Skip to main content
NCBI home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1996 Sep;16(9):4985–4995. doi: 10.1128/mcb.16.9.4985

Role of the Rab3A-binding domain in targeting of rabphilin-3A to vesicle membranes of PC12 cells.

C J McKiernan 1, P F Stabila 1, I G Macara 1
PMCID: PMC231500  PMID: 8756657

Abstract

Rab3A is a small GTPase implicated in the docking of secretory vesicles in neuroendocrine cells. A putative downstream target for Rab3A, rabphilin-3A, is located exclusively on secretory vesicle membranes. It contains near its C terminus two C2 domains that bind Ca2+ in a phospholipid-dependent manner and an N-terminal, Rab3A-binding domain that includes a Cys-rich region. We have determined that the Cys-rich domain binds two Zn2+ ions and is necessary but not sufficient for efficient binding of rabphilin to Rab3A. A minimal Rab3A-binding domain consists of residues 45 to 170 of rabphilin. HA1-tagged Rab3A and a green fluorescent protein (GFP)-rabphilin fusion were used to examine the roles of Rab3A and of rabphilin domains in the subcellular localization of these proteins. A Rab3A mutant (T54A) that does not bind rabphifin in vitro colocalized with the GFP-rabphilin fusion, indicating that Rab3A targeting is independent of its interaction with rabphilin. Deletion of the C2 domains of rabphilin reduced membrane association of GFP-rabphilin but did not cause mistargeting of the membrane-associated fraction. However, disruption of the zinc fingers, which drastically reduced Rab3A binding, did not reduce membrane association. These results suggest that the C2 domains are required for efficient membrane attachment of rabphilin in PC12 cells and that Rab3A binding may act to target the protein to the correct membrane.

Full Text

The Full Text of this article is available as a PDF (2.7 MB).

