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. 1989 Oct;8(10):2849–2853. doi: 10.1002/j.1460-2075.1989.tb08432.x

Permeabilization of the plasma membrane by deletion mutants of diphtheria toxin.

H Stenmark 1, S McGill 1, S Olsnes 1, K Sandvig 1
PMCID: PMC401337  PMID: 2479538

Abstract

Diphtheria toxin B-fragment binds to cell-surface receptors and facilitates translocation of the enzymatically active A-fragment to the cytosol. In this process the B-fragment inserts into the plasma membrane and induces formation of cation-selective channels. We examined the ability of a number of diphtheria toxin-derived molecules translated in vitro to permeabilize cells. Two proteins consisting of the whole B-fragment and small parts of the A-fragment, and one protein comprising most of the B-fragment alone, were more efficient than full-length toxin in permeabilizing the plasma membrane to monovalent cations. Two shorter B-fragment-derived proteins, with 3 and 10 kd N-terminal deletions, permeabilized the cells to sulfate and sucrose in addition to monovalent cations. The relationship between channel formation and toxin translocation is discussed.

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Selected References

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  1. Carroll S. F., Barbieri J. T., Collier R. J. Diphtheria toxin: purification and properties. Methods Enzymol. 1988;165:68–76. doi: 10.1016/s0076-6879(88)65014-2. [DOI] [PubMed] [Google Scholar]
  2. Donovan J. J., Simon M. I., Draper R. K., Montal M. Diphtheria toxin forms transmembrane channels in planar lipid bilayers. Proc Natl Acad Sci U S A. 1981 Jan;78(1):172–176. doi: 10.1073/pnas.78.1.172. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Draper R. K., Simon M. I. The entry of diphtheria toxin into the mammalian cell cytoplasm: evidence for lysosomal involvement. J Cell Biol. 1980 Dec;87(3 Pt 1):849–854. doi: 10.1083/jcb.87.3.849. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Dumont M. E., Richards F. M. The pH-dependent conformational change of diphtheria toxin. J Biol Chem. 1988 Feb 5;263(4):2087–2097. [PubMed] [Google Scholar]
  5. Eisenberg D., Schwarz E., Komaromy M., Wall R. Analysis of membrane and surface protein sequences with the hydrophobic moment plot. J Mol Biol. 1984 Oct 15;179(1):125–142. doi: 10.1016/0022-2836(84)90309-7. [DOI] [PubMed] [Google Scholar]
  6. Greenfield L., Bjorn M. J., Horn G., Fong D., Buck G. A., Collier R. J., Kaplan D. A. Nucleotide sequence of the structural gene for diphtheria toxin carried by corynebacteriophage beta. Proc Natl Acad Sci U S A. 1983 Nov;80(22):6853–6857. doi: 10.1073/pnas.80.22.6853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kagan B. L., Finkelstein A., Colombini M. Diphtheria toxin fragment forms large pores in phospholipid bilayer membranes. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4950–4954. doi: 10.1073/pnas.78.8.4950. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kayser G., Lambotte P., Falmagne P., Capiau C., Zanen J., Ruysschaert J. M. A CNBR peptide located in the middle region of diphtheria toxin fragment B induces conductance change in lipid bilayers. Possible role of an amphipathic helical segment. Biochem Biophys Res Commun. 1981 Mar 31;99(2):358–363. doi: 10.1016/0006-291x(81)91753-8. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Lambotte P., Falmagne P., Capiau C., Zanen J., Ruysschaert J. M., Dirkx J. Primary structure of diphtheria toxin fragment B: structural similarities with lipid-binding domains. J Cell Biol. 1980 Dec;87(3 Pt 1):837–840. doi: 10.1083/jcb.87.3.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. McGill S., Stenmark H., Sandvig K., Olsnes S. Membrane interactions of diphtheria toxin analyzed using in vitro synthesized mutants. EMBO J. 1989 Oct;8(10):2843–2848. doi: 10.1002/j.1460-2075.1989.tb08431.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Moskaug J. O., Sandvig K., Olsnes S. Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol. J Biol Chem. 1988 Feb 15;263(5):2518–2525. [PubMed] [Google Scholar]
