Synaptotagmins: Why So Many? (2002)

transmembrane region synaptotagmins 1–13 solubleN-ethylmaleimide-sensitive factor attachment protein receptor adaptor protein 2 Synaptotagmins constitute a family of membrane-trafficking proteins that are characterized by an N-terminal TMR,1 a variable linker, and two C-terminal C2-domains (1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Synaptotagmin 1 (Syt 1) was identified as p65 in a monoclonal antibody screen for synaptic proteins (2Matthew W.D. Tsavaler L. Reichardt L.F. J. Cell Biol. 1981; 91: 257-269Crossref PubMed Scopus (423) Google Scholar) and proposed as a potential Ca2+ sensor for regulated exocytosis when its cloning revealed the presence of two C2-domains (3Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (645) Google Scholar). Twelve additional synaptotagmins were subsequently discovered (Fig. 1 and TableI; Refs. 4Geppert M. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1991; 266: 13548-13552Abstract Full Text PDF PubMed Google Scholar, 5Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar, 6Hilbush B.S. Morgan J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8195-8199Crossref PubMed Scopus (77) Google Scholar, 7Li C. Ullrich B. Zhang Z.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (539) Google Scholar, 8Hudson A.W. Birnbaum M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5895-5899Crossref PubMed Scopus (78) Google Scholar, 9Craxton M. Goedert M. FEBS Lett. 1995; 361: 196-200Crossref PubMed Scopus (67) Google Scholar, 10Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 11von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 12Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar, 13von Poser C. Südhof T.C. Eur. J. Cell Biol. 2000; 80: 41-47Crossref Scopus (35) Google Scholar, 14Fukuda M. Mikoshiba K. Biochem. J. 2001; 354: 249-257Crossref PubMed Scopus (39) Google Scholar). Extensive work showed that Syts 1 and 2 likely function as Ca2+ sensors in synaptic vesicle exocytosis (15Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. Südhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1191) Google Scholar, 16Fernandez-Chacon R. Konigstorffer Gerber S.H. Garcia J. Matos M.F. Stevens C.F. Brose N. Rizo J. Rosenmund C. Südhof T.C. Nature. 2001; 410: 41-49Crossref PubMed Scopus (725) Google Scholar, 17Nonet M.L. Grundahl K. Meyer B.J. Rand J.B. Cell. 1993; 73: 1291-1305Abstract Full Text PDF PubMed Scopus (458) Google Scholar, 18DiAntonio A. Parfitt K.D. Schwarz T.L. Cell. 1993; 73: 1281-1290Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 19Littleton J.T. Stern M. Schulze K. Perin M. Bellen H.J. Cell. 1993; 74: 1125-1134Abstract Full Text PDF PubMed Scopus (377) Google Scholar). Much less is known about the other synaptotagmins, although many of them are abundantly co-expressed with Syts 1 and 2 in brain and are evolutionarily conserved. The most abundant of these "other" synaptotagmins, Syts 3 and 7, are localized on the plasma membrane opposite to synaptic vesicles and exhibit distinct Ca2+ affinities, suggesting that plasma membrane and vesicular synaptotagmins may function as complementary Ca2+ sensors in exocytosis with a hierarchy of Ca2+ affinities (20Butz S. Fernandez-Chacon R. Schmitz F. Jahn R. Südhof T.C. J. Biol. Chem. 1999; 274: 18290-18296Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 21Sugita S. Han W. Butz S. Liu X. Fernandez-Chacon R. Lao Y. Südhof T.C. Neuron. 2001; 30: 459-473Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 22Sugita S. Shin O.-K. Han W. Lao Y. Südhof T.C. EMBO J. 2002; (in press)PubMed Google Scholar). In the present short review, I shall discuss the synaptotagmin family as a whole and evaluate possible biological reasons for the co-expression of multiple isoforms.Table IProperties of synaptotagminsFormExpression pattern and localizationFunctionSpecial propertiesRef.Class 1, Syts 1 and 2Brain and endocranium; localized to synaptic vesicles and secretory granulesCa2+ sensor for fast exocytosisN-Glycosylated N terminus2Matthew W.D. Tsavaler L. Reichardt L.F. J. Cell Biol. 1981; 91: 257-269Crossref PubMed Scopus (423) Google Scholar–4Geppert M. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1991; 266: 13548-13552Abstract Full Text PDF PubMed Google Scholar, 15Geppert M. Goda Y. Hammer R.E. Li C. Rosahl T.W. Stevens C.F. Südhof T.C. Cell. 1994; 79: 717-727Abstract Full Text PDF PubMed Scopus (1191) Google Scholar, 16Fernandez-Chacon R. Konigstorffer Gerber S.H. Garcia J. Matos M.F. Stevens C.F. Brose N. Rizo J. Rosenmund C. Südhof T.C. Nature. 2001; 410: 41-49Crossref PubMed Scopus (725) Google Scholar,23Perin M.S. Brose N. Jahn R. Südhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google ScholarClass 2, Syt 7Brain >> ubiquitous; localized to active zone and plasma membraneCa2+ sensor for exocytosis?>12 splice forms7Li C. Ullrich B. Zhang Z.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (539) Google Scholar, 21Sugita S. Han W. Butz S. Liu X. Fernandez-Chacon R. Lao Y. Südhof T.C. Neuron. 2001; 30: 459-473Abstract Full Text Full Text PDF PubMed Scopus (180) Google ScholarClass 3, Syts 3, 5, 6, and 10Brain >> ubiquitous; localized to active zone and plasma membraneCa2+ sensor for exocytosis?Disulfide bonds at N terminus5Mizuta M. Inagaki N. Nemoto Y. Matsukura S. Takahashi M. Seino S. J. Biol. Chem. 1994; 269: 11675-11678Abstract Full Text PDF PubMed Google Scholar, 7Li C. Ullrich B. Zhang Z.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (539) Google Scholar, 10Babity J.M. Armstrong J.N. Plumier J.C. Currie R.W. Robertson H.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 2638-2641Crossref PubMed Scopus (73) Google Scholar, 24Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google ScholarClass 4, Syts 4 and 11Brain >> ubiquitous1-aBased only on RNA blotting experiments.; localization controversialUnknownAsp → Ser substitution in C2A-domain6Hilbush B.S. Morgan J.I. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 8195-8199Crossref PubMed Scopus (77) Google Scholar, 11von Poser C. Ichtchenko K. Shao X. Rizo J. Südhof T.C. J. Biol. Chem. 1997; 272: 14314-14319Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar, 79Berton F. Cornet V. Iborra C. Garrido J. Dargent B. Fukuda M. Seagar M. Marqueze B. Eur. J. Neurosci. 2000; 12: 1294-1302Crossref PubMed Scopus (45) Google ScholarClass 5, Syt 9Brain > ubiquitous1-aBased only on RNA blotting experiments.; localized to synaptic vesicles?UnknownAlso named Syt58Hudson A.W. Birnbaum M.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5895-5899Crossref PubMed Scopus (78) Google Scholar, 9Craxton M. Goedert M. FEBS Lett. 1995; 361: 196-200Crossref PubMed Scopus (67) Google ScholarClass 6, Syts 8, 12, and 13Brain >> ubiquitous1-aBased only on RNA blotting experiments.UnknownNo consensus Ca2+-binding sites7Li C. Ullrich B. Zhang Z.Z. Anderson R.G.W. Brose N. Südhof T.C. Nature. 1995; 375: 594-599Crossref PubMed Scopus (539) Google Scholar, 12Thompson C.C. J. Neurosci. 1996; 16: 7832-7840Crossref PubMed Google Scholar–14Fukuda M. Mikoshiba K. Biochem. J. 2001; 354: 249-257Crossref PubMed Scopus (39) Google Scholar1-a Based only on RNA blotting experiments. Open table in a new tab We classify as a synaptotagmin any protein that has an N-terminal TMR, a central linker, and two C-terminal C2-domains (Fig.1). Based on their sequences and properties, the 13 vertebrate synaptotagmins can be grouped into 6 classes (Table I). Fig. 1 displays the domain structures of synaptotagmins and the positions of the introns in the corresponding human genes. 2T. C. Südhof, unpublished observation. The N-terminal sequences preceding the TMRs are usually short (4–62 residues) and exhibit significant sequence similarity only within a given class. Syts 1 and 2 are the only synaptotagmins with an N-glycosylated N terminus (4Geppert M. Archer III, B.T. Südhof T.C. J. Biol. Chem. 1991; 266: 13548-13552Abstract Full Text PDF PubMed Google Scholar, 23Perin M.S. Brose N. Jahn R. Südhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar), and Syts 3, 5, 6, and 10 are the only synaptotagmins with disulfide-bonded cysteines at the N terminus (24Fukuda M. Kanno E. Mikoshiba K. J. Biol. Chem. 1999; 274: 31421-31427Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). The TMRs of synaptotagmins also lack significant sequence similarity. In all synapotagmins except for Syt 13, an intron is placed just N-terminal to the TMR,2 and all synaptotagmins except for Syt 12 contain cysteine residues at the cytoplasmic boundary of the TMR. At least in Syts 1 and 7, some of these cysteines are palmitoylated (25Chapman E.R. Blasi J. An S. Brose N. Johnston P.A. Südhof T.C. Jahn R. Biochem. Biophys. Res. Commun. 