Selected References

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

  1. Ahmed S., Kozma R., Lee J., Monfries C., Harden N., Lim L. The cysteine-rich domain of human proteins, neuronal chimaerin, protein kinase C and diacylglycerol kinase binds zinc. Evidence for the involvement of a zinc-dependent structure in phorbol ester binding. Biochem J. 1991 Nov 15;280(Pt 1):233–241. doi: 10.1042/bj2800233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. 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]
  3. 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: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  4. Brennwald P., Novick P. Interactions of three domains distinguishing the Ras-related GTP-binding proteins Ypt1 and Sec4. Nature. 1993 Apr 8;362(6420):560–563. doi: 10.1038/362560a0. [DOI] [PubMed] [Google Scholar]
  5. Brondyk W. H., McKiernan C. J., Burstein E. S., Macara I. G. Mutants of Rab3A analogous to oncogenic Ras mutants. Sensitivity to Rab3A-GTPase activating protein and Rab3A-guanine nucleotide releasing factor. J Biol Chem. 1993 May 5;268(13):9410–9415. [PubMed] [Google Scholar]
  6. Brondyk W. H., McKiernan C. J., Fortner K. A., Stabila P., Holz R. W., Macara I. G. Interaction cloning of Rabin3, a novel protein that associates with the Ras-like GTPase Rab3A. Mol Cell Biol. 1995 Mar;15(3):1137–1143. doi: 10.1128/mcb.15.3.1137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Burstein E. S., Brondyk W. H., Macara I. G. Amino acid residues in the Ras-like GTPase Rab3A that specify sensitivity to factors that regulate the GTP/GDP cycling of Rab3A. J Biol Chem. 1992 Nov 15;267(32):22715–22718. [PubMed] [Google Scholar]
  8. Burstein E. S., Macara I. G. Interactions of the ras-like protein p25rab3A with Mg2+ and guanine nucleotides. Biochem J. 1992 Mar 1;282(Pt 2):387–392. doi: 10.1042/bj2820387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Burstein E., Macara I. G. The ras-like protein p25rab3A is partially cytosolic and is expressed only in neural tissue. Mol Cell Biol. 1989 Nov;9(11):4807–4811. doi: 10.1128/mcb.9.11.4807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Chalfie M., Tu Y., Euskirchen G., Ward W. W., Prasher D. C. Green fluorescent protein as a marker for gene expression. Science. 1994 Feb 11;263(5148):802–805. doi: 10.1126/science.8303295. [DOI] [PubMed] [Google Scholar]
  11. Chung S. H., Takai Y., Holz R. W. Evidence that the Rab3a-binding protein, rabphilin3a, enhances regulated secretion. Studies in adrenal chromaffin cells. J Biol Chem. 1995 Jul 14;270(28):16714–16718. doi: 10.1074/jbc.270.28.16714. [DOI] [PubMed] [Google Scholar]
  12. Darchen F., Senyshyn J., Brondyk W. H., Taatjes D. J., Holz R. W., Henry J. P., Denizot J. P., Macara I. G. The GTPase Rab3a is associated with large dense core vesicles in bovine chromaffin cells and rat PC12 cells. J Cell Sci. 1995 Apr;108(Pt 4):1639–1649. doi: 10.1242/jcs.108.4.1639. [DOI] [PubMed] [Google Scholar]
  13. Dunn B., Stearns T., Botstein D. Specificity domains distinguish the Ras-related GTPases Ypt1 and Sec4. Nature. 1993 Apr 8;362(6420):563–565. doi: 10.1038/362563a0. [DOI] [PubMed] [Google Scholar]
  14. Farnsworth C. C., Kawata M., Yoshida Y., Takai Y., Gelb M. H., Glomset J. A. C terminus of the small GTP-binding protein smg p25A contains two geranylgeranylated cysteine residues and a methyl ester. Proc Natl Acad Sci U S A. 1991 Jul 15;88(14):6196–6200. doi: 10.1073/pnas.88.14.6196. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Ferro-Novick S., Jahn R. Vesicle fusion from yeast to man. Nature. 1994 Jul 21;370(6486):191–193. doi: 10.1038/370191a0. [DOI] [PubMed] [Google Scholar]
  16. Freedman L. P., Luisi B. F. On the mechanism of DNA binding by nuclear hormone receptors: a structural and functional perspective. J Cell Biochem. 1993 Feb;51(2):140–150. doi: 10.1002/jcb.240510205. [DOI] [PubMed] [Google Scholar]
  17. Fujita Y., Sasaki T., Araki K., Takahashi K., Imazumi K., Kato M., Matsuura Y., Takai Y. GDP/GTP exchange reaction-stimulating activity of Rabphilin-3A for Rab3A small GTP-binding protein. FEBS Lett. 1994 Oct 10;353(1):67–70. doi: 10.1016/0014-5793(94)01015-3. [DOI] [PubMed] [Google Scholar]
  18. Fykse E. M., Li C., Südhof T. C. Phosphorylation of rabphilin-3A by Ca2+/calmodulin- and cAMP-dependent protein kinases in vitro. J Neurosci. 1995 Mar;15(3 Pt 2):2385–2395. doi: 10.1523/JNEUROSCI.15-03-02385.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Geppert M., Bolshakov V. Y., Siegelbaum S. A., Takei K., De Camilli P., Hammer R. E., Südhof T. C. The role of Rab3A in neurotransmitter release. Nature. 1994 Jun 9;369(6480):493–497. doi: 10.1038/369493a0. [DOI] [PubMed] [Google Scholar]
  20. Heim R., Cubitt A. B., Tsien R. Y. Improved green fluorescence. Nature. 1995 Feb 23;373(6516):663–664. doi: 10.1038/373663b0. [DOI] [PubMed] [Google Scholar]
