Cell-mediated reduction and incomplete membrane translocation of diphtheria toxin mutants with internal disulfides in the A fragment.
Active diphtheria toxin consists of two fragments, A and B, joined by a disulfide bond. The B fragment binds to cell surface receptors and aids in the translocation of the enzymatically active A fragment to the cytosol. Normally, the toxin A fragment enters the cytosol from acidic endosomes, but translocation can also be induced at the level of the plasma membrane by exposing cells with surface-bound toxin to low pH. Recently, we showed that disulfide bonds introduced into the A fragment by mutation are inhibitory for translocation. In the present work, we found that although the complete translocation of the A fragment is blocked, three mutant toxins underwent reduction of the interfragment disulfide bond upon low pH exposure, whereas the internal disulfide in the A fragment remained intact. In the case of two of these mutants, the A fragment was released into the extracellular medium upon exposure of cell-bound toxin to low pH. The pH profile for the release of the mutant A fragments was the same as for translocation of wild-type A fragment to the cytosol, and the release was inhibited by conditions that interfere with A fragment translocation. In the case of the third mutant, which remained cell-associated upon reduction of the interfragment disulfide bond, a translocation intermediate was detected. The results show that the reduction of the interfragment disulfide bond can occur in the absence of complete translocation of the A fragment to the cytosol, and they indicate that the reduction takes place at an early stage in the translocation process. Our findings suggest that the translocation of the A fragment across the membrane is initiated at the C terminus.
Falnes
P O
PO
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Olsnes
S
S
D000818:Animals; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002522:Chlorocebus aethiops; D004167:Diphtheria Toxin / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D004220:Disulfides; D004705:Endocytosis; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008961:Models, Structural; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017354:Point Mutation; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014158:Transcription, Genetic; D014709:Vero Cells
D004167:Diphtheria Toxin; D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C029795:diphtheria toxin fragment A