INHIBITION OF MEMBRANE TRANSLOCATION OF DIPHTHERIA-TOXIN-A FRAGMENT BY INTERNAL DISULFIDE BRIDGES

Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cel l surface receptors through its B-fragment, and after endocytotic upta ke, the low endosomal pH triggers translocation of the A-fragment acro ss the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Fo ur of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was obse rved both when the toxin was endocytosed and when translocation was in duced by exposing surface-bound toxin to low pH. The data indicate tha t extensive unfolding of the A-fragment is required for translocation.