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Synaptotagmins: Why So Many?*

  • Thomas C. Südhof
    Correspondence
    To whom correspondence should be addressed: Center for Basic Neuroscience, Dept. of Molecular Genetics, Howard Hughes Medical Inst., University of Texas Southwestern Medical Center, 6000 Harry Hines Blvd. NA4.118, Dallas, TX 75390-9111. Tel.: 214-648-1976; Fax: 214-648-1879
    Affiliations
    Center for Basic Neuroscience, Department of Molecular Genetics, and Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9111
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  • Author Footnotes
    * This minireview will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002.
Open AccessPublished:December 05, 2001DOI:https://doi.org/10.1074/jbc.R100052200
      TMR
      transmembrane region
      Syt 1–Syt 13
      synaptotagmins 1–13
      SNARE
      solubleN-ethylmaleimide-sensitive factor attachment protein receptor
      AP-2
      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 (
      • Südhof T.C.
      • Rizo J.
      ). Synaptotagmin 1 (Syt 1) was identified as p65 in a monoclonal antibody screen for synaptic proteins (
      • Matthew W.D.
      • Tsavaler L.
      • Reichardt L.F.
      ) and proposed as a potential Ca2+ sensor for regulated exocytosis when its cloning revealed the presence of two C2-domains (
      • Perin M.S.
      • Fried V.A.
      • Mignery G.A.
      • Jahn R.
      • Südhof T.C.
      ). Twelve additional synaptotagmins were subsequently discovered (Fig. 1 and TableI; Refs.
      • Geppert M.
      • Archer III, B.T.
      • Südhof T.C.
      ,
      • Mizuta M.
      • Inagaki N.
      • Nemoto Y.
      • Matsukura S.
      • Takahashi M.
      • Seino S.
      ,
      • Hilbush B.S.
      • Morgan J.I.
      ,
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Hudson A.W.
      • Birnbaum M.J.
      ,
      • Craxton M.
      • Goedert M.
      ,
      • Babity J.M.
      • Armstrong J.N.
      • Plumier J.C.
      • Currie R.W.
      • Robertson H.A.
      ,
      • von Poser C.
      • Ichtchenko K.
      • Shao X.
      • Rizo J.
      • Südhof T.C.
      ,
      • Thompson C.C.
      ,
      • von Poser C.
      • Südhof T.C.
      ,
      • Fukuda M.
      • Mikoshiba K.
      ). Extensive work showed that Syts 1 and 2 likely function as Ca2+ sensors in synaptic vesicle exocytosis (
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      ,
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Nonet M.L.
      • Grundahl K.
      • Meyer B.J.
      • Rand J.B.
      ,
      • DiAntonio A.
      • Parfitt K.D.
      • Schwarz T.L.
      ,
      • Littleton J.T.
      • Stern M.
      • Schulze K.
      • Perin M.
      • Bellen H.J.
      ). 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 (
      • Butz S.
      • Fernandez-Chacon R.
      • Schmitz F.
      • Jahn R.
      • Südhof T.C.
      ,
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ,
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ). 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.
      Figure thumbnail gr1
      Figure 1Domain structures of synaptotagmins 1–13: relation of protein domains to the intron/exon organization of the human genes. Each diagram shows a single or several closely related synaptotagmins as identified on the left. Arrows indicate positions of introns in the corresponding human genes as identified in the human genome sequence.2The numbers next to the arrows describe the position in the codon at which the coding sequence is interrupted by the intron (0, at the codon junction; 1 and 2, after the first and second codon position, respectively). The N-terminal TMR is marked with a T, and the C2A- and C2B-domains are labeled.
      Table IProperties of synaptotagmins
      FormExpression 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 terminus
      • Matthew W.D.
      • Tsavaler L.
      • Reichardt L.F.
      • Geppert M.
      • Archer III, B.T.
      • Südhof T.C.
      ,
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      ,
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Perin M.S.
      • Brose N.
      • Jahn R.
      • Südhof T.C.
      Class 2, Syt 7Brain >> ubiquitous; localized to active zone and plasma membraneCa2+ sensor for  exocytosis?>12 splice forms
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      Class 3, Syts 3, 5, 6, and 10Brain >> ubiquitous; localized to active zone and plasma membraneCa2+ sensor for  exocytosis?Disulfide bonds at N terminus
      • Mizuta M.
      • Inagaki N.
      • Nemoto Y.
      • Matsukura S.
      • Takahashi M.
      • Seino S.
      ,
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Babity J.M.
      • Armstrong J.N.
      • Plumier J.C.
      • Currie R.W.
      • Robertson H.A.
      ,
      • Fukuda M.
      • Kanno E.
      • Mikoshiba K.
      Class 4, Syts 4 and 11Brain >> ubiquitous
      Based only on RNA blotting experiments.
      ; localization controversial
      UnknownAsp → Ser substitution  in C2A-domain
      • Hilbush B.S.
      • Morgan J.I.
      ,
      • von Poser C.
      • Ichtchenko K.
      • Shao X.
      • Rizo J.
      • Südhof T.C.
      ,
      • Berton F.
      • Cornet V.
      • Iborra C.
      • Garrido J.
      • Dargent B.
      • Fukuda M.
      • Seagar M.
      • Marqueze B.
      Class 5, Syt 9Brain > ubiquitous
      Based only on RNA blotting experiments.
      ; localized to synaptic vesicles?
      UnknownAlso named Syt5
      • Hudson A.W.
      • Birnbaum M.J.
      ,
      • Craxton M.
      • Goedert M.
      Class 6, Syts 8, 12, and 13Brain >> ubiquitous
      Based only on RNA blotting experiments.
      UnknownNo consensus Ca2+-binding sites
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Thompson C.C.
      • Fukuda M.
      • Mikoshiba K.
      1-a Based only on RNA blotting experiments.