  13. Olsnes S., Carvajal E., Sandvig K. Interactions between diphtheria toxin entry and anion transport in vero cells. III. Effect on toxin binding and anion transport of tumor-promoting phorbol esters, vanadate, fluoride, and salicylate. J Biol Chem. 1986 Feb 5;261(4):1562–1569. [PubMed] [Google Scholar]
  14. Olsnes S., Moskaug J. O., Stenmark H., Sandvig K. Diphtheria toxin entry: protein translocation in the reverse direction. Trends Biochem Sci. 1988 Sep;13(9):348–351. doi: 10.1016/0968-0004(88)90105-3. [DOI] [PubMed] [Google Scholar]
  15. Olsnes S., Sandvig K. Interactions between diphtheria toxin entry and anion transport in Vero cells. I. Anion antiport in Vero cells. J Biol Chem. 1986 Feb 5;261(4):1542–1552. [PubMed] [Google Scholar]
  16. Papini E., Sandoná D., Rappuoli R., Montecucco C. On the membrane translocation of diphtheria toxin: at low pH the toxin induces ion channels on cells. EMBO J. 1988 Nov;7(11):3353–3359. doi: 10.1002/j.1460-2075.1988.tb03207.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Papini E., Schiavo G., Tomasi M., Colombatti M., Rappuoli R., Montecucco C. Lipid interaction of diphtheria toxin and mutants with altered fragment B. 2. Hydrophobic photolabelling and cell intoxication. Eur J Biochem. 1987 Dec 15;169(3):637–644. doi: 10.1111/j.1432-1033.1987.tb13655.x. [DOI] [PubMed] [Google Scholar]
  18. Pappenheimer A. M., Jr Diphtheria toxin. Annu Rev Biochem. 1977;46:69–94. doi: 10.1146/annurev.bi.46.070177.000441. [DOI] [PubMed] [Google Scholar]
  19. Ramsay G., Montgomery D., Berger D., Freire E. Energetics of diphtheria toxin membrane insertion and translocation: calorimetric characterization of the acid pH induced transition. Biochemistry. 1989 Jan 24;28(2):529–533. doi: 10.1021/bi00428a018. [DOI] [PubMed] [Google Scholar]
  20. Sandvig K., Olsnes S. Diphtheria toxin entry into cells is facilitated by low pH. J Cell Biol. 1980 Dec;87(3 Pt 1):828–832. doi: 10.1083/jcb.87.3.828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Sandvig K., Olsnes S. Diphtheria toxin-induced channels in Vero cells selective for monovalent cations. J Biol Chem. 1988 Sep 5;263(25):12352–12359. [PubMed] [Google Scholar]
  22. Sandvig K., Olsnes S. Rapid entry of nicked diphtheria toxin into cells at low pH. Characterization of the entry process and effects of low pH on the toxin molecule. J Biol Chem. 1981 Sep 10;256(17):9068–9076. [PubMed] [Google Scholar]
  23. Stenmark H., Olsnes S., Sandvig K. Requirement of specific receptors for efficient translocation of diphtheria toxin A fragment across the plasma membrane. J Biol Chem. 1988 Sep 15;263(26):13449–13455. [PubMed] [Google Scholar]
  24. Zhao J. M., London E. Conformation and model membrane interactions of diphtheria toxin fragment A. J Biol Chem. 1988 Oct 25;263(30):15369–15377. [PubMed] [Google Scholar]
  25. Zhao J. M., London E. Similarity of the conformation of diphtheria toxin at high temperature to that in the membrane-penetrating low-pH state. Proc Natl Acad Sci U S A. 1986 Apr;83(7):2002–2006. doi: 10.1073/pnas.83.7.2002. [DOI] [PMC free article] [PubMed] [Google Scholar]

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