  21. Holz R. W., Brondyk W. H., Senter R. A., Kuizon L., Macara I. G. Evidence for the involvement of Rab3A in Ca(2+)-dependent exocytosis from adrenal chromaffin cells. J Biol Chem. 1994 Apr 8;269(14):10229–10234. [PubMed] [Google Scholar]
  22. Johannes L., Lledo P. M., Roa M., Vincent J. D., Henry J. P., Darchen F. The GTPase Rab3a negatively controls calcium-dependent exocytosis in neuroendocrine cells. EMBO J. 1994 May 1;13(9):2029–2037. doi: 10.1002/j.1460-2075.1994.tb06476.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kato M., Sasaki T., Imazumi K., Takahashi K., Araki K., Shirataki H., Matsuura Y., Ishida A., Fujisawa H., Takai Y. Phosphorylation of Rabphilin-3A by calmodulin-dependent protein kinase II. Biochem Biophys Res Commun. 1994 Dec 30;205(3):1776–1784. doi: 10.1006/bbrc.1994.2875. [DOI] [PubMed] [Google Scholar]
  24. 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]
  25. Kishida S., Shirataki H., Sasaki T., Kato M., Kaibuchi K., Takai Y. Rab3A GTPase-activating protein-inhibiting activity of Rabphilin-3A, a putative Rab3A target protein. J Biol Chem. 1993 Oct 25;268(30):22259–22261. [PubMed] [Google Scholar]
  26. Li C., Takei K., Geppert M., Daniell L., Stenius K., Chapman E. R., Jahn R., De Camilli P., Südhof T. C. Synaptic targeting of rabphilin-3A, a synaptic vesicle Ca2+/phospholipid-binding protein, depends on rab3A/3C. Neuron. 1994 Oct;13(4):885–898. doi: 10.1016/0896-6273(94)90254-2. [DOI] [PubMed] [Google Scholar]
  27. Lupas A., Van Dyke M., Stock J. Predicting coiled coils from protein sequences. Science. 1991 May 24;252(5009):1162–1164. doi: 10.1126/science.252.5009.1162. [DOI] [PubMed] [Google Scholar]
  28. McKiernan C. J., Brondyk W. H., Macara I. G. The Rab3A GTPase interacts with multiple factors through the same effector domain. Mutational analysis of cross-linking of Rab3A to a putative target protein. J Biol Chem. 1993 Nov 15;268(32):24449–24452. [PubMed] [Google Scholar]
  29. Mizoguchi A., Yano Y., Hamaguchi H., Yanagida H., Ide C., Zahraoui A., Shirataki H., Sasaki T., Takai Y. Localization of Rabphilin-3A on the synaptic vesicle. Biochem Biophys Res Commun. 1994 Aug 15;202(3):1235–1243. doi: 10.1006/bbrc.1994.2063. [DOI] [PubMed] [Google Scholar]
  30. Olofsson B., Chardin P., Touchot N., Zahraoui A., Tavitian A. Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues. Oncogene. 1988 Aug;3(2):231–234. [PubMed] [Google Scholar]
  31. Quest A. F., Bardes E. S., Bell R. M. A phorbol ester binding domain of protein kinase C gamma. Deletion analysis of the Cys2 domain defines a minimal 43-amino acid peptide. J Biol Chem. 1994 Jan 28;269(4):2961–2970. [PubMed] [Google Scholar]
  32. Quest A. F., Bell R. M. The regulatory region of protein kinase C gamma. Studies of phorbol ester binding to individual and combined functional segments expressed as glutathione S-transferase fusion proteins indicate a complex mechanism of regulation by phospholipids, phorbol esters, and divalent cations. J Biol Chem. 1994 Aug 5;269(31):20000–20012. [PubMed] [Google Scholar]
  33. Schaffner W., Weissmann C. A rapid, sensitive, and specific method for the determination of protein in dilute solution. Anal Biochem. 1973 Dec;56(2):502–514. doi: 10.1016/0003-2697(73)90217-0. [DOI] [PubMed] [Google Scholar]
  34. Shirataki H., Kaibuchi K., Sakoda T., Kishida S., Yamaguchi T., Wada K., Miyazaki M., Takai Y. Rabphilin-3A, a putative target protein for smg p25A/rab3A p25 small GTP-binding protein related to synaptotagmin. Mol Cell Biol. 1993 Apr;13(4):2061–2068. doi: 10.1128/mcb.13.4.2061. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. 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]
  36. Shirataki H., Yamamoto T., Hagi S., Miura H., Oishi H., Jin-no Y., Senbonmatsu T., Takai Y. Rabphilin-3A is associated with synaptic vesicles through a vesicle protein in a manner independent of Rab3A. J Biol Chem. 1994 Dec 30;269(52):32717–32720. [PubMed] [Google Scholar]
  37. Stahl B., Chou J. H., Li C., Südhof T. C., Jahn R. Rab3 reversibly recruits rabphilin to synaptic vesicles by a mechanism analogous to raf recruitment by ras. EMBO J. 1996 Apr 15;15(8):1799–1809. [PMC free article] [PubMed] [Google Scholar]
  38. Wilson R., Ainscough R., Anderson K., Baynes C., Berks M., Bonfield J., Burton J., Connell M., Copsey T., Cooper J. 2.2 Mb of contiguous nucleotide sequence from chromosome III of C. elegans. Nature. 1994 Mar 3;368(6466):32–38. doi: 10.1038/368032a0. [DOI] [PubMed] [Google Scholar]
  39. Yamaguchi T., Shirataki H., Kishida S., Miyazaki M., Nishikawa J., Wada K., Numata S., Kaibuchi K., Takai Y. Two functionally different domains of rabphilin-3A, Rab3A p25/smg p25A-binding and phospholipid- and Ca(2+)-binding domains. J Biol Chem. 1993 Dec 25;268(36):27164–27170. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES