Skip to main content
PMC home page
Infection and Immunity logoLink to Infection and Immunity
. 1995 Mar;63(3):825–832. doi: 10.1128/iai.63.3.825-832.1995

Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism.

Y Xu 1, J T Barbieri 1
PMCID: PMC173077  PMID: 7868253

Abstract

Pertussis toxin (PT)-catalyzed ADP-ribosylation of target proteins in intact Chinese hamster ovary (CHO) cells was evaluated with an in vitro ADP-ribosylation assay. In this assay, a postnuclear supernatant was prepared from CHO cells and used as a source of PT-sensitive target proteins for in vitro [32P[ADP-ribosylation. The postnuclear supernatant contained three proteins that were ADP-ribosylated in vitro, with apparent molecular masses of 50, 45, and 42 kDa. The 42- and 45-kDa proteins were membrane associated, while the 50-kDa protein was soluble. Following PT treatment of CHO cells, the 42- and 45-kDa proteins were not available for in vitro ADP-ribosylation, while the soluble 50-kDa protein remained available for in vitro ADP-ribosylation. The decrease in the availability of the 42- and 45-kDa proteins to in vitro ADP-ribosylation was proportional to the PT concentration and time of incubation with CHO cells. Western immunoblot analysis showed that extracts from PT-treated CHO cells and control CHO cells possessed equivalent amounts of two proteins that were recognized by anti-Gi protein antiserum. The two proteins recognized by anti-Gi protein antiserum from PT-treated cells migrated with higher apparent molecular weights than the two proteins from control cells. This was consistent with the in vivo ADP-ribosylation of the two proteins by PT.(ABSTRACT TRUNCATED AT 250 WORDS)

Full Text

The Full Text of this article is available as a PDF (291.9 KB).