      Structures of Synaptotagmins

      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.
      T. 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 (
      • Geppert M.
      • Archer III, B.T.
      • Südhof T.C.
      ,
      • Perin M.S.
      • Brose N.
      • Jahn R.
      • Südhof T.C.
      ), and Syts 3, 5, 6, and 10 are the only synaptotagmins with disulfide-bonded cysteines at the N terminus (
      • Fukuda M.
      • Kanno E.
      • Mikoshiba K.
      ). 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 (
      • Chapman E.R.
      • Blasi J.
      • An S.
      • Brose N.
      • Johnston P.A.
      • Südhof T.C.
      • Jahn R.
      ,
      • Veit M.
      • Sollner T.H.
      • Rothman J.E.
      ,
      • von Poser C.
      • Zhang J.Z.
      • Mineo C.
      • Ding W.
      • Ying Y.
      • Südhof T.C.
      • Anderson R.G.W.
      ). Synaptotagmins form constitutive dimers via their TMRs in a reaction that may require palmitoylation (
      • Perin M.S.
      • Brose N.
      • Jahn R.
      • Südhof T.C.
      ,
      • von Poser C.
      • Zhang J.Z.
      • Mineo C.
      • Ding W.
      • Ying Y.
      • Südhof T.C.
      • Anderson R.G.W.
      ). 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 (
      • Struhl G.
      • Adachi A.
      ). 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 (
      • Davletov B.
      • Sontag J.M.
      • Hata Y.
      • Petrenko A.G.
      • Fykse E.M.
      • Jahn R.
      • Südhof T.C.
      ,
      • Hilfiker S.
      • Pieribone V.A.
      • Nordstedt C.
      • Greengard P.
      • Czernik A.J.
      ).
      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 (
      • Coussens L.
      • Parker P.J.
      • Rhee L.
      • Yang-Feng T.L.
      • Chen E.
      • Waterfield M.D.
      • Francke U.
      • Ullrich A.
      ) and were first shown in Syt 1 to constitute Ca2+-binding modules (
      • Davletov B.
      • Südhof T.C.
      ,
      • Rizo J.
      • Südhof T.C.
      ,
      • Nalefski E.A.
      • Falke J.J.
      ). 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 (
      • Sutton A.B.
      • Davletov B.A.
      • Berghuis A.M.
      • Südhof T.C.
      • Sprang S.R.
      ,
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ). C2A-domains generally bind three Ca2+ ions, whereas C2B-domains bind only two Ca2+ ions (Fig. 3) (Refs.
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Garcia J.
      • Nittler M.P.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ; 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 (
      • Sutton A.B.
      • Davletov B.A.
      • Berghuis A.M.
      • Südhof T.C.
      • Sprang S.R.
      ,
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Garcia J.
      • Nittler M.P.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ). 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 (
      • Südhof T.C.
      • Rizo J.
      ).
      Figure thumbnail gr3
      Figure 3Geometry of synaptotagmins during exocytosis. One class of synaptotagmins (Syts 1 and 2 and possibly Syts 4, 9, and 11) are located on synaptic vesicles or secretory granules, whereas a second set (Syts 3, 6, and 7 and possibly others) are targeted to the plasma membrane, creating a symmetrical arrangement of opposing Ca2+-sensors. All synaptotagmins are probably dimers via interactions in the TMR and/or disulfide bonds between the extracytoplasmic N-terminal regions (for Syts 3, 5, 6, and 10). In addition to Ca2+-dependent interactions of the C2-domains of synaptotagmins with phospholipids, at least some synaptotagmins also bind to themselves and each other via Ca2+-dependent interactions that may be mediated by the C2B-domains (modified from Ref.
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ).
      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.
      • Südhof T.C.
      • Rizo J.
      ). 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 (
      • Morris N.J.
      • Ross S.A.
      • Neveu J.M.
      • Lane W.S.
      • Lienhard G.E.
      ,
      • Achanzar W.E.
      • Ward S.
      ,
      • Britton S.
      • Freeman T.
      • Vafiadaki E.
      • Keers S.
      • Harrison R.
      • Bushby K.
      • Bashir R.
      ,
      • Bashir 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.
      ,
      • Liu 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.
      ,
      • Yasunaga S.
      • Grati M.
      • Chardenoux S.
      • Smith T.N.
      • Friedman T.B.
      • Lalwani A.K.
      • Wilcox E.R.
      • Petit C.
      ).

