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. 2002 Jul 1;365(Pt 1):173–180. doi: 10.1042/BJ20011877

Alternative splicing isoforms of synaptotagmin VII in the mouse, rat and human.

Mitsunori Fukuda 1, Yukie Ogata 1, Chika Saegusa 1, Eiko Kanno 1, Katsuhiko Mikoshiba 1
PMCID: PMC1222667  PMID: 12071850

Abstract

Synaptotagmin VII (Syt VII) has been proposed to regulate several different types of Ca2+-dependent exocytosis, but its subcellular localization (lysosome or plasma membrane) and the number of alternative splicing isoforms of Syt VII (single or multiple forms) are matters of controversy. In the present study, we show by reverse transcriptase-PCR analysis that mouse Syt VII has one major isoform (Syt VIIalpha), the original Syt VII, and two minor isoforms (Syt VIIbeta and Syt VIIgamma), which contain unique insertions (of 44 and 116 amino acids respectively) in the spacer domain between the transmembrane and C2 domains of Syt VIIalpha. Similar results were obtained with respect to rat and human Syt VII mRNA expression. An antibody against the N-terminal domain of mouse Syt VII [anti-(Syt VII-N)], which specifically recognized recombinant Syt VII but not other Syt isoforms expressed in COS-7 cells, recognized two major, closely co-migrating bands (p58 and p60) and minor bands of approx. 65 kDa in mouse brain. Immunoaffinity purification of proteins that bind the anti-(Syt VII-N) antibody, and peptide sequence analysis revealed that: (i) the major p58 and p60 bands are identified as adenylate cyclase-associated protein 2; (ii) actin-binding protein is localized at the plasma membrane; and (iii) Syt VIIalpha (65 kDa) is the major Syt VII isoform, but with a much lower expression level than previously thought. It was also shown that FLAG-Syt VII-green-fluorescence-protein fusion protein stably expressed in PC12 cells is localized in the perinuclear region (co-localization with TGN38 protein, even after brefeldin A treatment) and in the tips of neurites (co-localization with Syt I), and not in the plasma membrane.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Butz S., Fernandez-Chacon R., Schmitz F., Jahn R., Südhof T. C. The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles. J Biol Chem. 1999 Jun 25;274(26):18290–18296. doi: 10.1074/jbc.274.26.18290. [DOI] [PubMed] [Google Scholar]
  2. Chapman E. R., Blasi J., An S., Brose N., Johnston P. A., Südhof T. C., Jahn R. Fatty acylation of synaptotagmin in PC12 cells and synaptosomes. Biochem Biophys Res Commun. 1996 Aug 5;225(1):326–332. doi: 10.1006/bbrc.1996.1174. [DOI] [PubMed] [Google Scholar]
  3. Craxton M. Genomic analysis of synaptotagmin genes. Genomics. 2001 Sep;77(1-2):43–49. doi: 10.1006/geno.2001.6619. [DOI] [PubMed] [Google Scholar]
  4. Craxton M., Goedert M. Alternative splicing of synaptotagmins involving transmembrane exon skipping. FEBS Lett. 1999 Nov 5;460(3):417–422. doi: 10.1016/s0014-5793(99)01382-4. [DOI] [PubMed] [Google Scholar]
  5. Elferink L. A., Peterson M. R., Scheller R. H. A role for synaptotagmin (p65) in regulated exocytosis. Cell. 1993 Jan 15;72(1):153–159. doi: 10.1016/0092-8674(93)90059-y. [DOI] [PubMed] [Google Scholar]
  6. Erba H. P., Gunning P., Kedes L. Nucleotide sequence of the human gamma cytoskeletal actin mRNA: anomalous evolution of vertebrate non-muscle actin genes. Nucleic Acids Res. 1986 Jul 11;14(13):5275–5294. doi: 10.1093/nar/14.13.5275. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Fukuda M., Aruga J., Niinobe M., Aimoto S., Mikoshiba K. Inositol-1,3,4,5-tetrakisphosphate binding to C2B domain of IP4BP/synaptotagmin II. J Biol Chem. 1994 Nov 18;269(46):29206–29211. [PubMed] [Google Scholar]
