Abstract
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.
Highlights
Translocation is a necessary and rate-limiting step the A chain of DT (made of two subunits, A and Blinked by for diphtheria toxin (DT) cytotoxicityW. e 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: '261-DT/humanCEM cells and '261-transferrin-DT/mouseBW cells
We show that DT receptors pres- rate is much lower from plasma ent on resistant cells block the translocation membrane vesicles than from endosomes? we thought that of the toxin and are responsible for the resistance of DT translocation remained to be demonstrated and analyzed these cellsto DT
We show in this paper that DT translocation can be observed in a cell-free system using functional endosomes puri
Summary
Translocation is a necessary and rate-limiting step the A chain of DT (made of two subunits, A and Blinked by for diphtheria toxin (DT) cytotoxicityW. e 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: '261-DT/humanCEM cells and '261-transferrin-DT/mouseBW cells. Translocation is a necessary and rate-limiting step the A chain of DT (made of two subunits, A and Blinked by for diphtheria toxin (DT) cytotoxicityW. Onlythe A chain of the toxin was found to escape a disulfide bridge) inactivates protein synthesisleading to cell death (3). Another action of DT through deoxyribonuclease activity has been reported, but thecontribution of this enzymatic activity in cytotoxicity is still being discussed (4). We show that DT receptors pres- rate (which is pH independent) is much lower from plasma ent on resistant (mouse) cells block the translocation membrane vesicles than from endosomes? We show in this paper that DT translocation can be observed in a cell-free system using functional endosomes puri-
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