title: Low pH-induced release of diphtheria toxin A-fragment in Vero cells. Biochemical evidence for transfer to the cytosol.
abstract: When Vero cells with surface-bound 125I-labeled, nicked diphtheria toxin were exposed to pH 4.5, two polypeptides of Mr 20,000 and 25,000 became protected against externally applied Pronase E. The 20-kDa polypeptide appears to be the toxin A-fragment, whereas the 25-kDa polypeptide must be derived from the B-fragment. Permeabilization of the cells with saponin allowed efflux of the 20-kDa fragment to occur, whereas most of the 25-kDa polypeptide remained associated with the cells. A number of compounds and conditions which protect cells against diphtheria toxin prevented the protection against Pronase E. Protection of the 25-kDa polypeptide occurred even when the transmembrane proton gradient (delta pH) was dissipated by acidification of the cytosol, whereas protection and release of the A-fragment were prevented under these conditions. Electrical depolarization and ATP depletion of the cells did not inhibit protection and release of the A-fragment. The data indicate that delta pH is required for the transfer of the A-fragment to the cytosol, whereas the insertion of part of the B-fragment into the membrane occurs at low pH, even in the absence of a delta pH.
authors.affiliation: Institute for Cancer Research, Norwegian Radium Hospital, Oslo.
authors.forename: J O
authors.initials: JO
authors.lastname: Moskaug
authors.forename: K
authors.initials: K
authors.lastname: Sandvig
authors.forename: S
authors.initials: S
authors.lastname: Olsnes
mesh_terms: D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003600:Cytosol / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008253:Macaca mulatta; D008274:Magnesium / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011402:Pronase / Q000378:metabolism; D013696:Temperature
chemical_list: D004167:Diphtheria Toxin; D010446:Peptide Fragments; C029795:diphtheria toxin fragment A; D000246:Adenosine Diphosphate Ribose; D000255:Adenosine Triphosphate; D011402:Pronase; D008274:Magnesium; D002118:Calcium