT3 and p65C, respectively, vs. 57 and 51 Downloaded by guest on October 2, 2021 8198 Neurobiology: Hilbush and Morgan Proc. Natl. Acad. Sci. USA 91 (1994)

A TMR C2A C2B

394 R 1 14 38 148 263 282 -42

3y.- I p E 3EXN Ewes L o I I p65A iE D N I I I VlB :: i 9 7e Syt-2 E PN A To L I. I I~ R 3sXAT I -I 204 p 6;5Cc AK H QVNC Y T T VK LKL Ku ly *R ~~286 Sy- -3 E Q 3TflFL E BN DEK AFV KE QG M XM T I 3 N 3

Syl. - i 11T L IS II E FK i N -'-4 -, p65A VfS. N L 4 Syt-2 -:Aai-oT T ; I _Q Su I K . N 2 4-S I- K p65B T S V 3 1 Qi |C 9T WT 26 6 p65C I T BEQ ;lNE ~ |L TV L D N L L E ,-41, ;y v TS|;Tl YG i p H I Q '-D--e RDA L I .r L [IT I--S

Lyt.1 T FlH V . T F p65A T F H V . T F 3:3F Syt - 2 TLflQ P I T I _ 308 46 5-. K LEL QQ LB T 320- FS S E D TT F 3A Ew|LT T 43 t, t -- ma I E L S D C: K M L V C Q H EBP nSu S

SY". - I 14 '3ss ~~V V I p65A N V 6 Sit - 2 N V 366 L C 384 p6 5B 1.: J.VN^ p6 5C AS C DE" N NME N :A 463< a-9^tV VIA .. .:7%T- ..t H A KIS LDVow *cB S LEFc.I S L-i S E: R. °7 iv

S;yt i D V Y S TEA T Q V3 A M A T K p65A D V YES T AA T Q Px A T Cv mK .. ,yt-2 E I S AT T S K P A L LNC p65B EnD ;tB I 93S 3A S 13T | S K P L V A G LKD P65C N E C R N A T D P G R E NE *_* Es Q I E S583Y M T _ FiiA ,yt- S EV RLV L ATAE SGG G K I C DF M *-- D)

