Selective translocation of the A chain of diphtheria toxin across the membrane of purified endosomes.
Translocation is a necessary and rate-limiting step for diphtheria toxin (DT) cytotoxicity. We have reconstituted DT translocation in a cell-free system using endosomes purified from lymphocytes and have demonstrated this using two different probe/cell systems, which provided identical results: 125I-DT/human CEM cells and 125I-transferrin-DT/mouse BW cells. The cell-free DT translocation process was found to be dependent on the presence of the pH gradient endosome (pH 5.3)/cytosol (pH 7). Among the pH equilibrating agents, nigericin (5 microM) was found to be the most effective, inhibiting DT translocation by 88%. An optimum pH value of 7 on the cytosolic side of the membrane (pH gradient approximately 1.7) was determined. ATP per se is not required for DT translocation. 125I-DT translocation was 3-fold more active from late than from early endosomes, probably because of their slightly more acidic pH. Only the A chain of the toxin was found to escape from either 125I-DT/CEM or 125I-transferrin-DT/BW endosomes. Translocation of control endosome labels (125I-transferrin and 125I-horseradish peroxidase) was never observed. We also show that DT receptors present on resistant (mouse) cells block the translocation of the toxin and are responsible for the resistance of these cells to DT.
Beaumelle
B
B
URA 530 Centre National de la Recherche Scientifique, Département Biologie Santé, Université Montpellier, France.
Bensammar
L
L
Bienvenüe
A
A
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism; D002474:Cell-Free System; D004167:Diphtheria Toxin / Q000235:genetics* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007124:Immunoenzyme Techniques; D007457:Iodine Radioisotopes; D008214:Lymphocytes / Q000378:metabolism; D051379:Mice; D014168:Transferrin / Q000378:metabolism
D004167:Diphtheria Toxin; D007457:Iodine Radioisotopes; D014168:Transferrin; D000246:Adenosine Diphosphate Ribose; D000255:Adenosine Triphosphate