The diphtheria toxin channel-forming T domain translocates its own NH2- terminal region across planar bilayers

Lisa Senzel, Paul D. Huynh, Karen S. Jakes, R. John Collier, Alan Finkelstein

Research output: Contribution to journalArticlepeer-review

55 Scopus citations


The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1-201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322-378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH2 terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH2-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH2 terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH2-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH2 terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH2 terminus be translocated across the membrane from the cis to the trans side, even though the NH2 terminus is >100 residues removed from the channel-forming part of the molecule.

Original languageEnglish (US)
Pages (from-to)317-324
Number of pages8
JournalJournal of General Physiology
Issue number3
StatePublished - Sep 1998


  • Channel gating
  • Histidine tag
  • Nickel binding
  • Streptavidin
  • Trypsin

ASJC Scopus subject areas

  • Physiology


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