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. 1996 Feb;64(2):593–599. doi: 10.1128/iai.64.2.593-599.1996

Pertussis toxin-catalyzed ADP-ribosylation of Gi-2 and Gi-3 in CHO cells is modulated by inhibitors of intracellular trafficking.

Y Xu 1, J T Barbieri 1
PMCID: PMC173806  PMID: 8550212

Abstract

In previous studies, an in vitro ADP-ribosylation assay was developed to quantitatively evaluate the in vivo ADP-ribosylation of eukaryotic target proteins in intact Chinese hamster ovary (CHO) cells by pertussis toxin (PT). Immunoblot analysis identified the two PT-sensitive target proteins in CHO cells as Gi-2 and Gi-3. In this in vitro ADP-ribosylation assay, the ability of PT and ADP-ribosylate Gi-2 and Gi-3 intact CHO cells was not inhibited by cytochalasin D but was inhibited by chloroquine, monensin, and nocodazole. These data implicated the involvement of a cytochalasin D-independent endocytic mechanism, a pH-sensitive step, and microtubules in the ADP-ribosylation of Gi-2 and Gi-3 by PT in intact CHO cells. Preincubation of CHO cells with cycloheximide, at concentrations that reduced protein synthesis by > 95%, did not inhibit the ability of PT to ADP-ribosylate Gi-2 and Gi-3. Control experiments showed that these agents did not affect either the ability of PT to directly ADP-ribosylate the heterotrimeric G protein, Gt, or the binding of PT to CHO cells, except that monensin slightly inhibited the binding of PT to CHO cells. These results are consistent with a model in which PT is internalized by receptor-mediated endocytosis, probably via a cytochalasin D-independent pathway, which involves intracellular trafficking through late endosomes and the Golgi apparatus. An alternative model predicts the presence of a eukaryotic factor that traffics within cells via this pathway and is required by PT to ADP-ribosylate Gi proteins.

