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. 1993 Apr;13(4):2061–2068. doi: 10.1128/mcb.13.4.2061

Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin.

H Shirataki 1, K Kaibuchi 1, T Sakoda 1, S Kishida 1, T Yamaguchi 1, K Wada 1, M Miyazaki 1, Y Takai 1
PMCID: PMC359527  PMID: 8384302

Abstract

In a previous study (H. Shirataki, K. Kaibuchi, T. Yamaguchi, K. Wada, H. Horiuchi, and Y. Takai, J. Biol. Chem. 267:10946-10949, 1992), we highly purified from bovine brain crude membranes the putative target protein for smg p25A/rab3A p25, a ras p21-related small GTP-binding protein implicated in neurotransmitter release. In this study, we have isolated and sequenced the cDNA of this protein from a bovine brain cDNA library. The cDNA had an open reading frame encoding a protein of 704 amino acids with a calculated M(r) of 77,976. We tentatively refer to this protein as rabphilin-3A. Structural analysis of rabphilin-3A revealed the existence of two copies of an internal repeat that were homologous to the C2 domain of protein kinase C as described for synaptotagmin, which is known to be localized in the membrane of the synaptic vesicle and to bind to membrane phospholipid in a Ca(2+)-dependent manner. The isolated cDNA was expressed in COS7 cells, and the encoded protein was recognized with an anti-rabphilin-3A polyclonal antibody and was identical in size with rabphilin-3A purified from bovine brain by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, both rabphilin-3A purified from bovine brain and recombinant rabphilin-3A made a complex with the GTP gamma S-bound form of rab3A p25 but not with the GDP-bound form of rab3A p25. Immunoblot and Northern (RNA) blot analyses showed that rabphilin-3A was highly expressed in bovine and rat brains. These results indicate that rabphilin-3A is a novel protein that has C2 domains and selectively interacts with the GTP-bound form of rab3A p25.