  8. Fukuda M., Ibata K., Mikoshiba K. A unique spacer domain of synaptotagmin IV is essential for Golgi localization. J Neurochem. 2001 May;77(3):730–740. doi: 10.1046/j.1471-4159.2001.00266.x. [DOI] [PubMed] [Google Scholar]
  9. Fukuda M., Kanno E., Mikoshiba K. Conserved N-terminal cysteine motif is essential for homo- and heterodimer formation of synaptotagmins III, V, VI, and X. J Biol Chem. 1999 Oct 29;274(44):31421–31427. doi: 10.1074/jbc.274.44.31421. [DOI] [PubMed] [Google Scholar]
  10. Fukuda M., Kanno E., Ogata Y., Mikoshiba K. Mechanism of the SDS-resistant synaptotagmin clustering mediated by the cysteine cluster at the interface between the transmembrane and spacer domains. J Biol Chem. 2001 Aug 20;276(43):40319–40325. doi: 10.1074/jbc.M105356200. [DOI] [PubMed] [Google Scholar]
  11. Fukuda M., Kojima T., Aruga J., Niinobe M., Mikoshiba K. Functional diversity of C2 domains of synaptotagmin family. Mutational analysis of inositol high polyphosphate binding domain. J Biol Chem. 1995 Nov 3;270(44):26523–26527. doi: 10.1074/jbc.270.44.26523. [DOI] [PubMed] [Google Scholar]
  12. Fukuda M., Mikoshiba K. A novel alternatively spliced variant of synaptotagmin VI lacking a transmembrane domain. Implications for distinct functions of the two isoforms. J Biol Chem. 1999 Oct 29;274(44):31428–31434. doi: 10.1074/jbc.274.44.31428. [DOI] [PubMed] [Google Scholar]
  13. Fukuda M., Mikoshiba K. Calcium-dependent and -independent hetero-oligomerization in the synaptotagmin family. J Biochem. 2000 Oct;128(4):637–645. doi: 10.1093/oxfordjournals.jbchem.a022796. [DOI] [PubMed] [Google Scholar]
  14. Fukuda M., Mikoshiba K. Characterization of KIAA1427 protein as an atypical synaptotagmin (Syt XIII). Biochem J. 2001 Mar 1;354(Pt 2):249–257. doi: 10.1042/0264-6021:3540249. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Fukuda M., Mikoshiba K. Distinct self-oligomerization activities of synaptotagmin family. Unique calcium-dependent oligomerization properties of synaptotagmin VII. J Biol Chem. 2000 Sep 8;275(36):28180–28185. doi: 10.1074/jbc.M001376200. [DOI] [PubMed] [Google Scholar]
  16. Fukuda M., Mikoshiba K. Mechanism of the calcium-dependent multimerization of synaptotagmin VII mediated by its first and second C2 domains. J Biol Chem. 2001 May 23;276(29):27670–27676. doi: 10.1074/jbc.M100851200. [DOI] [PubMed] [Google Scholar]
  17. Fukuda M., Mikoshiba K. Synaptotagmin-like protein 1-3: a novel family of C-terminal-type tandem C2 proteins. Biochem Biophys Res Commun. 2001 Mar;281(5):1226–1233. doi: 10.1006/bbrc.2001.4512. [DOI] [PubMed] [Google Scholar]
  18. Fukuda M., Mikoshiba K. The N-terminal cysteine cluster is essential for membrane targeting of B/K protein. Biochem J. 2001 Dec 1;360(Pt 2):441–448. doi: 10.1042/0264-6021:3600441. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fukuda M., Mikoshiba K. The function of inositol high polyphosphate binding proteins. Bioessays. 1997 Jul;19(7):593–603. doi: 10.1002/bies.950190710. [DOI] [PubMed] [Google Scholar]
  20. Fukuda M., Yamamoto A., Mikoshiba K. Formation of crystalloid endoplasmic reticulum induced by expression of synaptotagmin lacking the conserved WHXL motif in the C terminus. Structural importance of the WHXL motif in the C2B domain. J Biol Chem. 2001 Aug 30;276(44):41112–41119. doi: 10.1074/jbc.M106209200. [DOI] [PubMed] [Google Scholar]