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Selected References

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  1. Armstrong G. D., Peppler M. S. Maintenance of biological activity of pertussis toxin radioiodinated while bound to fetuin-agarose. Infect Immun. 1987 May;55(5):1294–1299. doi: 10.1128/iai.55.5.1294-1299.1987. [DOI] [PMC free article] [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. Bhatnagar R., Friedlander A. M. Protein synthesis is required for expression of anthrax lethal toxin cytotoxicity. Infect Immun. 1994 Jul;62(7):2958–2962. doi: 10.1128/iai.62.7.2958-2962.1994. [DOI] [PMC free article] [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. Cooper J. A. Effects of cytochalasin and phalloidin on actin. J Cell Biol. 1987 Oct;105(4):1473–1478. doi: 10.1083/jcb.105.4.1473. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. De Brabander M., Nuydens R., Geerts H., Hopkins C. R. Dynamic behavior of the transferrin receptor followed in living epidermoid carcinoma (A431) cells with nanovid microscopy. Cell Motil Cytoskeleton. 1988;9(1):30–47. doi: 10.1002/cm.970090105. [DOI] [PubMed] [Google Scholar]
  7. 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]
  8. 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]
  9. Goldstein J. L., Brown M. S., Anderson R. G., Russell D. W., Schneider W. J. Receptor-mediated endocytosis: concepts emerging from the LDL receptor system. Annu Rev Cell Biol. 1985;1:1–39. doi: 10.1146/annurev.cb.01.110185.000245. [DOI] [PubMed] [Google Scholar]
  10. Gordon V. M., Young W. W., Jr, Lechler S. M., Gray M. C., Leppla S. H., Hewlett E. L. Adenylate cyclase toxins from Bacillus anthracis and Bordetella pertussis. Different processes for interaction with and entry into target cells. J Biol Chem. 1989 Sep 5;264(25):14792–14796. [PubMed] [Google Scholar]
  11. Gruenberg J., Griffiths G., Howell K. E. Characterization of the early endosome and putative endocytic carrier vesicles in vivo and with an assay of vesicle fusion in vitro. J Cell Biol. 1989 Apr;108(4):1301–1316. doi: 10.1083/jcb.108.4.1301. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Herman B., Albertini D. F. A time-lapse video image intensification analysis of cytoplasmic organelle movements during endosome translocation. J Cell Biol. 1984 Feb;98(2):565–576. doi: 10.1083/jcb.98.2.565. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Janicot M., Fouque F., Desbuquois B. Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes. J Biol Chem. 1991 Jul 15;266(20):12858–12865. [PubMed] [Google Scholar]
  15. Kartenbeck J., Stukenbrok H., Helenius A. Endocytosis of simian virus 40 into the endoplasmic reticulum. J Cell Biol. 1989 Dec;109(6 Pt 1):2721–2729. doi: 10.1083/jcb.109.6.2721. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. 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]
  17. 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]
  18. 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]
  19. Leppla S., Dorland R. B., Middlebrook J. L. Inhibition of diphtheria toxin degradation and cytotoxic action by chloroquine. J Biol Chem. 1980 Mar 25;255(6):2247–2250. [PubMed] [Google Scholar]
  20. Marnell M. H., Stookey M., Draper R. K. Monensin blocks the transport of diphtheria toxin to the cell cytoplasm. J Cell Biol. 1982 Apr;93(1):57–62. doi: 10.1083/jcb.93.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Matteoni R., Kreis T. E. Translocation and clustering of endosomes and lysosomes depends on microtubules. J Cell Biol. 1987 Sep;105(3):1253–1265. doi: 10.1083/jcb.105.3.1253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Montesano R., Roth J., Robert A., Orci L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature. 1982 Apr 15;296(5858):651–653. doi: 10.1038/296651a0. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Pesonen M., Simons K. Transepithelial transport of a viral membrane glycoprotein implanted into the apical plasma membrane of Madin-Darby canine kidney cells. II. Immunological quantitation. J Cell Biol. 1983 Sep;97(3):638–643. doi: 10.1083/jcb.97.3.638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Pressman B. C. Biological applications of ionophores. Annu Rev Biochem. 1976;45:501–530. doi: 10.1146/annurev.bi.45.070176.002441. [DOI] [PubMed] [Google Scholar]
  26. Sakai T., Yamashina S., Ohnishi S. Microtubule-disrupting drugs blocked delivery of endocytosed transferrin to the cytocenter, but did not affect return of transferrin to plasma membrane. J Biochem. 1991 Apr;109(4):528–533. doi: 10.1093/oxfordjournals.jbchem.a123415. [DOI] [PubMed] [Google Scholar]
  27. Sandvig K., Olsnes S., Pihl A. Inhibitory effect of ammonium chloride and chloroquine on the entry of the toxic lectin modeccin into HeLa cells. Biochem Biophys Res Commun. 1979 Sep 27;90(2):648–655. doi: 10.1016/0006-291x(79)91284-1. [DOI] [PubMed] [Google Scholar]
  28. Sandvig K., Tønnessen T. I., Olsnes S. Ability of inhibitors of glycosylation and protein synthesis to sensitize cells to abrin, ricin, Shigella toxin, and Pseudomonas toxin. Cancer Res. 1986 Dec;46(12 Pt 1):6418–6422. [PubMed] [Google Scholar]
  29. Sandvig K., van Deurs B. Selective modulation of the endocytic uptake of ricin and fluid phase markers without alteration in transferrin endocytosis. J Biol Chem. 1990 Apr 15;265(11):6382–6388. [PubMed] [Google Scholar]
  30. 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]
  31. Sundan A., Sandvig K., Olsnes S. Calmodulin antagonists sensitize cells to pseudomonas toxin. J Cell Physiol. 1984 Apr;119(1):15–22. doi: 10.1002/jcp.1041190104. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Tran D., Carpentier J. L., Sawano F., Gorden P., Orci L. Ligands internalized through coated or noncoated invaginations follow a common intracellular pathway. Proc Natl Acad Sci U S A. 1987 Nov;84(22):7957–7961. doi: 10.1073/pnas.84.22.7957. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Wiechelman K. J., Braun R. D., Fitzpatrick J. D. Investigation of the bicinchoninic acid protein assay: identification of the groups responsible for color formation. Anal Biochem. 1988 Nov 15;175(1):231–237. doi: 10.1016/0003-2697(88)90383-1. [DOI] [PubMed] [Google Scholar]
  35. Xu Y., Barbieri J. T. Pertussis toxin-mediated ADP-ribosylation of target proteins in Chinese hamster ovary cells involves a vesicle trafficking mechanism. Infect Immun. 1995 Mar;63(3):825–832. doi: 10.1128/iai.63.3.825-832.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]

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