Role of anions in low pH-induced translocation of diphtheria toxin.
Previous work has shown that when Vero cells with surface-bound diphtheria toxin are exposed to low pH, toxin entry across the plasma membrane is induced and that this entry involves two steps, insertion of the B-fragment of the toxin into the membrane and translocation of the enzymatically active A-fragment to the cytosol. Here we have studied the role of permeant anions in this process. It was found that when the B-fragment was inserted into the membrane, part of it, a 25-kDa polypeptide, was shielded from externally added Pronase. This insertion did not require permeant anions. The translocation of the A-fragment was monitored by measuring either its ability to inhibit protein synthesis in the cells or the appearance of radioactively labeled 21-kDa fragment after treatment of the cells with externally applied Pronase. The translocation of the A-fragment was dependent on the presence of permeant anions in the medium. However, when the cells were depleted of Cl- by incubation in Cl- free buffer at high pH, translocation of the A-fragment did not require permeant anions in the medium. The possibility that translocation of the A-fragment is inhibited by an outward directed chloride gradient rather than by the absence of chloride is discussed.
Moskaug
J O
JO
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Sandvig
K
K
Olsnes
S
S
D000818:Animals; D000838:Anions; D001692:Biological Transport; D002462:Cell Membrane / Q000378:metabolism*; D002712:Chlorides / Q000008:administration & dosage / Q000494:pharmacology*; D003600:Cytosol / Q000378:metabolism; D004167:Diphtheria Toxin / Q000378:metabolism* / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005942:Gluconates / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008353:Mannitol / Q000494:pharmacology; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism; D011402:Pronase / Q000494:pharmacology; D014176:Protein Biosynthesis; D013431:Sulfates / Q000494:pharmacology; D014709:Vero Cells
D000838:Anions; D002712:Chlorides; D004167:Diphtheria Toxin; D005942:Gluconates; D010446:Peptide Fragments; D013431:Sulfates; D008353:Mannitol; D011402:Pronase; C030691:gluconic acid