Membrane translocation of diphtheria toxin A-fragment: role of carboxy-terminal region.
The C-terminal end of diphtheria toxin A-fragment was altered and the consequences for toxicity and translocation of the A-fragment to the cytosol were studied. Mutations and deletions in the protease-sensitive, disulfide-bridged region linking the two functional parts of the toxin, the A- and B-fragments, reduced the toxicity of the protein as such, but when the mutant toxins were cleaved ("nicked") by trypsin before being added to cells, the toxicity was restored. Prevention of disulfide formation by removal of Cys186 resulted in complete loss of toxicity. To circumvent the nicking step, toxin was formed by reconstitution from separate A- and B-fragments where the A-fragments varied in the C-terminal sequences. The amino acids C-terminal to Cys186 were found not to be required for translocation. Furthermore, both charged and uncharged residues near the C-terminal end were compatible with translocation. The data indicate that the C-terminal amino acid sequence is not decisive for translocation of diphtheria toxin A-fragment to the cytosol.
Ariansen
S
S
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Afanasiev
B N
BN
Moskaug
J O
JO
Stenmark
H
H
Madshus
I H
IH
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D004563:Electrochemistry; D004926:Escherichia coli / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000378:metabolism; D014709:Vero Cells
D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; D014357:Trypsin