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Selected References

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

  1. Aebersold R. H., Leavitt J., Saavedra R. A., Hood L. E., Kent S. B. Internal amino acid sequence analysis of proteins separated by one- or two-dimensional gel electrophoresis after in situ protease digestion on nitrocellulose. Proc Natl Acad Sci U S A. 1987 Oct;84(20):6970–6974. doi: 10.1073/pnas.84.20.6970. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Araki S., Kaibuchi K., Sasaki T., Hata Y., Takai Y. Role of the C-terminal region of smg p25A in its interaction with membranes and the GDP/GTP exchange protein. Mol Cell Biol. 1991 Mar;11(3):1438–1447. doi: 10.1128/mcb.11.3.1438. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Araki S., Kikuchi A., Hata Y., Isomura M., Takai Y. Regulation of reversible binding of smg p25A, a ras p21-like GTP-binding protein, to synaptic plasma membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J Biol Chem. 1990 Aug 5;265(22):13007–13015. [PubMed] [Google Scholar]
  4. Balch W. E. Small GTP-binding proteins in vesicular transport. Trends Biochem Sci. 1990 Dec;15(12):473–477. doi: 10.1016/0968-0004(90)90301-q. [DOI] [PubMed] [Google Scholar]
  5. Barbacid M. ras genes. Annu Rev Biochem. 1987;56:779–827. doi: 10.1146/annurev.bi.56.070187.004023. [DOI] [PubMed] [Google Scholar]
  6. Bennett M. K., Calakos N., Kreiner T., Scheller R. H. Synaptic vesicle membrane proteins interact to form a multimeric complex. J Cell Biol. 1992 Feb;116(3):761–775. doi: 10.1083/jcb.116.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Bennett M. K., Calakos N., Scheller R. H. Syntaxin: a synaptic protein implicated in docking of synaptic vesicles at presynaptic active zones. Science. 1992 Jul 10;257(5067):255–259. doi: 10.1126/science.1321498. [DOI] [PubMed] [Google Scholar]
  8. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  9. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  10. Brose N., Petrenko A. G., Südhof T. C., Jahn R. Synaptotagmin: a calcium sensor on the synaptic vesicle surface. Science. 1992 May 15;256(5059):1021–1025. doi: 10.1126/science.1589771. [DOI] [PubMed] [Google Scholar]
  11. Chin G. J., Goldman S. A. Purification of squid synaptic vesicles and characterization of the vesicle-associated proteins synaptobrevin and Rab3A. Brain Res. 1992 Jan 31;571(1):89–96. doi: 10.1016/0006-8993(92)90512-8. [DOI] [PubMed] [Google Scholar]
  12. Fischer von Mollard G., Mignery G. A., Baumert M., Perin M. S., Hanson T. J., Burger P. M., Jahn R., Südhof T. C. rab3 is a small GTP-binding protein exclusively localized to synaptic vesicles. Proc Natl Acad Sci U S A. 1990 Mar;87(5):1988–1992. doi: 10.1073/pnas.87.5.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Fischer von Mollard G., Südhof T. C., Jahn R. A small GTP-binding protein dissociates from synaptic vesicles during exocytosis. Nature. 1991 Jan 3;349(6304):79–81. doi: 10.1038/349079a0. [DOI] [PubMed] [Google Scholar]
  14. Fukumoto Y., Kaibuchi K., Hori Y., Fujioka H., Araki S., Ueda T., Kikuchi A., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for the rho proteins, ras p21-like small GTP-binding proteins. Oncogene. 1990 Sep;5(9):1321–1328. [PubMed] [Google Scholar]
  15. Geppert M., Archer B. T., 3rd, Südhof T. C. Synaptotagmin II. A novel differentially distributed form of synaptotagmin. J Biol Chem. 1991 Jul 25;266(21):13548–13552. [PubMed] [Google Scholar]
  16. Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
  17. Kaibuchi K., Fukumoto Y., Oku N., Hori Y., Yamamoto T., Toyoshima K., Takai Y. Activation of the serum response element and 12-O-tetradecanoylphorbol-13-acetate response element by the activated c-raf-1 protein in a manner independent of protein kinase C. J Biol Chem. 1989 Dec 15;264(35):20855–20858. [PubMed] [Google Scholar]
  18. Kanehisa M. I. Los Alamos sequence analysis package for nucleic acids and proteins. Nucleic Acids Res. 1982 Jan 11;10(1):183–196. doi: 10.1093/nar/10.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kikuchi A., Yamashita T., Kawata M., Yamamoto K., Ikeda K., Tanimoto T., Takai Y. Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes. J Biol Chem. 1988 Feb 25;263(6):2897–2904. [PubMed] [Google Scholar]
  20. Kozak M. An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs. Nucleic Acids Res. 1987 Oct 26;15(20):8125–8148. doi: 10.1093/nar/15.20.8125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kyte J., Doolittle R. F. A simple method for displaying the hydropathic character of a protein. J Mol Biol. 1982 May 5;157(1):105–132. doi: 10.1016/0022-2836(82)90515-0. [DOI] [PubMed] [Google Scholar]
  22. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  23. Leveque C., Hoshino T., David P., Shoji-Kasai Y., Leys K., Omori A., Lang B., el Far O., Sato K., Martin-Moutot N. The synaptic vesicle protein synaptotagmin associates with calcium channels and is a putative Lambert-Eaton myasthenic syndrome antigen. Proc Natl Acad Sci U S A. 1992 Apr 15;89(8):3625–3629. doi: 10.1073/pnas.89.8.3625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lowe A. W., Madeddu L., Kelly R. B. Endocrine secretory granules and neuronal synaptic vesicles have three integral membrane proteins in common. J Cell Biol. 1988 Jan;106(1):51–59. doi: 10.1083/jcb.106.1.51. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matsui Y., Kikuchi A., Araki S., Hata Y., Kondo J., Teranishi Y., Takai Y. Molecular cloning and characterization of a novel type of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol Cell Biol. 1990 Aug;10(8):4116–4122. doi: 10.1128/mcb.10.8.4116. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsui Y., Kikuchi A., Kondo J., Hishida T., Teranishi Y., Takai Y. Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain. J Biol Chem. 1988 Aug 15;263(23):11071–11074. [PubMed] [Google Scholar]
  27. Matteoli M., Takei K., Cameron R., Hurlbut P., Johnston P. A., Südhof T. C., Jahn R., De Camilli P. Association of Rab3A with synaptic vesicles at late stages of the secretory pathway. J Cell Biol. 1991 Nov;115(3):625–633. doi: 10.1083/jcb.115.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Matthew W. D., Tsavaler L., Reichardt L. F. Identification of a synaptic vesicle-specific membrane protein with a wide distribution in neuronal and neurosecretory tissue. J Cell Biol. 1981 Oct;91(1):257–269. doi: 10.1083/jcb.91.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mizoguchi A., Arakawa M., Masutani M., Tamekane A., Yamaguchi H., Minami N., Takai Y., Ide C. Localization of smg p25A/rab3A p25, a small GTP-binding protein, at the active zone of the rat neuromuscular junction. Biochem Biophys Res Commun. 1992 Aug 14;186(3):1345–1352. doi: 10.1016/s0006-291x(05)81554-2. [DOI] [PubMed] [Google Scholar]
  30. Mizoguchi A., Kim S., Ueda T., Kikuchi A., Yorifuji H., Hirokawa N., Takai Y. Localization and subcellular distribution of smg p25A, a ras p21-like GTP-binding protein, in rat brain. J Biol Chem. 1990 Jul 15;265(20):11872–11879. [PubMed] [Google Scholar]
  31. Mizoguchi A., Kim S., Ueda T., Takai Y. Tissue distribution of smg p25A, a ras p21-like GTP-binding protein, studied by use of a specific monoclonal antibody. Biochem Biophys Res Commun. 1989 Aug 15;162(3):1438–1445. doi: 10.1016/0006-291x(89)90835-8. [DOI] [PubMed] [Google Scholar]
  32. Motoike T., Sano K., Tsuneishi S., Nakamura H., Kushima Y., Hatanaka H., Takai Y. Expression and localization of smg p25A (= rab3A) in cultured rat hippocampal cells. Neurosci Lett. 1991 Dec 16;134(1):109–112. doi: 10.1016/0304-3940(91)90519-y. [DOI] [PubMed] [Google Scholar]
  33. Motoike T., Sano K., Tsuneishi S., Nakamura H., Takai Y. Expression of smg p25A, a ras p21-like small GTP-binding protein, during postnatal development of rat cerebellum. Brain Res Dev Brain Res. 1990 Dec 15;57(2):279–289. doi: 10.1016/0165-3806(90)90054-3. [DOI] [PubMed] [Google Scholar]
  34. Nishizuka Y. The molecular heterogeneity of protein kinase C and its implications for cellular regulation. Nature. 1988 Aug 25;334(6184):661–665. doi: 10.1038/334661a0. [DOI] [PubMed] [Google Scholar]
  35. Padfield P. J., Balch W. E., Jamieson J. D. A synthetic peptide of the rab3a effector domain stimulates amylase release from permeabilized pancreatic acini. Proc Natl Acad Sci U S A. 1992 Mar 1;89(5):1656–1660. doi: 10.1073/pnas.89.5.1656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Perin M. S., Brose N., Jahn R., Südhof T. C. Domain structure of synaptotagmin (p65) J Biol Chem. 1991 Jan 5;266(1):623–629. [PubMed] [Google Scholar]
  37. Perin M. S., Fried V. A., Mignery G. A., Jahn R., Südhof T. C. Phospholipid binding by a synaptic vesicle protein homologous to the regulatory region of protein kinase C. Nature. 1990 May 17;345(6272):260–263. doi: 10.1038/345260a0. [DOI] [PubMed] [Google Scholar]
