Skip to main content
NCBI home page
The EMBO Journal logoLink to The EMBO Journal
. 1989 Apr;8(4):1087–1092. doi: 10.1002/j.1460-2075.1989.tb03477.x

The mammalian G protein rhoC is ADP-ribosylated by Clostridium botulinum exoenzyme C3 and affects actin microfilaments in Vero cells.

P Chardin 1, P Boquet 1, P Madaule 1, M R Popoff 1, E J Rubin 1, D M Gill 1
PMCID: PMC400918  PMID: 2501082

Abstract

Clostridium botulinum C3 is a recently discovered exoenzyme that ADP-ribosylates a eukaryotic GTP-binding protein of the ras superfamily. We show now that the bacterially-expressed product of the human rhoC gene is ADP-ribosylated by C3 and corresponds in size, charge and behavior to the dominant C3 substrate of eukaryotic cells. C3 treatment of Vero cells results in the disappearance of microfilaments and in actinomorphic shape changes without any apparent direct effect upon actin. Thus the ADP-ribosylation of a rho protein seems to be responsible for microfilament disassembly and we infer that the unmodified form of a rho protein may be involved in cytoskeletal control.

Full text

PDF
1087

Images in this article

1088Image
on p.1088
1088Image
on p.1088
1089Image
on p.1089

Selected References

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

  1. Adam-Vizi V., Rosener S., Aktories K., Knight D. E. Botulinum toxin-induced ADP-ribosylation and inhibition of exocytosis are unrelated events. FEBS Lett. 1988 Oct 10;238(2):277–280. doi: 10.1016/0014-5793(88)80496-4. [DOI] [PubMed] [Google Scholar]
  2. Aktories K., Rösener S., Blaschke U., Chhatwal G. S. Botulinum ADP-ribosyltransferase C3. Purification of the enzyme and characterization of the ADP-ribosylation reaction in platelet membranes. Eur J Biochem. 1988 Mar 1;172(2):445–450. doi: 10.1111/j.1432-1033.1988.tb13908.x. [DOI] [PubMed] [Google Scholar]
  3. Ames G. F., Nikaido K. Two-dimensional gel electrophoresis of membrane proteins. Biochemistry. 1976 Feb 10;15(3):616–623. doi: 10.1021/bi00648a026. [DOI] [PubMed] [Google Scholar]
  4. 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]
  5. 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]
  6. 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]
  7. Goud B., Salminen A., Walworth N. C., Novick P. J. A GTP-binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell. 1988 Jun 3;53(5):753–768. doi: 10.1016/0092-8674(88)90093-1. [DOI] [PubMed] [Google Scholar]
  8. Janmey P. A., Stossel T. P. Modulation of gelsolin function by phosphatidylinositol 4,5-bisphosphate. Nature. 1987 Jan 22;325(6102):362–364. doi: 10.1038/325362a0. [DOI] [PubMed] [Google Scholar]
  9. Kahn R. A., Goddard C., Newkirk M. Chemical and immunological characterization of the 21-kDa ADP-ribosylation factor of adenylate cyclase. J Biol Chem. 1988 Jun 15;263(17):8282–8287. [PubMed] [Google Scholar]
  10. Kikuchi A., Yamamoto K., Fujita T., Takai Y. ADP-ribosylation of the bovine brain rho protein by botulinum toxin type C1. J Biol Chem. 1988 Nov 5;263(31):16303–16308. [PubMed] [Google Scholar]
  11. 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]
  12. 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]
  13. Madaule P., Axel R., Myers A. M. Characterization of two members of the rho gene family from the yeast Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1987 Feb;84(3):779–783. doi: 10.1073/pnas.84.3.779. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Matsuoka I., Syoto B., Kurihara K., Kubo S., Syuto B. ADP-ribosylation of specific membrane proteins in pheochromocytoma and primary-cultured brain cells by botulinum neurotoxins type C and D. FEBS Lett. 1987 Jun 1;216(2):295–299. doi: 10.1016/0014-5793(87)80709-3. [DOI] [PubMed] [Google Scholar]
  15. Mitchell M. J., Laughon B. E., Lin S. Biochemical studies on the effect of Clostridium difficile toxin B on actin in vivo and in vitro. Infect Immun. 1987 Jul;55(7):1610–1615. doi: 10.1128/iai.55.7.1610-1615.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Novick P., Botstein D. Phenotypic analysis of temperature-sensitive yeast actin mutants. Cell. 1985 Feb;40(2):405–416. doi: 10.1016/0092-8674(85)90154-0. [DOI] [PubMed] [Google Scholar]
  17. O'Farrell P. H. High resolution two-dimensional electrophoresis of proteins. J Biol Chem. 1975 May 25;250(10):4007–4021. [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Ohishi I., Tsuyama S. ADP-ribosylation of nonmuscle actin with component I of C2 toxin. Biochem Biophys Res Commun. 1986 Apr 29;136(2):802–806. doi: 10.1016/0006-291x(86)90511-5. [DOI] [PubMed] [Google Scholar]
  20. Popoff M. R., Boquet P. Clostridium spiroforme toxin is a binary toxin which ADP-ribosylates cellular actin. Biochem Biophys Res Commun. 1988 May 16;152(3):1361–1368. doi: 10.1016/s0006-291x(88)80435-2. [DOI] [PubMed] [Google Scholar]
  21. Popoff M. R., Rubin E. J., Gill D. M., Boquet P. Actin-specific ADP-ribosyltransferase produced by a Clostridium difficile strain. Infect Immun. 1988 Sep;56(9):2299–2306. doi: 10.1128/iai.56.9.2299-2306.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Rubin E. J., Gill D. M., Boquet P., Popoff M. R. Functional modification of a 21-kilodalton G protein when ADP-ribosylated by exoenzyme C3 of Clostridium botulinum. Mol Cell Biol. 1988 Jan;8(1):418–426. doi: 10.1128/mcb.8.1.418. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Rösener S., Chhatwal G. S., Aktories K. Botulinum ADP-ribosyltransferase C3 but not botulinum neurotoxins C1 and D ADP-ribosylates low molecular mass GTP-binding proteins. FEBS Lett. 1987 Nov 16;224(1):38–42. doi: 10.1016/0014-5793(87)80418-0. [DOI] [PubMed] [Google Scholar]
  24. Schmitt H. D., Puzicha M., Gallwitz D. Study of a temperature-sensitive mutant of the ras-related YPT1 gene product in yeast suggests a role in the regulation of intracellular calcium. Cell. 1988 May 20;53(4):635–647. doi: 10.1016/0092-8674(88)90579-x. [DOI] [PubMed] [Google Scholar]
  25. 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]
  26. Segev N., Botstein D. The ras-like yeast YPT1 gene is itself essential for growth, sporulation, and starvation response. Mol Cell Biol. 1987 Jul;7(7):2367–2377. doi: 10.1128/mcb.7.7.2367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Segev N., Mulholland J., Botstein D. The yeast GTP-binding YPT1 protein and a mammalian counterpart are associated with the secretion machinery. Cell. 1988 Mar 25;52(6):915–924. doi: 10.1016/0092-8674(88)90433-3. [DOI] [PubMed] [Google Scholar]
  28. Shapland C., Lowings P., Lawson D. Identification of new actin-associated polypeptides that are modified by viral transformation and changes in cell shape. J Cell Biol. 1988 Jul;107(1):153–161. doi: 10.1083/jcb.107.1.153. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. 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]
  30. Tucker J., Sczakiel G., Feuerstein J., John J., Goody R. S., Wittinghofer A. Expression of p21 proteins in Escherichia coli and stereochemistry of the nucleotide-binding site. EMBO J. 1986 Jun;5(6):1351–1358. doi: 10.1002/j.1460-2075.1986.tb04366.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vandekerckhove J., Schering B., Bärmann M., Aktories K. Clostridium perfringens iota toxin ADP-ribosylates skeletal muscle actin in Arg-177. FEBS Lett. 1987 Dec 10;225(1-2):48–52. doi: 10.1016/0014-5793(87)81129-8. [DOI] [PubMed] [Google Scholar]
  32. Wedel N., Toselli P., Pothoulakis C., Faris B., Oliver P., Franzblau C., LaMont T. Ultrastructural effects of Clostridium difficile toxin B on smooth muscle cells and fibroblasts. Exp Cell Res. 1983 Oct 15;148(2):413–422. doi: 10.1016/0014-4827(83)90163-5. [DOI] [PubMed] [Google Scholar]
  33. 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]
  34. Yeramian P., Chardin P., Madaule P., Tavitian A. Nucleotide sequence of human rho cDNA clone 12. Nucleic Acids Res. 1987 Feb 25;15(4):1869–1869. doi: 10.1093/nar/15.4.1869. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. de Boer H. A., Comstock L. J., Vasser M. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc Natl Acad Sci U S A. 1983 Jan;80(1):21–25. doi: 10.1073/pnas.80.1.21. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The EMBO Journal are provided here courtesy of Nature Publishing Group

RESOURCES