1996; 225: 326-332Crossref PubMed Scopus (59) Google Scholar, 26Veit M. Sollner T.H. Rothman J.E. FEBS Lett. 1996; 385: 119-123Crossref PubMed Scopus (202) Google Scholar, 27von Poser C. Zhang J.Z. Mineo C. Ding W. Ying Y. Südhof T.C. Anderson R.G.W. J. Biol. Chem. 2000; 275: 30916-30924Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). Synaptotagmins form constitutive dimers via their TMRs in a reaction that may require palmitoylation (23Perin M.S. Brose N. Jahn R. Südhof T.C. J. Biol. Chem. 1991; 266: 623-629Abstract Full Text PDF PubMed Google Scholar, 27von Poser C. Zhang J.Z. Mineo C. Ding W. Ying Y. Südhof T.C. Anderson R.G.W. J. Biol. Chem. 2000; 275: 30916-30924Abstract Full Text Full Text PDF PubMed Scopus (73) Google Scholar). One possible reason for the constitutive dimerization of synaptotagmins is to protect them from cleavage by γ-secretase since γ-secretase promiscuously cleaves monomeric but not dimeric membrane proteins containing a short extracellular stub (28Struhl G. Adachi A. Mol. Cell. 2000; 6: 625-636Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar). The size of the linker between the TMR and the C2-domains varies from 43 residues for Syt 8 to 417 residues for the longest splice variant of Syt 7 (Fig. 1). At least in Syt 1, the linker is phosphorylated by multiple protein kinases (29Davletov B. Sontag J.M. Hata Y. Petrenko A.G. Fykse E.M. Jahn R. Südhof T.C. J. Biol. Chem. 1993; 268: 6816-6822Abstract Full Text PDF PubMed Google Scholar,30Hilfiker S. Pieribone V.A. Nordstedt C. Greengard P. Czernik A.J. J. Neurochem. 1999; 73: 921-932Crossref PubMed Scopus (86) Google Scholar). The C-terminal C2-domains of synaptotagmins, referred to as the C2A- and C2B-domains, account for the majority of their sequences and represent their only homologous sequences (Fig. 1). C2-domains are widespread modules of 130–140 residues that were initially defined as the second constant sequence (hence "C2") in protein kinase C isoforms (31Coussens L. Parker P.J. Rhee L. Yang-Feng T.L. Chen E. Waterfield M.D. Francke U. Ullrich A. Science. 1986; 233: 859-866Crossref PubMed Scopus (748) Google Scholar) and were first shown in Syt 1 to constitute Ca2+-binding modules (32Davletov B. Südhof T.C. J. Biol. Chem. 1993; 268: 26386-26390Abstract Full Text PDF PubMed Google Scholar, 33Rizo J. Südhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 34Nalefski E.A. Falke J.J. Protein Sci. 1996; 5: 2375-2390Crossref PubMed Scopus (681) Google Scholar). Atomic structures revealed that the synaptotagmin C2A- and C2B-domains are similarly composed of a β-sandwich containing eight β-strands, with flexible loops emerging from the top and the bottom (35Sutton A.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1994; 80: 929-938Abstract Full Text PDF Scopus (598) Google Scholar, 36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar). C2A-domains generally bind three Ca2+ ions, whereas C2B-domains bind only two Ca2+ ions (Fig. 3) (Refs. 36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 37Ubach J. Garcia J. Nittler M.P. Südhof T.C. Rizo J. Nat. Cell Biol. 1999; 1: 106-112Crossref PubMed Scopus (64) Google Scholar, 38Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (243) Google Scholar; but see below for exceptions). All C2B-domains contain a bottom α-helix between the 7th and 8th β-strands that is absent from C2A-domains (35Sutton A.B. Davletov B.A. Berghuis A.M. Südhof T.C. Sprang S.R. Cell. 1994; 80: 929-938Abstract Full Text PDF Scopus (598) Google Scholar, 36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 37Ubach J. Garcia J. Nittler M.P. Südhof T.C. Rizo J. Nat. Cell Biol. 1999; 1: 106-112Crossref PubMed Scopus (64) Google Scholar, 38Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (243) Google Scholar). The fact that the differences between the C2A- and C2B-domains are conserved indicates a common ancestry and suggests that the C2-domains are functionally specialized in all synaptotagmins (1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). In addition to synaptotagmins, proteins such as rabphilin and Doc2s contain similar C2A- and C2B-domains but are not classified as synaptotagmins because they lack TMRs (reviewed in Ref. 1Südhof T.C. Rizo J. Neuron. 