  38. Perin M. S., Johnston P. A., Ozcelik T., Jahn R., Francke U., Südhof T. C. Structural and functional conservation of synaptotagmin (p65) in Drosophila and humans. J Biol Chem. 1991 Jan 5;266(1):615–622. [PubMed] [Google Scholar]
  39. Plutner H., Schwaninger R., Pind S., Balch W. E. Synthetic peptides of the Rab effector domain inhibit vesicular transport through the secretory pathway. EMBO J. 1990 Aug;9(8):2375–2383. doi: 10.1002/j.1460-2075.1990.tb07412.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Reichardt L. F., Kelly R. B. A molecular description of nerve terminal function. Annu Rev Biochem. 1983;52:871–926. doi: 10.1146/annurev.bi.52.070183.004255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sano K., Kikuchi A., Matsui Y., Teranishi Y., Takai Y. Tissue-specific expression of a novel GTP-binding protein (smg p25A) mRNA and its increase by nerve growth factor and cyclic AMP in rat pheochromocytoma PC-12 cells. Biochem Biophys Res Commun. 1989 Jan 31;158(2):377–385. doi: 10.1016/s0006-291x(89)80058-0. [DOI] [PubMed] [Google Scholar]
  43. Sano K., Motoike T., Nakamura H., Matsui Y., Teranishi Y., Takai Y. Differential expression of the N-myc, c-fos, and smg p25A genes in human neuroblastoma cells during neuronal and Schwannian differentiation. Brain Res Mol Brain Res. 1991 Jan;9(1-2):149–152. doi: 10.1016/0169-328x(91)90140-s. [DOI] [PubMed] [Google Scholar]
  44. Sasaki T., Kaibuchi K., Kabcenell A. K., Novick P. J., Takai Y. A mammalian inhibitory GDP/GTP exchange protein (GDP dissociation inhibitor) for smg p25A is active on the yeast SEC4 protein. Mol Cell Biol. 1991 May;11(5):2909–2912. doi: 10.1128/mcb.11.5.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Sasaki T., Kikuchi A., Araki S., Hata Y., Isomura M., Kuroda S., Takai Y. Purification and characterization from bovine brain cytosol of a protein that inhibits the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP-binding protein. J Biol Chem. 1990 Feb 5;265(4):2333–2337. [PubMed] [Google Scholar]
  46. Shirataki H., Kaibuchi K., Yamaguchi T., Wada K., Horiuchi H., Takai Y. A possible target protein for smg-25A/rab3A small GTP-binding protein. J Biol Chem. 1992 Jun 5;267(16):10946–10949. [PubMed] [Google Scholar]
  47. Südhof T. C., Jahn R. Proteins of synaptic vesicles involved in exocytosis and membrane recycling. Neuron. 1991 May;6(5):665–677. doi: 10.1016/0896-6273(91)90165-v. [DOI] [PubMed] [Google Scholar]
  48. Takai Y., Kaibuchi K., Kikuchi A., Kawata M. Small GTP-binding proteins. Int Rev Cytol. 1992;133:187–230. doi: 10.1016/s0074-7696(08)61861-6. [DOI] [PubMed] [Google Scholar]
  49. Takebe Y., Seiki M., Fujisawa J., Hoy P., Yokota K., Arai K., Yoshida M., Arai N. SR alpha promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the R-U5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol Cell Biol. 1988 Jan;8(1):466–472. doi: 10.1128/mcb.8.1.466. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Touchot N., Chardin P., Tavitian A. Four additional members of the ras gene superfamily isolated by an oligonucleotide strategy: molecular cloning of YPT-related cDNAs from a rat brain library. Proc Natl Acad Sci U S A. 1987 Dec;84(23):8210–8214. doi: 10.1073/pnas.84.23.8210. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Ueda T., Takeyama Y., Ohmori T., Ohyanagi H., Saitoh Y., Takai Y. Purification and characterization from rat liver cytosol of a GDP dissociation inhibitor (GDI) for liver 24K G, a ras p21-like GTP-binding protein, with properties similar to those of smg p25A GDI. Biochemistry. 1991 Jan 29;30(4):909–917. doi: 10.1021/bi00218a005. [DOI] [PubMed] [Google Scholar]
  53. Ushkaryov Y. A., Petrenko A. G., Geppert M., Südhof T. C. Neurexins: synaptic cell surface proteins related to the alpha-latrotoxin receptor and laminin. Science. 1992 Jul 3;257(5066):50–56. doi: 10.1126/science.1621094. [DOI] [PubMed] [Google Scholar]
  54. Wendland B., Miller K. G., Schilling J., Scheller R. H. Differential expression of the p65 gene family. Neuron. 1991 Jun;6(6):993–1007. doi: 10.1016/0896-6273(91)90239-v. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Yanisch-Perron C., Vieira J., Messing J. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene. 1985;33(1):103–119. doi: 10.1016/0378-1119(85)90120-9. [DOI] [PubMed] [Google Scholar]

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