INTERMEDIATES IN TRANSLOCATION OF DIPHTHERIA-TOXIN ACROSS THE PLASMA-MEMBRANE

Active diphtheria toxin consists of two parts, fragments A and B. Frag ment A has enzymatic activity and inhibits protein synthesis. Fragment B binds to cellular receptors, and upon exposure to low pH it inserts into the membrane and facilitates translocation of the A fragment int o the cytosol, concomitantly with formation of cation-selective channe ls. Reduction of the interfragment disulfide bridge is required for re lease of fragment A and intoxication. In cells treated with N-ethylmal eimide (NEM), which inhibits reduction of the disulfide bridge, fragme nt A was translocated to the cytosol but not released from fragment B. In the presence of NEM a peptide larger than fragment A was protected against extracellularly added Pronase. This peptide (M(r) similar to 24,000) was released to the supernatant fraction of saponin-treated ce lls. This indicates that fragment A, which is 21 kDa, is covalently at tached via a disulfide bond to an N-terminal (M(r) similar to 3,000) p iece of fragment B. The 24-kDa fragment disappeared upon reduction, an d the 21-kDa fragment A appeared instead. NEM did not prevent channel activity by fragment B in the context of full-length toxin, demonstrat ing that channel formation occurs in spite of inhibited reduction of t he disulfide bond. Thus, channel formation is not dependent on release of fragment A from the toxin-receptor complex.