Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1988 Jan;8(1):418–426. doi: 10.1128/mcb.8.1.418

Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum.

E J Rubin 1, D M Gill 1, P Boquet 1, M R Popoff 1
PMCID: PMC363141  PMID: 3122025

Abstract

Exoenzyme C3 from Clostridium botulinum types C and D specifically ADP-ribosylated a 21-kilodalton cellular protein, p21.bot. Guanyl nucleotides protected the substrate against denaturation, which implies that p21.bot is a G protein. When introduced into the interior of cells, purified exoenzyme C3 ADP-ribosylated intracellular p21.bot and changed its function. NIH 3T3, PC12, and other cells rapidly underwent temporary morphological alterations that were in certain respects similar to those seen after microinjection of cloned ras proteins. When injected into Xenopus oocytes, C3 induced migration of germinal vesicles and potentiated the cholera toxin-sensitive augmentation of germinal vesicle breakdown by progesterone, also as caused by ras proteins. Nevertheless, p21.bot was immunologically distinct from p21ras.

Full text

PDF
418

Images in this article

420Image
on p.420
421Image
on p.421
421Image
on p.421
423Image
on p.423

Selected References

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

  1. Aktories K., Bärmann M., Ohishi I., Tsuyama S., Jakobs K. H., Habermann E. Botulinum C2 toxin ADP-ribosylates actin. Nature. 1986 Jul 24;322(6077):390–392. doi: 10.1038/322390a0. [DOI] [PubMed] [Google Scholar]
  2. Aktories K., Weller U., Chhatwal G. S. Clostridium botulinum type C produces a novel ADP-ribosyltransferase distinct from botulinum C2 toxin. FEBS Lett. 1987 Feb 9;212(1):109–113. doi: 10.1016/0014-5793(87)81566-1. [DOI] [PubMed] [Google Scholar]
  3. Bar-Sagi D., Feramisco J. R. Microinjection of the ras oncogene protein into PC12 cells induces morphological differentiation. Cell. 1985 Oct;42(3):841–848. doi: 10.1016/0092-8674(85)90280-6. [DOI] [PubMed] [Google Scholar]
  4. Birchmeier C., Broek D., Wigler M. ras proteins can induce meiosis in Xenopus oocytes. Cell. 1985 Dec;43(3 Pt 2):615–621. doi: 10.1016/0092-8674(85)90233-8. [DOI] [PubMed] [Google Scholar]
  5. Burnette W. N. "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 1981 Apr;112(2):195–203. doi: 10.1016/0003-2697(81)90281-5. [DOI] [PubMed] [Google Scholar]
  6. Chardin P., Tavitian A. The ral gene: a new ras related gene isolated by the use of a synthetic probe. EMBO J. 1986 Sep;5(9):2203–2208. doi: 10.1002/j.1460-2075.1986.tb04485.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  8. Eklund M. W., Poysky F. T., Reed S. M., Smith C. A. Bacteriophage and the toxigenicity of Clostridium botulinum type C. Science. 1971 Apr 30;172(3982):480–482. doi: 10.1126/science.172.3982.480. [DOI] [PubMed] [Google Scholar]
  9. Evans T., Brown M. L., Fraser E. D., Northup J. K. Purification of the major GTP-binding proteins from human placental membranes. J Biol Chem. 1986 May 25;261(15):7052–7059. [PubMed] [Google Scholar]
  10. Feramisco J. R., Gross M., Kamata T., Rosenberg M., Sweet R. W. Microinjection of the oncogene form of the human H-ras (T-24) protein results in rapid proliferation of quiescent cells. Cell. 1984 Aug;38(1):109–117. doi: 10.1016/0092-8674(84)90531-2. [DOI] [PubMed] [Google Scholar]
  11. Fodge D. W., Rubin H. Glucose utilization, pH reduction and density dependent inhibition in cultures of chick embryo fibroblasts. J Cell Physiol. 1975 Jun;85(3):635–642. doi: 10.1002/jcp.1040850316. [DOI] [PubMed] [Google Scholar]
  12. Furth M. E., Davis L. J., Fleurdelys B., Scolnick E. M. Monoclonal antibodies to the p21 products of the transforming gene of Harvey murine sarcoma virus and of the cellular ras gene family. J Virol. 1982 Jul;43(1):294–304. doi: 10.1128/jvi.43.1.294-304.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gibbs J. B., Sigal I. S., Poe M., Scolnick E. M. Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5704–5708. doi: 10.1073/pnas.81.18.5704. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Gill D. M., Meren R. A second guanyl nucleotide-binding site associated with adenylate cyclase. Distinct nucleotides activate adenylate cyclase and permit ADP-ribosylation by cholera toxin. J Biol Chem. 1983 Oct 10;258(19):11908–11914. [PubMed] [Google Scholar]