      Genes and Alternative Splicing of Synaptotagmins

      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 (
      • Perin M.S.
      • Fried V.A.
      • Mignery G.A.
      • Jahn R.
      • Südhof T.C.
      ) 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 (
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ). 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 (
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ). The alternative splicing of Syt 7 is developmentally and regionally regulated, suggesting that it regulates the coupling of the C2-domains to the TMR (
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ).
      PCR experiments suggested that for many synaptotagmins, variants lacking TMRs are produced by “exon skipping” (
      • Craxton M.
      • Goedert M.
      ,
      • Fukuda M.
      • Mikoshiba K.
      ). 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.

      Synaptotagmin C2-Domains

      The synaptotagmin C2-domains are functionally polarized in that Ca2+ exclusively binds to the top loops but not the bottom loops (
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Garcia J.
      • Nittler M.P.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ,
      • Shao X.
      • Davletov B.A.
      • Sutton R.B.
      • Südhof T.C.
      • Rizo J.
      ,
      • Shao X.
      • Li C.
      • Fernandez I.
      • Zhang X.
      • Südhof T.C.
      • Rizo J.
      ). 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 (
      • Fukuda M.
      • Moreira J.E.
      • Liu V.
      • Sugimori M.
      • Mikoshiba K.
      • Llinas R.R.
      ) 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 (
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ) that is unlikely to unfold under physiological conditions. Another example is the AD3 mutant inDrosophila (
      • DiAntonio A.
      • Parfitt K.D.
      • Schwarz T.L.
      ,
      • Littleton J.T.
      • Stern M.
      • Schulze K.
      • Perin M.
      • Bellen H.J.
      ). 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 (
      • Fukuda M.
      • Kabayama H.
      • Mikoshiba K.
      ,
      • Littleton J.T.
      • Bai J.
      • Vyas B.
      • Desai R.
      • Baltus A.E.
      • Garment M.B.
      • Carlson S.D.
      • Ganetzky B.
      • Chapman E.R.
      ) are thus difficult to interpret. Similar reservations apply to other studies of synaptotagmins that involve sequences with conserved secondary structures.

      Ca2+ and Phospholipid Binding

      C2-domains bind Ca2+ exclusively via their top loops (
      • Rizo J.
      • Südhof T.C.
      ,
      • Shao X.
      • Davletov B.A.
      • Sutton R.B.
      • Südhof T.C.
      • Rizo J.
      ). Fig. 2shows a model of the Ca2+-binding sites of the Syt 1 C2-domains (
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ). The Ca2+-binding sites of C2-domains are unusual because Ca2+ ions are coordinated by residues that are widely separated in the primary sequence and because the coordination spheres for the Ca2+ions are incomplete (
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Shao X.
      • Davletov B.A.
      • Sutton R.B.
      • Südhof T.C.
      • Rizo J.
      ). The intrinsic Ca2+ affinities of the C2-domains are low (K d >1 mm and >0.3 mm for the Syt 1 C2A- and C2B-domains, respectively (
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      )). Ca2+binding to C2-domains (except for the unusual piccolo/aczonin C2-domain (
      • Gerber S.H.
      • Garcia J.
      • Rizo J.
      • Südhof T.C.
      )) does not cause significant conformational changes (
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Garcia J.
      • Nittler M.P.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ), suggesting that C2-domains function as electrostatic switches whereby Ca2+ binding triggers interactions by altering their surface charge (
      • Shao X.
      • Li C.
      • Fernandez I.
      • Zhang X.
      • Südhof T.C.
      • Rizo J.
      ).
      Figure thumbnail gr2
      Figure 2Structure of the Ca2+-binding sites of the C2A- and C2B-domains of synaptotagmins. The diagram shows a generic model for synaptotagmin Ca2+-binding sites that is based on the structure of the C2A- and C2B-domains of Syt 1 (
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ,
      • Ubach J.
      • Zhang X.
      • Shao X.
      • Südhof T.C.
      • Rizo J.
      ). The Ca2+-binding sites at the top of the synaptotagmin C2-domains are formed by loops 1 (right blue line) and 3 (left blue line). The C2A-domain ligates three Ca2+ions via five aspartate and one serine residue, whereas the C2B-domain lacks the binding site for Ca3 and ligates only two Ca2+ ions. The model is proposed for Syts 1–7 and 9–11, whereas Syts 8, 12, and 13 lack almost all of the Ca2+-binding residues. In Syts 4 and 11, one of five Ca2+-binding aspartates in the C2A-domain is substituted for a serine residue, suggesting that this C2A-domain may only bind one or two Ca2+ ions (
      • von Poser C.
      • Ichtchenko K.
      • Shao X.
      • Rizo J.
      • Südhof T.C.
      ).
      The C2-domains of Syt 1 bind phospholipids as a function of Ca2+ with apparent Ca2+ affinities (1–20 μm Ca2+) that are 100–10,000-fold the intrinsic Ca2+ affinities (
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Fernandez I.
      • Arac D.
      • Ubach J.
      • Gerber S.H.
      • Shin O.
      • Gao Y.
      • Anderson R.G.W.
      • Südhof T.C.
      • Rizo J.
      ). This dramatic increase probably occurs because the docked phospholipids on top of the C2-domains provide additional coordination sites for the incomplete Ca2+-binding spheres (
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Zhang X.
      • Rizo J.
      • Südhof T.C.
      ). It is likely that phospholipid binding by most synaptotagmins is functionally important but not all synaptotagmins bind Ca2+ and phospholipids. For example, Syts 4 and 11 have an evolutionarily conserved substitution in the Ca2+-binding site of the C2A-domain (Fig.2; Ref.
      • von Poser C.
      • Ichtchenko K.
      • Shao X.
      • Rizo J.
      • Südhof T.C.
      ) that prevents binding of a full complement of Ca2+ ions. In Syts 8, 12, and 13, the top loops of the C2A- and the C2B-domains lack almost all of the aspartate and glutamate residues involved in Ca2+binding.

