The diphtheria toxin channel-forming T-domain translocates its own NH2-terminal region and the catalytic domain across planar phospholipid bilayers.
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 (residues 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. Here we show that in association with channel formation by the T-domain, its hydrophilic 63-amino acid NH2-terminal region (residues 202-264) as well as the entire catalytic A fragment (residues 1-201) cross the lipid bilayer. The phenomenon that enabled us to demonstrate this was the rapid closure of channels at cis negative voltages when a histidine tag was placed at various positions in the NH2-terminal region of the T-domain or in the A fragment; the inhibition of this effect by trans nickel established that the histidine tag was present on the trans side of the membrane. Thus, all of the machinery necessary to translocate the A fragment across membranes is built into the 114 residues at the carboxy-terminal end of the T-domain (residues 265-378), without the requirement of any proteins in the plasma membrane (e.g., toxin receptor) or of any other cellular components.
Finkelstein
A
A
Department of Physiology & Biophysics, Albert Einstein College of Medicine, Bronx, NY 10461, USA. finkelst@aecom.yu.edu
Oh
K J
KJ
Senzel
L
L
Gordon
M
M
Blaustein
R O
RO
Collier
R J
RJ
D000595:Amino Acid Sequence; D001692:Biological Transport; D020134:Catalytic Domain; D004167:Diphtheria Toxin / Q000737:chemistry / Q000378:metabolism*; D015640:Ion Channel Gating; D008051:Lipid Bilayers / Q000378:metabolism*; D008969:Molecular Sequence Data; D010743:Phospholipids / Q000378:metabolism*
D004167:Diphtheria Toxin; D008051:Lipid Bilayers; D010743:Phospholipids