Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes

H. Zhan, J. L. Elliott, W. H. Shen, P. D. Huynh, A. Finkelstein, R. J. Collier

Research output: Contribution to journalArticle

14 Citations (Scopus)

Abstract

Translocation of the catalytic domain of diphtheria toxin (DT) across the endosomal membrane to the cytoplasm of mammalian cells requires the low- pH-dependent insertion of a hydrophobic helical hairpin (TH8-TH9) that is buried within the T domain of the native protein. Mutations of Pro345, which terminates helix TH8, have been reported to block toxicity for Vero cells. We found that mutant toxins in which Pro345 had been replaced by Cys, Glu, or Gly were profoundly defective at low pH in forming channels in planar phospholipid bilayers and in permeabilizing phospholipid vesicles to entrapped fluorophores. Experiments with isolated T domain containing a polarity-sensitive fluorophore attached to Cys at position 332 suggest that the P345E mutation blocks membrane insertion. None of the Pro345 mutations shifted the pH-dependence of binding in solution of the hydrophobic fluorophore, 2-ptoluidinyl-naphthalene 7-sulfonate. The results indicate that proline at position 345 is required for the T domain to insert into phospholipid bilayers or to adopt a functional conformation within the bilayer.

Original languageEnglish (US)
Pages (from-to)173-181
Number of pages9
JournalJournal of Membrane Biology
Volume167
Issue number2
DOIs
StatePublished - 1999

Fingerprint

Diphtheria Toxin
Proline
Phospholipids
Mutation
Membranes
Vero Cells
Catalytic Domain
Cytoplasm

Keywords

  • Diphtheria toxin
  • Membrane insertion
  • Mutagenesis
  • Proline
  • Site-specific labeling
  • Transmembrane domain

ASJC Scopus subject areas

  • Biophysics
  • Physiology
  • Cell Biology

Cite this

Zhan, H., Elliott, J. L., Shen, W. H., Huynh, P. D., Finkelstein, A., & Collier, R. J. (1999). Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes. Journal of Membrane Biology, 167(2), 173-181. https://doi.org/10.1007/s002329900481

Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes. / Zhan, H.; Elliott, J. L.; Shen, W. H.; Huynh, P. D.; Finkelstein, A.; Collier, R. J.

In: Journal of Membrane Biology, Vol. 167, No. 2, 1999, p. 173-181.

Research output: Contribution to journalArticle

Zhan, H, Elliott, JL, Shen, WH, Huynh, PD, Finkelstein, A & Collier, RJ 1999, 'Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes', Journal of Membrane Biology, vol. 167, no. 2, pp. 173-181. https://doi.org/10.1007/s002329900481
Zhan, H. ; Elliott, J. L. ; Shen, W. H. ; Huynh, P. D. ; Finkelstein, A. ; Collier, R. J. / Effects of mutations in proline 345 on insertion of diphtheria toxin into model membranes. In: Journal of Membrane Biology. 1999 ; Vol. 167, No. 2. pp. 173-181.
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