      Ca2+-dependent Self-association

      The recombinant C2B-domains, but not C2A-domains, of most synaptotagmins pull down native Syt 1 and Syt 2 from brain in a Ca2+-dependent manner (
      • Sugita S.
      • Hata Y.
      • Südhof T.C.
      ,
      • Chapman E.R.
      • An S.
      • Edwardson J.M.
      • Jahn R.
      ,
      • Damer C.K.
      • Creutz C.E.
      ,
      • Osborne S.L.
      • Herreros J.
      • Bastiaens P.I.
      • Schiavo G.
      ). This activity suggests a potential mechanism by which C2B-domains could mediate Ca2+-dependent polymerization of synaptotagmins during exocytosis. Some studies detected Ca2+-dependent multimerization of recombinant C2-domains (
      • Damer C.K.
      • Creutz C.E.
      ,
      • Osborne S.L.
      • Herreros J.
      • Bastiaens P.I.
      • Schiavo G.
      ,
      • Desai R.C.
      • Vyas B.
      • Earles C.A.
      • Littleton J.T.
      • Kowalchyck J.A.
      • Martin T.F.
      • Chapman E.R.
      ), whereas others did not (
      • Sugita S.
      • Hata Y.
      • Südhof T.C.
      ,
      • Chapman E.R.
      • An S.
      • Edwardson J.M.
      • Jahn R.
      ). Structural characterization of the Syt 1 C2B-domain revealed that the electrophoretically pure C2B-domain contains stoichiometric amounts of bacterial contaminants (probably polynucleotides and lipids) that are tightly attached to a polybasic sequence (LKKKKT) on the C2B-domain, cause domain aggregation, and are only removed by harsh washes (
      • Ubach J.
      • Lao Y.
      • Fernandez I.
      • Arac D.
      • Südhof T.C.
      • Rizo J.
      ). C2B-domain that was cleared of bacterial contaminants exhibited competent Ca2+ binding but no self-association even at millimolar Ca2+ (
      • Ubach J.
      • Lao Y.
      • Fernandez I.
      • Arac D.
      • Südhof T.C.
      • Rizo J.
      ), but native Syt 1 formed Ca2+-dependent high molecular weight complexes, indicating that Ca2+-dependent self-association (
      • Sugita S.
      • Hata Y.
      • Südhof T.C.
      ,
      • Chapman E.R.
      • An S.
      • Edwardson J.M.
      • Jahn R.
      ) may prove relevant but utilize a more complex mechanism.

      Interactions with SNAREs

      Most synaptotagmins bind to syntaxin 1 in a Ca2+-dependent manner (
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ), suggesting an intersection of the mechanism of fusion (via the SNARE protein syntaxin) with the Ca2+-dependent regulation of fusion (via Syt 1) (reviewed in Ref.
      • Südhof T.C.
      ). However, the precise nature of this interaction is unclear. Binding of Syt 1 to syntaxin, SNAP-25 (another SNARE protein), and assembled SNARE complexes has been reported (
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Littleton J.T.
      • Bai J.
      • Vyas B.
      • Desai R.
      • Baltus A.E.
      • Garment M.B.
      • Carlson S.D.
      • Ganetzky B.
      • Chapman E.R.
      ,
      • Kee Y.
      • Scheller R.H.
      ,
      • Gerona R.R.
      • Larsen E.C.
      • Kowalchyk J.A.
      • Martin T.F.
      ,
      • Fernandez I.
      • Ubach J.
      • Dulubova I.
      • Zhang X.
      • Südhof T.C.
      • Rizo J.
      ). Synaptotagmin-binding sites on syntaxin have been localized on the N-terminal Habc domain (
      • Fernandez I.
      • Ubach J.
      • Dulubova I.
      • Zhang X.
      • Südhof T.C.
      • Rizo J.
      ) and the C-terminal SNARE motif (
      • Kee Y.
      • Scheller R.H.
      ).

      Binding to Endocytosis Proteins

      All synaptotagmins tested (Syts 1–7) interact with AP-2 with high affinity (
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Zhang J.Z.
      • Davletov B.A.
      • Südhof T.C.
      • Anderson R.G.W.
      ), and binding is enhanced by hydrophobic peptides (
      • Haucke V.
      • De Camilli P.
      ). Transfection of truncated synaptotagmins inhibits clathrin coat assembly and endocytosis in fibroblasts, suggesting that synaptotagmins have a physiological role in endocytosis (
      • Veit M.
      • Sollner T.H.
      • Rothman J.E.
      ). In Drosophila, mutations in the stoned gene cause a defect in synaptic vesicle endocytosis and also a mislocalization and destabilization of Syt 1 (
      • Fergestad T.
      • Broadie K.
      ,
      • Phillips A.M.
      • Smith M.
      • Ramaswami M.
      • Kelly L.E.
      ). Mammalian stoned proteins may also interact with Syt 1, suggesting that this could be a general mechanism in synaptic vesicle endocytosis (
      • Martina J.A.
      • Bonangelino C.J.
      • Aguilar R.C.
      • Bonifacino J.S.
      ,
      • Walther K.
      • Krauss M.
      • Diril M.K.
      • Lemke S.
      • Ricotta D.
      • Honing S.
      • Kaiser S.
      • Haucke V.
      ).