  21. Fukuda Mitsunori, Kowalchyk Judith A., Zhang Xiaodong, Martin Thomas F. J., Mikoshiba Katsuhiko. Synaptotagmin IX regulates Ca2+-dependent secretion in PC12 cells. J Biol Chem. 2001 Dec 21;277(7):4601–4604. doi: 10.1074/jbc.C100588200. [DOI] [PubMed] [Google Scholar]
  22. Gao Z., Reavey-Cantwell J., Young R. A., Jegier P., Wolf B. A. Synaptotagmin III/VII isoforms mediate Ca2+-induced insulin secretion in pancreatic islet beta -cells. J Biol Chem. 2000 Nov 17;275(46):36079–36085. doi: 10.1074/jbc.M004284200. [DOI] [PubMed] [Google Scholar]
  23. Gottwald U., Brokamp R., Karakesisoglou I., Schleicher M., Noegel A. A. Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin. Mol Biol Cell. 1996 Feb;7(2):261–272. doi: 10.1091/mbc.7.2.261. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Gut A., Kiraly C. E., Fukuda M., Mikoshiba K., Wollheim C. B., Lang J. Expression and localisation of synaptotagmin isoforms in endocrine beta-cells: their function in insulin exocytosis. J Cell Sci. 2001 May;114(Pt 9):1709–1716. doi: 10.1242/jcs.114.9.1709. [DOI] [PubMed] [Google Scholar]
  25. Hubberstey A., Yu G., Loewith R., Lakusta C., Young D. Mammalian CAP interacts with CAP, CAP2, and actin. J Cell Biochem. 1996 Jun 1;61(3):459–466. doi: 10.1002/(SICI)1097-4644(19960601)61:3%3C459::AID-JCB13%3E3.0.CO;2-E. [DOI] [PubMed] [Google Scholar]
  26. Hudson A. W., Birnbaum M. J. Identification of a nonneuronal isoform of synaptotagmin. Proc Natl Acad Sci U S A. 1995 Jun 20;92(13):5895–5899. doi: 10.1073/pnas.92.13.5895. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ibata K., Fukuda M., Hamada T., Kabayama H., Mikoshiba K. Synaptotagmin IV is present at the Golgi and distal parts of neurites. J Neurochem. 2000 Feb;74(2):518–526. doi: 10.1046/j.1471-4159.2000.740518.x. [DOI] [PubMed] [Google Scholar]
  28. Kawai J., Shinagawa A., Shibata K., Yoshino M., Itoh M., Ishii Y., Arakawa T., Hara A., Fukunishi Y., Konno H. Functional annotation of a full-length mouse cDNA collection. Nature. 2001 Feb 8;409(6821):685–690. doi: 10.1038/35055500. [DOI] [PubMed] [Google Scholar]
  29. Kida Y., Sakaguchi M., Fukuda M., Mikoshiba K., Mihara K. Membrane topogenesis of a type I signal-anchor protein, mouse synaptotagmin II, on the endoplasmic reticulum. J Cell Biol. 2000 Aug 21;150(4):719–730. doi: 10.1083/jcb.150.4.719. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kishore B. K., Wade J. B., Schorr K., Inoue T., Mandon B., Knepper M. A. Expression of synaptotagmin VIII in rat kidney. Am J Physiol. 1998 Jul;275(1 Pt 2):F131–F142. doi: 10.1152/ajprenal.1998.275.1.F131. [DOI] [PubMed] [Google Scholar]
  31. Kwon O. J., Gainer H., Wray S., Chin H. Identification of a novel protein containing two C2 domains selectively expressed in the rat brain and kidney. FEBS Lett. 1996 Jan 8;378(2):135–139. doi: 10.1016/0014-5793(95)01430-6. [DOI] [PubMed] [Google Scholar]
  32. Li C., Ullrich B., Zhang J. Z., Anderson R. G., Brose N., Südhof T. C. Ca(2+)-dependent and -independent activities of neural and non-neural synaptotagmins. Nature. 1995 Jun 15;375(6532):594–599. doi: 10.1038/375594a0. [DOI] [PubMed] [Google Scholar]
  33. Marquèze B., Berton F., Seagar M. Synaptotagmins in membrane traffic: which vesicles do the tagmins tag? Biochimie. 2000 May;82(5):409–420. doi: 10.1016/s0300-9084(00)00220-0. [DOI] [PubMed] [Google Scholar]
