Self-translocation of diphtheria toxin across model membranes.
To understand the mechanism of diphtheria toxin membrane translocation, the toxin was entrapped within lipid vesicles, and its low pH-induced translocation across the lipid bilayer was measured. Proteolysis and resistance to guanidinium chloride denaturation were used to demonstrate that the toxin molecules were entrapped. Low pH-induced movement of entrapped toxin to the outer (trans) face of the bilayer was assayed by the binding of external streptavidin to biotin-labeled entrapped toxin. Complete translocation was quantified by the amount of protein released into the external medium. Using whole toxin, it was found that the A fragment was efficiently translocated, but the B fragment was not. This was true both in the low temperature (A domain folded) and high temperature (A domain unfolded) toxin conformations previously identified [Jiang J. X., Abrams, F. S., and London, E. (1991) Biochemistry 30, 3857-3864]. Remarkably, even isolated fragment A appeared to self-translocate under some conditions. Toxin-induced translocation may partly result from formation of a nonspecific toxin-induced pore. This idea is supported by the toxin-induced release of fluorescent dextrans coentrapped within the vesicles. However, low pH-induced exposure of entrapped toxin on the outside of the membrane was conformation dependent. Exposure was greatest for the high temperature conformation. This suggests the existence of a more specific translocation process. The nature and relationship of these processes, and their relative roles in translocation in vivo are discussed.
Jiang
J X
JX
Department of Biochemistry and Cell Biology, State University of New York, Stony Brook 11794-5215.
Chung
L A
LA
London
E
E
D001665:Binding Sites; D003911:Dextrans; D004167:Diphtheria Toxin / Q000737:chemistry*; D016650:Fluorescein-5-isothiocyanate / Q000031:analogs & derivatives; D005456:Fluorescent Dyes; D019791:Guanidine; D006146:Guanidines; D006863:Hydrogen-Ion Concentration; D008051:Lipid Bilayers; D008961:Models, Structural; D010446:Peptide Fragments / Q000302:isolation & purification; D010713:Phosphatidylcholines; D011487:Protein Conformation; D011489:Protein Denaturation; D013050:Spectrometry, Fluorescence; D014357:Trypsin
D003911:Dextrans; D004167:Diphtheria Toxin; D005456:Fluorescent Dyes; D006146:Guanidines; D008051:Lipid Bilayers; D010446:Peptide Fragments; D010713:Phosphatidylcholines; C015219:fluorescein isothiocyanate dextran; D014357:Trypsin; D016650:Fluorescein-5-isothiocyanate; D019791:Guanidine