      Binding of Inositol Phosphates

      The C2B-domains of most synaptotagmins bind to inositol polyphosphates via the short polybasic sequence that contains bacterial contaminants in recombinant C2B-domains (
      • Ibata K.
      • Fukuda M.
      • Mikoshiba K.
      ). Binding does not require the folded C2B-domain but is obtained with the isolated polybasic sequence. Inhibition of neurotransmitter release by inositol polyphosphates suggests that inositol phosphate binding to synaptotagmin physiologically regulates release (
      • Llinas R
      • Sugimori M.
      • Lang E.J.
      • Morita M.
      • Fukuda M.
      • Niinobe M.
      • Mikoshiba K.
      ). However, hundreds of synaptic proteins may bind to inositol phosphates, and the specific role of synaptotagmin is unknown.

      Functional Properties of Individual Synaptotagmins

      Class 1: Syts 1 and 2 as Vesicular Ca2+Sensors

      Syts 1 and 2 are localized to synaptic vesicles and secretory granules (
      • Matthew W.D.
      • Tsavaler L.
      • Reichardt L.F.
      ,
      • Perin M.S.
      • Fried V.A.
      • Mignery G.A.
      • Jahn R.
      • Südhof T.C.
      ,
      • Geppert M.
      • Archer III, B.T.
      • Südhof T.C.
      ,
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ), are closely related, and are differentially expressed in a largely non-overlapping pattern (
      • Geppert M.
      • Archer III, B.T.
      • Südhof T.C.
      ,
      • Ullrich B.
      • Li C.
      • Zhang J.Z.
      • McMahon H.
      • Anderson R.G.
      • Geppert M.
      • Südhof T.C.
      ,
      • Marqueze B.
      • Boudier J.A.
      • Mizuta M.
      • Inagaki N.
      • Seino S.
      • Seagar M.
      ,
      • Marqueze B.
      • Berton F.
      • Seagar M.
      ). Syts 1 and 2 probably function as an essential Ca2+sensor in fast synaptic vesicle exocytosis (
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      ) as shown by a point mutation that was introduced into the endogenous mouse genome (
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ). This mutation caused an approximately 2-fold decrease in the apparent Ca2+ affinity of Syt 1 and a similar decrease in the Ca2+ sensitivity of synaptic vesicle exocytosis, indicating that Ca2+ binding to Syt 1 is an essential step in triggering exocytosis. However, Syts 1 and 2 are not the only Ca2+ sensor because some Ca2+-dependent exocytosis remains in their absence (
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      ,
      • Nonet M.L.
      • Grundahl K.
      • Meyer B.J.
      • Rand J.B.
      ,
      • DiAntonio A.
      • Parfitt K.D.
      • Schwarz T.L.
      ). In addition to exocytosis, Syt 1 may also function in endocytosis as discussed above.

