IMMUNOCHEMICAL ANALYSIS SHOWS ALL 3 DOMAINS OF DIPHTHERIA-TOXIN PENETRATE ACROSS MODEL MEMBRANES

Diphtheria toxin undergoes membrane insertion and translocation across membranes when exposed to low pH, In this study, the translocation of the toxin has been investigated by the binding of antibodies to two p reparations of model membrane-inserted toxin, In one prep aration, tox in was added externally to model membrane vesicles and then inserted b y exposure to low pH, In the other preparation, toxin was entrapped in the vesicles at neutral pH, and then inserted by decreasing pH, At ne utral pH, externally added antibodies could not bind to entrapped toxi n, although they could bind to externally added native toxin, However, after low pH exposure, antibodies against all three toxin domains (ca talytic (C), transmembrane (T), and receptor-binding (R)) could bind t o entrapped toxin, and also to externally added membrane-inserted toxi n, The binding to the entrapped toxin shows that all three domains of the toxin translocate to the trans face of the membrane after exposure to low pH, The observation that antibodies bind to both external and entrapped preparations of toxin after low pH exposure shows that toxin inserts in a mixed orientation. A difference in antibody binding to l ow pH-treated toxin in which the C domain is folded (Lr' conformation) or unfolded (Lr '' conformation) was also observed, An increase in an tibody binding to C and T domains in the Lr '' conformation relative t o binding to the Lr' conformation was found for entrapped toxin, sugge sting that more of the C and T domains translocate across the bilayer in the Lr '' conformation. These results suggest all three toxin domai ns insert in the membrane bilayer and participate in translocation in vitro. The C and R domains lack classical transmembrane hydrophobic se quences, However, they possess sequences that have the potential to fo rm membrane-inserting beta-sheets.