1996; 17: 379-388Abstract Full Text Full Text PDF PubMed Scopus (394) Google Scholar). Furthermore, two other protein families (called Vp115 and ferlins) contain multiple C2-domains and a TMR similar to synaptotagmins but differ from synaptotagmins in that they have more than two C2-domains (39Morris N.J. Ross S.A. Neveu J.M. Lane W.S. Lienhard G.E. Biochim. Biophys. Acta. 1999; 1431: 525-530Crossref PubMed Scopus (18) Google Scholar, 40Achanzar W.E. Ward S. J. Cell Sci. 1997; 110: 1073-1081Crossref PubMed Google Scholar, 41Britton S. Freeman T. Vafiadaki E. Keers S. Harrison R. Bushby K. Bashir R. Genomics. 2000; 68: 313-321Crossref PubMed Scopus (59) Google Scholar, 42Bashir R. Britton S. Strachan T. Keers S. Vafiadaki E. Lako M. Richard I. Marchand S. Bourg N. Argov Z. Sadeh M. Mahjneh I. Marconi G. Passos-Bueno M.R. Moreira E.D Zatz M. Beckmann J.S. Bushby K. Nat. Genet. 1998; 20: 37-42Crossref PubMed Scopus (544) Google Scholar, 43Liu J. Aoki M. Illa I. Wu C. Fardeau M. Angelini C. Serrano C. Urtizberea J.A. Hentati F. Hamida M.B. Bohlega S. Culper E.J. Amato A.A. Bossie K. Oeltjen J. Bejaoui K. McKenna-Yasek D. Hosler B.A. Schurr E. Arahata K. de Jong P.J. Brown R.H. Nat. Genet. 1998; 20: 31-36Crossref PubMed Scopus (732) Google Scholar, 44Yasunaga S. Grati M. Chardenoux S. Smith T.N. Friedman T.B. Lalwani A.K. Wilcox E.R. Petit C. Am. J. Hum. Genet. 2000; 67: 591-600Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar). Analysis of the human genome revealed that synaptotagmin genes vary from <10 kilobases (Syts 8 and 9) to >60 kilobases (Syt 1), and are not physically linked.2 The exon/intron structures of synaptotagmin genes are nearly identical for synaptotagmins within a class but differ dramatically between classes. Some synaptotagmin genes are relatively simple (e.g. the Syt 4 and 11 genes contain only four exons), whereas others are complex (the Syt 7 gene with 14 exons; Fig. 1). Only a single exon (at the beginning of the C2B-domain) is shared among all synaptotagmins, making this exon a signature of synaptotagmin genes. Alternative splicing of the linker sequence between the TMR and the C2-domains of synaptotagmins and of the TMR itself have been reported. Alternative splicing of a short sequence in the linker of Syt 1 (3Perin M.S. Fried V.A. Mignery G.A. Jahn R. Südhof T.C. Nature. 1990; 345: 260-263Crossref PubMed Scopus (645) Google Scholar) probably represents a sliding exon/intron junction. Strikingly, the Syt 7 linker region is subject to extensive alternative splicing with dramatic changes in protein structure (21Sugita S. Han W. Butz S. Liu X. Fernandez-Chacon R. Lao Y. Südhof T.C. Neuron. 2001; 30: 459-473Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Five exons are variably included or excluded in the mRNA (Fig. 1), resulting in a 10-fold expansion or contraction of the linker. One of the five alternatively spliced exons features an evolutionarily conserved stop codon; insertion of this exon creates a truncated Syt 7 protein (21Sugita S. Han W. Butz S. Liu X. Fernandez-Chacon R. Lao Y. Südhof T.C. Neuron. 2001; 30: 459-473Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). The alternative splicing of Syt 7 is developmentally and regionally regulated, suggesting that it regulates the coupling of the C2-domains to the TMR (21Sugita S. Han W. Butz S. Liu X. Fernandez-Chacon R. Lao Y. Südhof T.C. Neuron. 2001; 30: 459-473Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). PCR experiments suggested that for many synaptotagmins, variants lacking TMRs are produced by "exon skipping" (45Craxton M. Goedert M. FEBS Lett. 1999; 460: 417-422Crossref PubMed Scopus (35) Google Scholar, 46Fukuda M. Mikoshiba K. J. Biol. Chem. 1999; 274: 31428-31434Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, the gene structures of these synaptotagmins show that exon skipping would create out-of-frame junctions between the N- and C-terminal exons (Fig.1). This result together with the fact that no soluble synaptotagmins were observed make the alternative splicing of synaptotagmin TMRs doubtful. The synaptotagmin C2-domains are functionally polarized in that Ca2+ exclusively binds to the top loops but not the bottom loops (36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 37Ubach J. Garcia J. Nittler M.P. Südhof T.C. Rizo J. Nat. Cell Biol. 1999; 1: 106-112Crossref PubMed Scopus (64) Google Scholar, 38Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (243) Google Scholar, 47Shao X. Davletov B.A. Sutton R.B. Südhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (292) Google Scholar, 48Shao X. Li C. Fernandez I. Zhang X. Südhof T.C. Rizo J. Neuron. 