  34. Martinez I., Chakrabarti S., Hellevik T., Morehead J., Fowler K., Andrews N. W. Synaptotagmin VII regulates Ca(2+)-dependent exocytosis of lysosomes in fibroblasts. J Cell Biol. 2000 Mar 20;148(6):1141–1149. doi: 10.1083/jcb.148.6.1141. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Matviw H., Yu G., Young D. Identification of a human cDNA encoding a protein that is structurally and functionally related to the yeast adenylyl cyclase-associated CAP proteins. Mol Cell Biol. 1992 Nov;12(11):5033–5040. doi: 10.1128/mcb.12.11.5033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Mizushima S., Nagata S. pEF-BOS, a powerful mammalian expression vector. Nucleic Acids Res. 1990 Sep 11;18(17):5322–5322. doi: 10.1093/nar/18.17.5322. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Osborne S. L., Herreros J., Bastiaens P. I., Schiavo G. Calcium-dependent oligomerization of synaptotagmins I and II. Synaptotagmins I and II are localized on the same synaptic vesicle and heterodimerize in the presence of calcium. J Biol Chem. 1999 Jan 1;274(1):59–66. doi: 10.1074/jbc.274.1.59. [DOI] [PubMed] [Google Scholar]
  38. Reddy A., Caler E. V., Andrews N. W. Plasma membrane repair is mediated by Ca(2+)-regulated exocytosis of lysosomes. Cell. 2001 Jul 27;106(2):157–169. doi: 10.1016/s0092-8674(01)00421-4. [DOI] [PubMed] [Google Scholar]
  39. Schiavo G., Osborne S. L., Sgouros J. G. Synaptotagmins: more isoforms than functions? Biochem Biophys Res Commun. 1998 Jul 9;248(1):1–8. doi: 10.1006/bbrc.1998.8527. [DOI] [PubMed] [Google Scholar]
  40. Smith D. B., Johnson K. S. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. doi: 10.1016/0378-1119(88)90005-4. [DOI] [PubMed] [Google Scholar]
  41. Sugimori M., Tong C. K., Fukuda M., Moreira J. E., Kojima T., Mikoshiba K., Llinás R. Presynaptic injection of syntaxin-specific antibodies blocks transmission in the squid giant synapse. Neuroscience. 1998 Sep;86(1):39–51. doi: 10.1016/s0306-4522(97)00689-1. [DOI] [PubMed] [Google Scholar]
  42. Sugita S., Han W., Butz S., Liu X., Fernández-Chacón R., Lao Y., Südhof T. C. Synaptotagmin VII as a plasma membrane Ca(2+) sensor in exocytosis. Neuron. 2001 May;30(2):459–473. doi: 10.1016/s0896-6273(01)00290-2. [DOI] [PubMed] [Google Scholar]
  43. Südhof T. C., Rizo J. Synaptotagmins: C2-domain proteins that regulate membrane traffic. Neuron. 1996 Sep;17(3):379–388. doi: 10.1016/s0896-6273(00)80171-3. [DOI] [PubMed] [Google Scholar]
  44. Thomas D. M., Ferguson G. D., Herschman H. R., Elferink L. A. Functional and biochemical analysis of the C2 domains of synaptotagmin IV. Mol Biol Cell. 1999 Jul;10(7):2285–2295. doi: 10.1091/mbc.10.7.2285. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Veit M., Söllner T. H., Rothman J. E. Multiple palmitoylation of synaptotagmin and the t-SNARE SNAP-25. FEBS Lett. 1996 Apr 29;385(1-2):119–123. doi: 10.1016/0014-5793(96)00362-6. [DOI] [PubMed] [Google Scholar]
  46. Vojtek A. B., Cooper J. A. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein. J Cell Sci. 1993 Jul;105(Pt 3):777–785. doi: 10.1242/jcs.105.3.777. [DOI] [PubMed] [Google Scholar]
  47. Vojtek A. B., Cooper J. A. Identification and characterization of a cDNA encoding mouse CAP: a homolog of the yeast adenylyl cyclase associated protein. J Cell Sci. 1993 Jul;105(Pt 3):777–785. doi: 10.1242/jcs.105.3.777. [DOI] [PubMed] [Google Scholar]
  48. Zelicof A., Gatica J., Gerst J. E. Molecular cloning and characterization of a rat homolog of CAP, the adenylyl cyclase-associated protein from Saccharomyces cerevisiae. J Biol Chem. 1993 Jun 25;268(18):13448–13453. [PubMed] [Google Scholar]

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