Insertion of diphtheria toxin B-fragment into the plasma membrane at low pH. Characterization and topology of inserted regions.
When the enzymatically active A-fragment of diphtheria toxin is translocated to the cytosol, the B-fragment inserts into the membrane in such a way that a 25-kDa polypeptide becomes shielded from proteases added to the external medium. We have attempted to determine the boundaries of this polypeptide within the toxin B-fragment as well as the topology of the B-fragment in the membrane. Chemical cleavage of the 25-kDa polypeptide with hydroxylamine and o-iodosobenzoic acid yielded fragments of sizes indicating that the 25-kDa polypeptide starts at residue approximately 300 and extends to the COOH-terminal end. Experiments where the toxin was labeled with [35S]cysteine at distinct positions of the B-fragment supported this conclusion. Treatment of cells with inserted B-fragment with L-1-tosyl-amido-2-phenylethyl chloromethyl ketone-treated trypsin and with V8 protease from Staphylococcus aureus yielded protected 27- and 30-kDa fragments in addition to 25 kDa, indicating that the region 240-264 is also at the outside. The topology of the inserted B-fragment is discussed.
Moskaug
J O
JO
Institute for Cancer Research, Norwegian Radium Hospital, Montebello, Oslo.
Stenmark
H
H
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002462:Cell Membrane / Q000378:metabolism*; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006898:Hydroxylamines / Q000494:pharmacology; D007463:Iodobenzoates / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D010957:Plasmids; D014357:Trypsin / Q000378:metabolism; D014709:Vero Cells
D004167:Diphtheria Toxin; D006898:Hydroxylamines; D007463:Iodobenzoates; D010446:Peptide Fragments; C039171:diphtheria toxin fragment B; C008154:2-iodosobenzoic acid; D014357:Trypsin