  15. Gill D. M., Pappenheimer A. M., Jr Structure-activity relationships in diphtheria toxin. J Biol Chem. 1971 Mar 10;246(5):1492–1495. [PubMed] [Google Scholar]
  16. Gilman A. G. G proteins and dual control of adenylate cyclase. Cell. 1984 Mar;36(3):577–579. doi: 10.1016/0092-8674(84)90336-2. [DOI] [PubMed] [Google Scholar]
  17. Hattori S., Yamashita T., Copeland T. D., Oroszlan S., Shih T. Y. Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding. J Biol Chem. 1986 Nov 5;261(31):14582–14586. [PubMed] [Google Scholar]
  18. Kahn R. A., Gilman A. G. The protein cofactor necessary for ADP-ribosylation of Gs by cholera toxin is itself a GTP binding protein. J Biol Chem. 1986 Jun 15;261(17):7906–7911. [PubMed] [Google Scholar]
  19. Lowe D. G., Capon D. J., Delwart E., Sakaguchi A. Y., Naylor S. L., Goeddel D. V. Structure of the human and murine R-ras genes, novel genes closely related to ras proto-oncogenes. Cell. 1987 Jan 16;48(1):137–146. doi: 10.1016/0092-8674(87)90364-3. [DOI] [PubMed] [Google Scholar]
  20. Madaule P., Axel R. A novel ras-related gene family. Cell. 1985 May;41(1):31–40. doi: 10.1016/0092-8674(85)90058-3. [DOI] [PubMed] [Google Scholar]
  21. Miyazaki S., Iwasaki M., Sakaguchi G. Clostridium botulinum type D toxin: purification, molecular structure, and some immunological properties. Infect Immun. 1977 Aug;17(2):395–401. doi: 10.1128/iai.17.2.395-401.1977. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oguma K., Yamaguchi T., Sudou K., Yokosawa N., Fujikawa Y. Biochemical classification of Clostridium botulinum type C and D strains and their nontoxigenic derivatives. Appl Environ Microbiol. 1986 Feb;51(2):256–260. doi: 10.1128/aem.51.2.256-260.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Ohashi Y., Narumiya S. ADP-ribosylation of a Mr 21,000 membrane protein by type D botulinum toxin. J Biol Chem. 1987 Feb 5;262(4):1430–1433. [PubMed] [Google Scholar]
  24. Ohishi I., Iwasaki M., Sakaguchi G. Purification and characterization of two components of botulinum C2 toxin. Infect Immun. 1980 Dec;30(3):668–673. doi: 10.1128/iai.30.3.668-673.1980. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Oka J., Ueda K., Hayaishi O. Snake venom phosphodiesterase: simple purification with Blue Sepharose and its application to poly(ADP-ribose) study. Biochem Biophys Res Commun. 1978 Feb 28;80(4):841–848. doi: 10.1016/0006-291x(78)91321-9. [DOI] [PubMed] [Google Scholar]
  26. Okada C. Y., Rechsteiner M. Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Cell. 1982 May;29(1):33–41. doi: 10.1016/0092-8674(82)90087-3. [DOI] [PubMed] [Google Scholar]
  27. Roa M., Boquet P. Interaction of tetanus toxin with lipid vesicles at low pH. Protection of specific polypeptides against proteolysis. J Biol Chem. 1985 Jun 10;260(11):6827–6835. [PubMed] [Google Scholar]
  28. Schmitt H. D., Wagner P., Pfaff E., Gallwitz D. The ras-related YPT1 gene product in yeast: a GTP-binding protein that might be involved in microtubule organization. Cell. 1986 Nov 7;47(3):401–412. doi: 10.1016/0092-8674(86)90597-0. [DOI] [PubMed] [Google Scholar]
  29. Shih T. Y., Weeks M. O., Gruss P., Dhar R., Oroszlan S., Scolnick E. M. Identification of a precursor in the biosynthesis of the p21 transforming protein of harvey murine sarcoma virus. J Virol. 1982 Apr;42(1):253–261. doi: 10.1128/jvi.42.1.253-261.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sigal I. S., Gibbs J. B., D'Alonzo J. S., Scolnick E. M. Identification of effector residues and a neutralizing epitope of Ha-ras-encoded p21. Proc Natl Acad Sci U S A. 1986 Jul;83(13):4725–4729. doi: 10.1073/pnas.83.13.4725. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Simpson L. L. Molecular pharmacology of botulinum toxin and tetanus toxin. Annu Rev Pharmacol Toxicol. 1986;26:427–453. doi: 10.1146/annurev.pa.26.040186.002235. [DOI] [PubMed] [Google Scholar]
  32. Stacey D. W., Kung H. F. Transformation of NIH 3T3 cells by microinjection of Ha-ras p21 protein. Nature. 1984 Aug 9;310(5977):508–511. doi: 10.1038/310508a0. [DOI] [PubMed] [Google Scholar]
  33. Tamir A., Tolkovsky A. M. Transient states of adenylate cyclase in brain membranes. J Neurochem. 1985 Apr;44(4):1006–1013. doi: 10.1111/j.1471-4159.1985.tb08719.x. [DOI] [PubMed] [Google Scholar]

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

RESOURCES