Selected References

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

  1. Armstrong G. D., Howard L. A., Peppler M. S. Use of glycosyltransferases to restore pertussis toxin receptor activity to asialoagalactofetuin. J Biol Chem. 1988 Jun 25;263(18):8677–8684. [PubMed] [Google Scholar]
  2. Barbieri J. T., Moloney B. K., Mende-Mueller L. M. Expression and secretion of the S-1 subunit and C180 peptide of pertussis toxin in Escherichia coli. J Bacteriol. 1989 Aug;171(8):4362–4369. doi: 10.1128/jb.171.8.4362-4369.1989. [DOI] [PMC free article] [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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  4. Brennan M. J., David J. L., Kenimer J. G., Manclark C. R. Lectin-like binding of pertussis toxin to a 165-kilodalton Chinese hamster ovary cell glycoprotein. J Biol Chem. 1988 Apr 5;263(10):4895–4899. [PubMed] [Google Scholar]
  5. Burns D. L., Kenimer J. G., Manclark C. R. Role of the A subunit of pertussis toxin in alteration of Chinese hamster ovary cell morphology. Infect Immun. 1987 Jan;55(1):24–28. doi: 10.1128/iai.55.1.24-28.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Burns D. L., Manclark C. R. Adenine nucleotides promote dissociation of pertussis toxin subunits. J Biol Chem. 1986 Mar 25;261(9):4324–4327. [PubMed] [Google Scholar]
  7. Correa-Sales C., Reid K., Maze M. Pertussis toxin-mediated ribosylation of G proteins blocks the hypnotic response to an alpha 2-agonist in the locus coeruleus of the rat. Pharmacol Biochem Behav. 1992 Nov;43(3):723–727. doi: 10.1016/0091-3057(92)90400-a. [DOI] [PubMed] [Google Scholar]
  8. Donta S. T., Beristain S., Tomicic T. K. Inhibition of heat-labile cholera and Escherichia coli enterotoxins by brefeldin A. Infect Immun. 1993 Aug;61(8):3282–3286. doi: 10.1128/iai.61.8.3282-3286.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Draper R. K., O'Keefe D. O., Stookey M., Graves J. Identification of a cold-sensitive step in the mechanism of modeccin action. J Biol Chem. 1984 Apr 10;259(7):4083–4088. [PubMed] [Google Scholar]
  10. Dunn W. A., Hubbard A. L., Aronson N. N., Jr Low temperature selectively inhibits fusion between pinocytic vesicles and lysosomes during heterophagy of 125I-asialofetuin by the perfused rat liver. J Biol Chem. 1980 Jun 25;255(12):5971–5978. [PubMed] [Google Scholar]
  11. Fishman P. H., Curran P. K. Brefeldin A inhibits protein synthesis in cultured cells. FEBS Lett. 1992 Dec 21;314(3):371–374. doi: 10.1016/0014-5793(92)81508-j. [DOI] [PubMed] [Google Scholar]
  12. FitzGerald D., Morris R. E., Saelinger C. B. Receptor-mediated internalization of Pseudomonas toxin by mouse fibroblasts. Cell. 1980 Oct;21(3):867–873. doi: 10.1016/0092-8674(80)90450-x. [DOI] [PubMed] [Google Scholar]
  13. Gerhardt M. A., Neubig R. R. Multiple Gi protein subtypes regulate a single effector mechanism. Mol Pharmacol. 1991 Nov;40(5):707–711. [PubMed] [Google Scholar]
  14. Griffiths G., Simons K. The trans Golgi network: sorting at the exit site of the Golgi complex. Science. 1986 Oct 24;234(4775):438–443. doi: 10.1126/science.2945253. [DOI] [PubMed] [Google Scholar]
  15. Hausman S. Z., Burns D. L. Interaction of pertussis toxin with cells and model membranes. J Biol Chem. 1992 Jul 5;267(19):13735–13739. [PubMed] [Google Scholar]
  16. Hewlett E. L., Sauer K. T., Myers G. A., Cowell J. L., Guerrant R. L. Induction of a novel morphological response in Chinese hamster ovary cells by pertussis toxin. Infect Immun. 1983 Jun;40(3):1198–1203. doi: 10.1128/iai.40.3.1198-1203.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hsia J. A., Moss J., Hewlett E. L., Vaughan M. ADP-ribosylation of adenylate cyclase by pertussis toxin. Effects on inhibitory agonist binding. J Biol Chem. 1984 Jan 25;259(2):1086–1090. [PubMed] [Google Scholar]
  18. Hudson T. H., Grillo F. G. Brefeldin-A enhancement of ricin A-chain immunotoxins and blockade of intact ricin, modeccin, and abrin. J Biol Chem. 1991 Oct 5;266(28):18586–18592. [PubMed] [Google Scholar]
  19. Kaslow H. R., Burns D. L. Pertussis toxin and target eukaryotic cells: binding, entry, and activation. FASEB J. 1992 Jun;6(9):2684–2690. doi: 10.1096/fasebj.6.9.1612292. [DOI] [PubMed] [Google Scholar]
  20. Katada T., Tamura M., Ui M. The A protomer of islet-activating protein, pertussis toxin, as an active peptide catalyzing ADP-ribosylation of a membrane protein. Arch Biochem Biophys. 1983 Jul 1;224(1):290–298. doi: 10.1016/0003-9861(83)90212-6. [DOI] [PubMed] [Google Scholar]
  21. Keen J. H., Maxfield F. R., Hardegree M. C., Habig W. H. Receptor-mediated endocytosis of diphtheria toxin by cells in culture. Proc Natl Acad Sci U S A. 1982 May;79(9):2912–2916. doi: 10.1073/pnas.79.9.2912. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kounnas M. Z., Morris R. E., Thompson M. R., FitzGerald D. J., Strickland D. K., Saelinger C. B. The alpha 2-macroglobulin receptor/low density lipoprotein receptor-related protein binds and internalizes Pseudomonas exotoxin A. J Biol Chem. 1992 Jun 25;267(18):12420–12423. [PubMed] [Google Scholar]
  23. Krueger K. M., Barbieri J. T. Molecular characterization of the in vitro activation of pertussis toxin by ATP. J Biol Chem. 1993 Jun 15;268(17):12570–12578. [PubMed] [Google Scholar]
  24. 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]
  25. Lencer W. I., Delp C., Neutra M. R., Madara J. L. Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. J Cell Biol. 1992 Jun;117(6):1197–1209. doi: 10.1083/jcb.117.6.1197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lenhard J. M., Mayorga L., Stahl P. D. Characterization of endosome-endosome fusion in a cell-free system using Dictyostelium discoideum. J Biol Chem. 1992 Jan 25;267(3):1896–1903. [PubMed] [Google Scholar]
  27. Lotti L. V., Torrisi M. R., Pascale M. C., Bonatti S. Immunocytochemical analysis of the transfer of vesicular stomatitis virus G glycoprotein from the intermediate compartment to the Golgi complex. J Cell Biol. 1992 Jul;118(1):43–50. doi: 10.1083/jcb.118.1.43. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Madshus I. H., Stenmark H., Sandvig K., Olsnes S. Entry of diphtheria toxin-protein A chimeras into cells. J Biol Chem. 1991 Sep 15;266(26):17446–17453. [PubMed] [Google Scholar]
  29. Merritt E. A., Sarfaty S., van den Akker F., L'Hoir C., Martial J. A., Hol W. G. Crystal structure of cholera toxin B-pentamer bound to receptor GM1 pentasaccharide. Protein Sci. 1994 Feb;3(2):166–175. doi: 10.1002/pro.5560030202. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Montecucco C., Tomasi M., Schiavo G., Rappuoli R. Hydrophobic photolabelling of pertussis toxin subunits interacting with lipids. FEBS Lett. 1986 Jan 6;194(2):301–304. doi: 10.1016/0014-5793(86)80105-3. [DOI] [PubMed] [Google Scholar]
  31. Moskaug J. O., Stenmark H., Olsnes S. Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions. J Biol Chem. 1991 Feb 5;266(4):2652–2659. [PubMed] [Google Scholar]
  32. Naglich J. G., Metherall J. E., Russell D. W., Eidels L. Expression cloning of a diphtheria toxin receptor: identity with a heparin-binding EGF-like growth factor precursor. Cell. 1992 Jun 12;69(6):1051–1061. doi: 10.1016/0092-8674(92)90623-k. [DOI] [PubMed] [Google Scholar]
  33. Nambiar M. P., Oda T., Chen C., Kuwazuru Y., Wu H. C. Involvement of the Golgi region in the intracellular trafficking of cholera toxin. J Cell Physiol. 1993 Feb;154(2):222–228. doi: 10.1002/jcp.1041540203. [DOI] [PubMed] [Google Scholar]
  34. Nogimori K., Tamura M., Yajima M., Hashimura N., Ishii S., Ui M. Structure-function relationship of islet-activating protein, pertussis toxin: biological activities of hybrid toxins reconstituted from native and methylated subunits. Biochemistry. 1986 Mar 25;25(6):1355–1363. doi: 10.1021/bi00354a025. [DOI] [PubMed] [Google Scholar]
  35. Ogata M., Chaudhary V. K., Pastan I., FitzGerald D. J. Processing of Pseudomonas exotoxin by a cellular protease results in the generation of a 37,000-Da toxin fragment that is translocated to the cytosol. J Biol Chem. 1990 Nov 25;265(33):20678–20685. [PubMed] [Google Scholar]
  36. Orlandi P. A., Curran P. K., Fishman P. H. Brefeldin A blocks the response of cultured cells to cholera toxin. Implications for intracellular trafficking in toxin action. J Biol Chem. 1993 Jun 5;268(16):12010–12016. [PubMed] [Google Scholar]
  37. Roerig S. C., Loh H. H., Law P. Y. Requirement of ADP-ribosylation for the pertussis toxin-induced alteration in electrophoretic mobility of G-proteins. Biochem Biophys Res Commun. 1991 Nov 14;180(3):1227–1232. doi: 10.1016/s0006-291x(05)81327-0. [DOI] [PubMed] [Google Scholar]
  38. Sandvig K., Olsnes S. Effect of temperature on the uptake, excretion and degradation of abrin and ricin by HeLa cells. Exp Cell Res. 1979 Jun;121(1):15–25. doi: 10.1016/0014-4827(79)90439-7. [DOI] [PubMed] [Google Scholar]
  39. Sandvig K., Prydz K., Hansen S. H., van Deurs B. Ricin transport in brefeldin A-treated cells: correlation between Golgi structure and toxic effect. J Cell Biol. 1991 Nov;115(4):971–981. doi: 10.1083/jcb.115.4.971. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Schweizer A., Fransen J. A., Matter K., Kreis T. E., Ginsel L., Hauri H. P. Identification of an intermediate compartment involved in protein transport from endoplasmic reticulum to Golgi apparatus. Eur J Cell Biol. 1990 Dec;53(2):185–196. [PubMed] [Google Scholar]
  41. Sekura R. D., Fish F., Manclark C. R., Meade B., Zhang Y. L. Pertussis toxin. Affinity purification of a new ADP-ribosyltransferase. J Biol Chem. 1983 Dec 10;258(23):14647–14651. [PubMed] [Google Scholar]
  42. Staddon J. M., Bouzyk M. M., Rozengurt E. A novel approach to detect toxin-catalyzed ADP-ribosylation in intact cells: its use to study the action of Pasteurella multocida toxin. J Cell Biol. 1991 Nov;115(4):949–958. doi: 10.1083/jcb.115.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Stein P. E., Boodhoo A., Armstrong G. D., Cockle S. A., Klein M. H., Read R. J. The crystal structure of pertussis toxin. Structure. 1994 Jan 15;2(1):45–57. doi: 10.1016/s0969-2126(00)00007-1. [DOI] [PubMed] [Google Scholar]
  44. Stow J. L., de Almeida J. B., Narula N., Holtzman E. J., Ercolani L., Ausiello D. A. A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells. J Cell Biol. 1991 Sep;114(6):1113–1124. doi: 10.1083/jcb.114.6.1113. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Tamura M., Nogimori K., Murai S., Yajima M., Ito K., Katada T., Ui M., Ishii S. Subunit structure of islet-activating protein, pertussis toxin, in conformity with the A-B model. Biochemistry. 1982 Oct 26;21(22):5516–5522. doi: 10.1021/bi00265a021. [DOI] [PubMed] [Google Scholar]
  46. Yoshida T., Chen C. C., Zhang M. S., Wu H. C. Disruption of the Golgi apparatus by brefeldin A inhibits the cytotoxicity of ricin, modeccin, and Pseudomonas toxin. Exp Cell Res. 1991 Feb;192(2):389–395. doi: 10.1016/0014-4827(91)90056-z. [DOI] [PubMed] [Google Scholar]

Articles from Infection and Immunity are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES