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. 1991 Jun 15;276(Pt 3):793–799. doi: 10.1042/bj2760793

Site specificity in the interactions of synapsin 1 with tubulin.

A F Bennett 1, N V Hayes 1, A J Baines 1
PMCID: PMC1151074  PMID: 1905928

Abstract

Synapsin 1 is one of a family of phosphoproteins located on small synaptic vesicles (SSV) in the presynaptic terminal, and probably plays a critical role in the process of neuronal exocytosis by providing regulated linkages between SSV and the cytoskeleton. Two forms of synapsin 1 are produced from a single gene by differential mRNA splicing: 1a, 706 amino acid residues, and 1b, 670 residues. Synapsin 1 has two structural domains, a globular N-terminal head domain and an elongated tail domain. Electron microscopy of nerve terminals in situ and reconstitution studies in vitro indicates that synapsin 1 can interact with microtubules, microfilaments and brain spectrin. In vitro, synapsin 1 can bundle microtubules. This could either occur by synapsin 1 being at least bivalent for microtubules, or by univalent synapsin 1 molecules aggregating to form complexes that are more than univalent. To resolve this question, we have taken the approach of preparing defined fragments of synapsin 1 from each structural domain and analysing them for tubulin-binding activity. Our results show that there are tubulin-binding sites in both head and tail domains. We conclude that synapsin 1 monomers should be able to cross-link microtubules.

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Selected References

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

  1. Aubert-Foucher E., Font B. Limited proteolysis of synapsin I. Identification of the region of the molecule responsible for its association with microtubules. Biochemistry. 1990 Jun 5;29(22):5351–5357. doi: 10.1021/bi00474a021. [DOI] [PubMed] [Google Scholar]
  2. Baines A. J. A role for repeated 18-amino-acid stretches in the sequence of synapsin I in tubulin binding? Biochem J. 1989 May 15;260(1):311–312. doi: 10.1042/bj2600311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baines A. J., Bennett V. Synapsin I is a microtubule-bundling protein. Nature. 1986 Jan 9;319(6049):145–147. doi: 10.1038/319145a0. [DOI] [PubMed] [Google Scholar]
  4. Baines A. J., Bennett V. Synapsin I is a spectrin-binding protein immunologically related to erythrocyte protein 4.1. 1985 May 30-Jun 5Nature. 315(6018):410–413. doi: 10.1038/315410a0. [DOI] [PubMed] [Google Scholar]
  5. Baines A. J. Synapsin I and the cytoskeleton. Nature. 1987 Apr 16;326(6114):646–646. doi: 10.1038/326646a0. [DOI] [PubMed] [Google Scholar]
  6. Benfenati F., Bähler M., Jahn R., Greengard P. Interactions of synapsin I with small synaptic vesicles: distinct sites in synapsin I bind to vesicle phospholipids and vesicle proteins. J Cell Biol. 1989 May;108(5):1863–1872. doi: 10.1083/jcb.108.5.1863. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Benfenati F., Greengard P., Brunner J., Bähler M. Electrostatic and hydrophobic interactions of synapsin I and synapsin I fragments with phospholipid bilayers. J Cell Biol. 1989 May;108(5):1851–1862. doi: 10.1083/jcb.108.5.1851. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bennett V., Baines A. J., Davis J. Purification of brain analogs of red blood cell membrane skeletal proteins: ankyrin, protein 4.1 (synapsin), spectrin, and spectrin subunits. Methods Enzymol. 1986;134:55–69. doi: 10.1016/0076-6879(86)34075-8. [DOI] [PubMed] [Google Scholar]
  9. Bähler M., Benfenati F., Valtorta F., Czernik A. J., Greengard P. Characterization of synapsin I fragments produced by cysteine-specific cleavage: a study of their interactions with F-actin. J Cell Biol. 1989 May;108(5):1841–1849. doi: 10.1083/jcb.108.5.1841. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Correas I., Padilla R., Avila J. The tubulin-binding sequence of brain microtubule-associated proteins, tau and MAP-2, is also involved in actin binding. Biochem J. 1990 Jul 1;269(1):61–64. doi: 10.1042/bj2690061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Czernik A. J., Pang D. T., Greengard P. Amino acid sequences surrounding the cAMP-dependent and calcium/calmodulin-dependent phosphorylation sites in rat and bovine synapsin I. Proc Natl Acad Sci U S A. 1987 Nov;84(21):7518–7522. doi: 10.1073/pnas.84.21.7518. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Davis J. Q., Bennett V. Brain ankyrin. A membrane-associated protein with binding sites for spectrin, tubulin, and the cytoplasmic domain of the erythrocyte anion channel. J Biol Chem. 1984 Nov 10;259(21):13550–13559. [PubMed] [Google Scholar]
  13. Dentler W. L., Granett S., Rosenbaum J. L. Ultrastructural localization of the high molecular weight proteins associated with in vitro-assembled brain microtubules. J Cell Biol. 1975 Apr;65(1):237–241. doi: 10.1083/jcb.65.1.237. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Drapeau G. R. Protease from Staphyloccus aureus. Methods Enzymol. 1976;45:469–475. doi: 10.1016/s0076-6879(76)45041-3. [DOI] [PubMed] [Google Scholar]
  15. Font B., Aubert-Foucher E. Detection by chemical cross-linking of bovine brain synapsin I self-association. Biochem J. 1989 Dec 15;264(3):893–899. doi: 10.1042/bj2640893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Goldenring J. R., Lasher R. S., Vallano M. L., Ueda T., Naito S., Sternberger N. H., Sternberger L. A., DeLorenzo R. J. Association of synapsin I with neuronal cytoskeleton. Identification in cytoskeletal preparations in vitro and immunocytochemical localization in brain of synapsin I. J Biol Chem. 1986 Jun 25;261(18):8495–8504. [PubMed] [Google Scholar]
  17. Greengard P. Neuronal phosphoproteins. Mediators of signal transduction. Mol Neurobiol. 1987 Spring-Summer;1(1-2):81–119. doi: 10.1007/BF02935265. [DOI] [PubMed] [Google Scholar]
  18. Hayes N. V., Bennett A. F., Baines A. J. Selective Ca2(+)-dependent interaction of calmodulin with the head domain of synapsin 1. Biochem J. 1991 Apr 1;275(Pt 1):93–97. doi: 10.1042/bj2750093. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Himmler A. Structure of the bovine tau gene: alternatively spliced transcripts generate a protein family. Mol Cell Biol. 1989 Apr;9(4):1389–1396. doi: 10.1128/mcb.9.4.1389. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hirokawa N., Sobue K., Kanda K., Harada A., Yorifuji H. The cytoskeletal architecture of the presynaptic terminal and molecular structure of synapsin 1. J Cell Biol. 1989 Jan;108(1):111–126. doi: 10.1083/jcb.108.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kelly R. B. The cell biology of the nerve terminal. Neuron. 1988 Aug;1(6):431–438. doi: 10.1016/0896-6273(88)90174-2. [DOI] [PMC free article] [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. Landis D. M., Hall A. K., Weinstein L. A., Reese T. S. The organization of cytoplasm at the presynaptic active zone of a central nervous system synapse. Neuron. 1988 May;1(3):201–209. doi: 10.1016/0896-6273(88)90140-7. [DOI] [PubMed] [Google Scholar]
  24. Landis D. M. Membrane and cytoplasmic structure at synaptic junctions in the mammalian central nervous system. J Electron Microsc Tech. 1988 Oct;10(2):129–151. doi: 10.1002/jemt.1060100203. [DOI] [PubMed] [Google Scholar]
  25. McCaffery C. A., DeGennaro L. J. Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I. EMBO J. 1986 Dec 1;5(12):3167–3173. doi: 10.1002/j.1460-2075.1986.tb04625.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Noble M., Lewis S. A., Cowan N. J. The microtubule binding domain of microtubule-associated protein MAP1B contains a repeated sequence motif unrelated to that of MAP2 and tau. J Cell Biol. 1989 Dec;109(6 Pt 2):3367–3376. doi: 10.1083/jcb.109.6.3367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Okabe T., Sobue K. Identification of a new 84/82 kDa calmodulin-binding protein, which also interacts with actin filaments, tubulin and spectrin, as synapsin I. FEBS Lett. 1987 Mar 9;213(1):184–188. doi: 10.1016/0014-5793(87)81488-6. [DOI] [PubMed] [Google Scholar]
  28. Olmsted J. B. Affinity purification of antibodies from diazotized paper blots of heterogeneous protein samples. J Biol Chem. 1981 Dec 10;256(23):11955–11957. [PubMed] [Google Scholar]
  29. Rayner D. A., Baines A. J. A novel component of the axonal cortical cytoskeleton, A60, defined by a monoclonal antibody. J Cell Sci. 1989 Nov;94(Pt 3):489–500. doi: 10.1242/jcs.94.3.489. [DOI] [PubMed] [Google Scholar]
  30. Steiner J. P., Gardner K., Baines A., Bennett V. Synapsin I: a regulated synaptic vesicle organizing protein. Brain Res Bull. 1987 Jun;18(6):777–785. doi: 10.1016/0361-9230(87)90216-4. [DOI] [PubMed] [Google Scholar]
  31. Steiner J. P., Ling E., Bennett V. Nearest neighbor analysis for brain synapsin I. Evidence from in vitro reassociation assays for association with membrane protein(s) and the Mr = 68,000 neurofilament subunit. J Biol Chem. 1987 Jan 15;262(2):905–914. [PubMed] [Google Scholar]
  32. Südhof T. C., Czernik A. J., Kao H. T., Takei K., Johnston P. A., Horiuchi A., Kanazir S. D., Wagner M. A., Perin M. S., De Camilli P. Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins. Science. 1989 Sep 29;245(4925):1474–1480. doi: 10.1126/science.2506642. [DOI] [PubMed] [Google Scholar]
  33. Südhof T. C. The structure of the human synapsin I gene and protein. J Biol Chem. 1990 May 15;265(14):7849–7852. [PubMed] [Google Scholar]
  34. Ueda T., Greengard P. Adenosine 3':5'-monophosphate-regulated phosphoprotein system of neuronal membranes. I. Solubilization, purification, and some properties of an endogenous phosphoprotein. J Biol Chem. 1977 Jul 25;252(14):5155–5163. [PubMed] [Google Scholar]
  35. Williams R. C., Jr, Detrich H. W., 3rd Separation of tubulin from microtubule-associated proteins on phosphocellulose. Accompanying alterations in concentrations of buffer components. Biochemistry. 1979 Jun 12;18(12):2499–2503. doi: 10.1021/bi00579a010. [DOI] [PubMed] [Google Scholar]

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