      Class 2: Syt 7 as an Active Zone Ca2+ Sensor

      Syt 7, together with Syt 3, is the most abundant synaptotagmin after Syts 1 and 2. Syt 7 is expressed predominantly in brain (
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ,
      • Ullrich B.
      • Li C.
      • Zhang J.Z.
      • McMahon H.
      • Anderson R.G.
      • Geppert M.
      • Südhof T.C.
      ,
      • Marqueze B.
      • Boudier J.A.
      • Mizuta M.
      • Inagaki N.
      • Seino S.
      • Seagar M.
      ) where it is primarily present on plasma membranes (
      • Butz S.
      • Fernandez-Chacon R.
      • Schmitz F.
      • Jahn R.
      • Südhof T.C.
      ,
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ). Confusingly, in cultured cells Syt 7 has been reported in lysosomes (
      • Martinez I.
      • Chakrabarti S.
      • Hellevik T.
      • Morehead J.
      • Fowler K.
      • Andrews N.W.
      ). However, the antibody used in that study did not detect Syt 7 in brain, making the localization of Syt 7 in non-neuronal cells uncertain. The C2-domains of Syt 7 have a 10–20-fold higher Ca2+ affinity than those of Syts 1 and 2 (
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ) and self-associate as a function of Ca2+ (
      • Fukuda M.
      • Mikoshiba K.
      ). In permeabilized PC12 cells, recombinant C2A- and C2B-domains of Syt 7 severely inhibit exocytosis (
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ). C2-domains of Syt 1, by contrast, had no effect consistent with the fact that Syt 1 is not essential for Ca2+-triggered exocytosis in PC12 cells as opposed to synapses (
      • Shoji-Kasai Y.
      • Yoshida A.
      • Sato K.
      • Hoshino T.
      • Ogura A.
      • Kondo S.
      • Fujimoto Y.
      • Kuwahara R.
      • Kato R.
      • Takahashi M.
      ). Inhibition by recombinant Syt 7 C2-domains required intact Ca2+-binding sites, suggesting that plasma membrane Syt 7 in PC12 cells functions as a Ca2+ sensor for exocytosis.

      Class 3: Syts 3, 5, 6, and 10 as Plasma Membrane Ca2+Sensors

      Similar to Syt 7, Syts 3 and 6 are localized to plasma membranes, have a 10-fold higher Ca2+ affinity than Syts 1 and 2, and probably function in Ca2+-triggered PC12 exocytosis (
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ,
      • Berton F.
      • Cornet V.
      • Iborra C.
      • Garrido J.
      • Dargent B.
      • Fukuda M.
      • Seagar M.
      • Marqueze B.
      ). Syt 10 was identified as a seizure-inducible mRNA and may have a function related to neuronal excitability (
      • Babity J.M.
      • Armstrong J.N.
      • Plumier J.C.
      • Currie R.W.
      • Robertson H.A.
      ).

      Class 4: Syts 4 and 11

      Syts 4 and 11 are encoded by the simplest genes among synaptotagmins (Fig. 1). Their characteristic feature is a conserved substitution of an aspartate for a serine residue in the C2A-domain (Fig. 2) that changes the Ca2+-binding properties of the C2A-domain (
      • von Poser C.
      • Ichtchenko K.
      • Shao X.
      • Rizo J.
      • Südhof T.C.
      ). Syt 4 is expressed at highest levels early during postnatal development (
      • Berton F.
      • Cornet V.
      • Iborra C.
      • Garrido J.
      • Dargent B.
      • Fukuda M.
      • Seagar M.
      • Marqueze B.
      ) but is strongly induced by seizures in adult brain (
      • Vician L.
      • Lim I.K.
      • Ferguson G.
      • Tocco G.
      • Baudry M.
      • Herschman H.R.
      ) and by increases in cAMP or Ca2+ in PC12 cells (
      • Ferguson G.D.
      • Thomas D.M.
      • Elferink L.A.
      • Herschman H.R.
      ). Immunocytochemistry of brain sections, cultured neurons, and transfected cells localized Syt 4 in the Golgi apparatus (
      • Berton F.
      • Cornet V.
      • Iborra C.
      • Garrido J.
      • Dargent B.
      • Fukuda M.
      • Seagar M.
      • Marqueze B.
      ,
      • Fukuda M.
      • Ibata K.
      • Mikoshiba K.
      ). In contrast, the Drosophila Syt 4 homolog was observed in synaptic vesicles (
      • Littleton J.T.
      • Serano T.L.
      • Rubin G.M.
      • Ganetzky B.
      • Chapman E.R.
      ).
      The function of Syts 4 and 11 is also uncertain. Microinjection of Syt 4 fragments into PC12 cells (
      • Thomas D.M.
      • Ferguson G.D.
      • Herschman H.R.
      • Elferink L.A.
      ) and overexpression ofDrosophila Syt 4 inhibited exocytosis (
      • Littleton J.T.
      • Serano T.L.
      • Rubin G.M.
      • Ganetzky B.
      • Chapman E.R.
      ). A knockout of Syt 4 in mice uncovered a subtle but interesting phenotype that is consistent with a modulatory role at the synapse (
      • Ferguson G.D.
      • Anagnostaras S.G.
      • Silva A.J.
      • Herschman H.R.
      ). Nothing is known about the expression and localization of Syt 11, but the similarity between Syts 4 and 11 suggests that they are redundant.