1997; 18: 133-142Abstract Full Text Full Text PDF PubMed Scopus (196) Google Scholar). Diverse activities have been reported for synaptotagmin C2-domains. In assessing potential interactions of C2-domains, structural implications are unfortunately often ignored in favor of "functional" interpretations. For example, experiments with peptide injections into nerve terminals (49Fukuda M. Moreira J.E. Liu V. Sugimori M. Mikoshiba K. Llinas R.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 14715-14719Crossref PubMed Scopus (51) Google Scholar) led to the conclusion that the characteristic C-terminal WHXL sequence of the Syt 1 C2B-domain functions in synaptic vesicle docking. In the folded C2B-domain, however, the WHXL sequence forms a stably bonded β-strand (36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar) that is unlikely to unfold under physiological conditions. Another example is the AD3 mutant inDrosophila (18DiAntonio A. Parfitt K.D. Schwarz T.L. Cell. 1993; 73: 1281-1290Abstract Full Text PDF PubMed Scopus (217) Google Scholar, 19Littleton J.T. Stern M. Schulze K. Perin M. Bellen H.J. Cell. 1993; 74: 1125-1134Abstract Full Text PDF PubMed Scopus (377) Google Scholar). The AD3 mutation changes a conserved β-strand of the C2B-domain and likely interferes with its folding. Claims about testing specific binding activities of the C2B-domain with the AD3 mutant domain (50Fukuda M. Kabayama H. Mikoshiba K. FEBS Lett. 2000; 482: 269-272Crossref PubMed Scopus (21) Google Scholar, 51Littleton J.T. Bai J. Vyas B. Desai R. Baltus A.E. Garment M.B. B. E.R. J. Neurosci. 2001; PubMed Google Scholar) are to to other of synaptotagmins that sequences with conserved C2-domains bind Ca2+ exclusively via their top loops J. Südhof T.C. J. Biol. Chem. 1998; 273: 15879-15882Abstract Full Text Full Text PDF PubMed Scopus (700) Google Scholar, 47Shao X. Davletov B.A. Sutton R.B. Südhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (292) Google Scholar). Fig. a of the Ca2+-binding of the Syt 1 C2-domains (36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 38Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (243) Google Scholar). The Ca2+-binding of C2-domains are because Ca2+ ions are by residues that are in the sequence and because the for the are R. Konigstorffer Gerber S.H. Garcia J. Matos M.F. Stevens C.F. Brose N. Rizo J. Rosenmund C. Südhof T.C. Nature. 2001; 410: 41-49Crossref PubMed Scopus (725) Google Scholar, 47Shao X. Davletov B.A. Sutton R.B. Südhof T.C. Rizo J. Science. 1996; 273: 248-251Crossref PubMed Scopus (292) Google Scholar). The Ca2+ affinities of the C2-domains are and for the Syt 1 C2A- and C2B-domains, R. Konigstorffer Gerber S.H. Garcia J. Matos M.F. Stevens C.F. Brose N. Rizo J. Rosenmund C. Südhof T.C. Nature. 2001; 410: 41-49Crossref PubMed Scopus (725) Google Scholar, 36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google to C2-domains for the S.H. Garcia J. Rizo J. Südhof T.C. EMBO J. 2001; 20: PubMed Scopus Google not significant changes (36Fernandez I. Arac D. Ubach J. Gerber S.H. Shin O. Gao Y. Anderson R.G.W. Südhof T.C. Rizo J. Neuron. 2001; 32: 1057-1069Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 37Ubach J. Garcia J. Nittler M.P. Südhof T.C. Rizo J. Nat. Cell Biol. 1999; 1: 106-112Crossref PubMed Scopus (64) Google Scholar, 38Ubach J. Zhang X. Shao X. Südhof T.C. Rizo J. EMBO J. 1998; 17: 3921-3930Crossref PubMed Scopus (243) Google Scholar), suggesting that C2-domains function as Ca2+ binding interactions by their X. Li C. Fernandez I. Zhang X. Südhof T.C. Rizo J. 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