FIG. 2. Domain structure ofmurine SYT3 and alignment ofC2 domain with rat and marine ray synaptotagmins. (A) Schematic representation of the overall structure of SYT3 is shown; boxes denote domains identified in synaptotagmins (21). The N-terminal region is shown as an open box followed by a proposed transmembrane region (TMR) indicated by diagonal bars. The two repeats (C2A and C2B) composing the C2 domain are shown as black boxes. An intervening region between the TMR and the C2 domain is indicated with a dark-stippled background, and the C-terminal domain is shown by a light-stippled background. Amino acid positions defining the domain boundaries are below. (B) Sequence comparison of C2 domains of rat and marine ray synaptotagmin isoforms with mouse SYT3. Alignment of amino acid sequences was generated by using PILEUP. Identical residues in four of six sequences are shown with black background; conservative amino acid changes are shown with dark gray shading, and moderately conservative amino acid changes are shown with light gray shading, using threshold values of 1.0 and 0.5, respectively (21). amino acids for p65A and p65B, respectively). In addition, the ulated release of catecholamines from dense-core vesicles. hydrophobic region is highly conserved among SYT1/SYT2 Although the phenotypic properties of clonal cell lines may and p65A/p65B homologs, whereas this conservation is not vary greatly among laboratories, our results indicate that found in either SYT3 or p65C. Although the hydrophobic SYT3, and therefore synaptotagmin function, may have been domain of SYT3 is most related to that of p65C, the level of present in these cells, thereby accounting for the observed amino acid identity in this region or in other conserved Ca2+-dependent release. domains is not sufficient to establish whether Syt3 is the Recent genetic and electrophysiological evidence suggests mammalian homolog of p65C and leaves open the possibility that synaptotagmins are presynaptic Ca2+ sensors (13-15, 23). that other synaptotagmin family members await identification. Therefore, the various isoforms might represent species that The existence of multiple synaptotagmin isoforms raises a respond to different ranges of intracellular Ca2+, thereby number of issues. The first is whether a given neuronal cell providing potential mechanisms for differential vesicle re- type contains a single, or several, form(s) of synaptotagmin. lease. For example, each isoform could have unique Ca2+- Analysis ofgrossly dissected brain regions reveals that while binding constants. Differences in the Ca2+-binding properties each synaptotagmin isoform has a distinctive pattern of of the synaptotagmins could be generated by physical inter- expression in the CNS, the distributions clearly overlap. actions between synaptotagmin isoforms. It is known that However, the resolution of Northern blot analysis is insuf- Ca2+ binding to SYT1 depends highly upon the state of ficient to determine whether individual neurons in the brain oligomerization of this synaptotagmin (9, 21). If it were pos- contain multiple synaptotagmins. To circumvent this limita- sible for SYT1 to form mixed oligomers with other synap- tion, synaptotagmin expression was determined in PC12 totagmin isoforms, this might modify the Ca2+-binding prop- cells, which represent a homogeneous population of cells. erties of the complex. Indeed, this type of situation would Both Syti and Syt3 are coexpressed in PC12 cells, thereby potentially provide for an enormous breadth in the Ca2+- suggesting that synaptotagmins can be present in particular sensing range. It is also conceivable that particular synap- combinations within a given cell type. Indeed, Syt3 tran- totagmins or combinations of synaptotagmins are present on scripts are more abundant than those for Syti in PC12 cells, distinct classes of vesicles. SYT1 has been demonstrated to indicating further that one isoform may predominate over exist on both synaptic vesicles and large dense-core vesicles another. The prevalence ofSyt3 transcripts in PC12 cells also (4). Therefore, rather than individual synaptotagmins being casts doubt on a recent study in which depolarization- vesicle-specific, the particular combination of isoforms local- induced transmitter release was assessed in SYT1-deficient ized to a given vesicle may be characteristic. Thus, it will be PC12 cells (12). In that study, the presence of Ca2+-evoked important to establish not only the anatomical and subcellular catecholamine release in the mutant cell line was interpreted distributions of all three synaptotagmin isoforms but also as evidence that synaptotagmins were not essential for reg- whether they are capable of heterodimerization. Downloaded by guest on October 2, 2021 Neurobiology: Hilbush and Morgan Proc. Natl. Acad. Sci. USA 91 (1994) 8199 additional synaptotagmin family members with unique pro- (n E tein-protein interactions or affinities to proteins in the fusion ,;a 5 00; complex raises the possibility that multiple regulatory com- - C CD I 0 -_ 0 .. c ponents are involved in initiating C a 0. vesicle release in the -x = z CL presynaptic terminal. This hypothesis may account for the 28S - * discrepancies between apparent loss of synaptotagmin func- syt-1 tion and the persistence of vesicular release in previous 18S - studies (12-15). Therefore, it will be important to determine the nature of the presynaptic components interacting with each synaptotagmin isoform and to further define the con- tributions that synaptotagmins make, not only singly but also combinatorially, to presynaptic function. Note Added in Proof. Recently, another synaptotagmin gene distinct _- syt-2 from that reported here was published as Syt III (29). To avoid confusion, we refer to our sequence in the data base as Syt4. We thank R. Wurzburger for assistance with DNA sequencing and oligonucleotide synthesis. B.S.H. was supported, in part, by a Na- tional Research Service Award Postdoctoral Fellowship NS 09053. 