      Class 5: Syt 9

      Syt 9 (also called Syt 5 (
      • Li C.
      • Ullrich B.
      • Zhang Z.Z.
      • Anderson R.G.W.
      • Brose N.
      • Südhof T.C.
      ,
      • Hudson A.W.
      • Birnbaum M.J.
      ,
      • Craxton M.
      • Goedert M.
      )) is synthesized at low abundance in brain and non-neuronal cells (
      • Hudson A.W.
      • Birnbaum M.J.
      ,
      • Craxton M.
      • Goedert M.
      ) and localized to synaptic vesicles (
      • Hudson A.W.
      • Birnbaum M.J.
      ).

      Class 6: Atypical Syts 8, 12, and 13 That Lack Consensus Ca2+-binding Sites

      Very little is also known about these synaptotagmins. Syt 12 was initially discovered as a thyroid hormone-regulated transcript (
      • Thompson C.C.
      ), indicating a specialized function in brain that could be related to thyroid-dependent developmental changes.

      A Working Model for Synaptotagmin Function

      Vertebrates express at least 13 synaptotagmins that are synthesized exclusively or predominantly in neurons and neuroendocrine cells. By virtue of their C2-domains, most synaptotagmins are membrane-bound Ca2+-signaling machines. The presence of distinct synaptotagmins on the membranes of synaptic vesicles and active zones, the membranes that have to fuse during neurotransmitter release, suggests a potential explanation for the existence of multiple synaptotagmins. The model in Fig. 3proposes that at least Syts 1, 2, 3, 6, and 7 perform complementary functions in Ca2+-triggered exocytosis whereby Ca2+ binding to each class of synaptotagmins contributes differently to triggering exocytosis. As Ca2+ sensors, all synaptotagmins share the same Ca2+ cooperativity and Ca2+-dependent phospholipid binding but have distinct Ca2+ binding properties. Vesicular synaptotagmins have a lower Ca2+ affinity and are more important in fast synaptic exocytosis than in endocrine exocytosis, the bulk of which is much slower (
      • Geppert M.
      • Goda Y.
      • Hammer R.E.
      • Li C.
      • Rosahl T.W.
      • Stevens C.F.
      • Südhof T.C.
      ,
      • Fernandez-Chacon R.
      • Konigstorffer
      • Gerber S.H.
      • Garcia J.
      • Matos M.F.
      • Stevens C.F.
      • Brose N.
      • Rizo J.
      • Rosenmund C.
      • Südhof T.C.
      ,
      • Voets T.
      • Moser T.
      • Lund P.E.
      • Chow R.H.
      • Geppert M.
      • Südhof T.C.
      • Neher E.
      ). Plasma membrane synaptotagmins, in contrast, have a higher Ca2+ affinity and may be more important for endocrine exocytosis (
      • Sugita S.
      • Han W.
      • Butz S.
      • Liu X.
      • Fernandez-Chacon R.
      • Lao Y.
      • Südhof T.C.
      ,
      • Sugita S.
      • Shin O.-K.
      • Han W.
      • Lao Y.
      • Südhof T.C.
      ). With these distinct properties, the combination and relative abundance of various synaptotagmins could contribute to shaping the characteristic Ca2+ responses of synapses.
      Although the model proposed for synaptotagmin function (Fig. 3) is attractive because it provides an explanation for the unprecedented Ca2+ control of neurotransmitter release, it is as yet only a working hypothesis. Questions abound. For example, is Ca2+ binding to a single type of synaptotagmin sufficient to trigger exocytosis, what is the functional relation between different C2-domains of synaptotagmins, and how do synaptotagmins precisely work in exocytosis? If synaptotagmins are Ca2+-signaling machines, why do some synaptotagmins lack Ca2+-binding sites? Another set of questions regards the potential role of synaptotagmins in endocytosis and the possibility that at least some of them may be expressed outside of neurons and endocrine cells. Addressing these questions will be essential for further progress.

      Acknowledgments

      I thank Drs. J. Rizo, R. Jahn, and E. Kavalali for innumerable discussions.

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