1. Stevens, C. (1993) Cell 72/Neuron 10 (Suppl.), 55-63. 2. Katz, B. (1969) in The Release ofNeuronal Transmitter Substances, syt-3 ed. Katz, B. (Thomas, Springfield, IL). 3. Geppert, M., Archer, B. T., III, & Sudhof, T. C. (1991) J. Biol. Chem. 266, 13548-13552. 4. Matthew, W. D., Tsavaler, L. & Reichardt, L. F. (1981) J. Cell Biol. 91, 257-269. 5. Perin, M. S., Fried, V. A., Mignery, G. A., Jahn, R. & Sudhof, T. C. (1990) Nature (London) 345, 260-263. 6. Wendland, B., Miller, K. G., Schilling, J. & Scheller, R. H. (1991) Total RNA Neuron 6, 993-1007. FIG. 3. Tissue distribution of synaptotagmin mRNA.mRNA.Total RNA 7. Nishizuka, Y. (1988) Nature (London) 334, 661-665. was prepared from various tissues and PC12 cells, as indicated. 8. Clark, J. D., Lin, L.-L., Driz, R. W., Ramesha, C. S., Sultzman, Samples (5 pig) were analyzed in triplicate on 0.8% agarose- L. A., Lin, A. Y., Milona, N. & Knopf, J. L. (1991) Cell 65, formaldehyde gels, blotted to nitrocellulose, a nd hybridized with 1043-1051. 32P-labeled cDNA probes specific for Syti, -2, and -3 (see Materials 9. Brose, N., Petrenko, A. G., Sudhof, T. C. & Jahn, R. (1992) and Methods). Blots were washed for 15 min at:55TC in 2x standard Science 256, 1021-1025. saline citrate (SSC), followed by 15 min at 600C in 0.5x SSC. 10. Elferink, L. A., Peterson, M. R. & Scheller, R. H. (1993) Cell 72, Positions of28S and 18S ribosomal RNA are indiicated at left, and the 153-159. position of the single mRNA species obtained Mvith each Syt cDNA 11. Bommert, K., Charlton, M. P., DeBello, W. M., Chin, G. J., Betz, probe is shown at right. A photograph of the ethidium bromide- H. & Augustine, G. J. (1993) Nature (London) 363, 163-165. stained gel is shown to indicate equal loading off RNA in each lane. 12. Shoji-Kasai, Y., Yoshida, A., Sato, K., Hoshino, T., Ogura, A., Kondo, S., Fujimoto, Y., Kuwahara, R., Kato, R. & Takahashi, M. (1992) Science 256, 1820-1823. A final issue concerns the function of s,ynapynaptotagminstotagins inin 13.13 Littleton, J. T., Stern, M., Schulze, K., Perin, M. & Bellen, H. J. vesicular release. Several studies have dremonstrated that (1993) Cell 74, 1125-1134. SYT1 interacts with multiple presynapticc membrane pro- 14. Nonet, M. L., Grundahl, K., Meyer, B. J. & Rand, J. B. (1993) Cell teins. These associations include the abilit3y of SYT1 to bind 73, 1291-1305. in vitro to (24), a proposed compotnent of the vesicle 15. DiAntonio, A., Parfitt, K. D. & Schwarz, T. L. (1993) Cell 73, thecytoplasmic1281-1290. docking and fusion apparatus (25), and wit].h,hthe cytoplasmic 16. Perin, M. S., Johnston, P. A., Ozcelik, T., Jahn, R., Francke, U. & domains of neurexins (26, 27). In additic )n, SYTi can be Sudhof, T. C. (1991) J. Biol. Chem. 266, 615-622. immunoprecipitated in a complex with Ca +2' channels (28). 17. Chomczynski, P. & Sacchi, N. (1987) Anal. Biochem. 162, 156-159. The physical association of this vesicle prc)tein with compo- 18. Morgan, J. I., Cohen, D. R., Hempstead, J. L. & Curran, T. (1987) nents localized to release sites in the presyrnaptic membrane Science 237, 192-197. 19. Pearson, W. & Lipman, D. (1988) Proc. Natd. Acad. Sci. USA 85, has led to the proposal that synaptotagmin]plays a regulatoryy2 44 24 8. role in vesicle docking and fusion (5, 6, 23). The existence of 20. Burgess, R. R. & Gribskov, M. (1988) Nucleic Acids Res. 14, 6745-6763. AA en 0 21. Perin, M. S., Brose, N., Jahn, R. & Sudhof, T. C. (1991) J. Biol. A , o BD ~ E E c E Chem. 266, 623-629. .c Es z a a 0.C, = 'D , X 22. Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, u a ns S., Yamaguchi, 0 .0 0 .0 nL s agU *=T., Wada, K., Miyazaki, M. & Takai, Y. (1993) Mol. Cell. Biol. 13, ELC 0 L tD - s m 2 - v*- _ 2061-2068. 23. DeBello, W. M., Betz, H. & Augustine, G. J. (1993) Cell 74, 947-950. 24. Bennett, M. K., Calakos, N. & Scheller, R. H. (1992) Science 257, 28S *|1,|| # 255-259. 18S 25. Soilner, T., Whiteheart, S. W., Brunner, M., Erdjument-Bromage, H., Geromanos, S., Tempst, P. & Rothman, J. E. (1993) Nature (London) 362, 318-324. 26. Hata, Y., Davletov, B., Petrenko, A. G., Jahn, R. & Sudhof, T. C. (1993) Neuron 10, 307-315. 27. Petrenko, A. G., Perin, M. S., Davletov, B. A., Ushkaryov, Y. A., Geppert, M. & Sudhof, T. C. (1991) Nature (London) 353, 65-68. 28. Leveque, C., Hoshino, T., David, P., Shoh-Kasai, Y., Leys, K., FIG. 4. Differential expression of Syt trans(cripts In the mouse Omori, A., Lang, B., El Far, O., Sato, K., Martin-Moutot, N., CNS. RNA samples (5 pug) from various brain iregions were run in Newsom-Davis, J., Takahashi, M. & Seagar, M. J. (1992) Proc. triplicate, and Syt expression was analyzed by: Northern blot using Natd. Acad. Sci. USA 89, 3625-3629. probes specific for Syt) (A), Syt2 (B), and Syt3 (C)ias described in Fig. 29. Mizuta, M., Inagaki, N., Nemoto, Y., Matsukura, S., Takahashi, 3 legend. Positions of28S and 18S ribosomal RNA are indicated at left. M. & Seino, S. (1994) J. Biol. Chem. 269, 11675-11678. Downloaded by guest on October 2, 2021