Inhibition of membrane translocation of diphtheria toxin A-fragment by internal disulfide bridges.
Fragment A of diphtheria toxin is translocated to the cytosol when the toxin is presented to receptor-positive cells. The toxin binds to cell surface receptors through its B-fragment, and after endocytotic uptake, the low endosomal pH triggers translocation of the A-fragment across the membrane. Translocation can also be induced at the level of the plasma membrane by exposure to low pH medium. Based on the diphtheria toxin crystal structure, we made five double cysteine mutants of the A-fragment, each expected to form an intramolecular disulfide bond. Four of the double cysteine mutants efficiently formed an intramolecular disulfide bridge, and these same mutants showed a strong reduction in their translocating ability. The inhibition of translocation was observed both when the toxin was endocytosed and when translocation was induced by exposing surface-bound toxin to low pH. The data indicate that extensive unfolding of the A-fragment is required for translocation.
Falnes
P O
PO
Department of Biochemistry, Norwegian Radium Hospital, Montebello, Oslo.
Choe
S
S
Madshus
I H
IH
Wilson
B A
BA
Olsnes
S
S
D000246:Adenosine Diphosphate Ribose / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004167:Diphtheria Toxin / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D004220:Disulfides / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism* / Q000633:toxicity; D014176:Protein Biosynthesis; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism / Q000633:toxicity; D014158:Transcription, Genetic; D014709:Vero Cells
D017931:DNA Primers; D004167:Diphtheria Toxin; D004220:Disulfides; D010446:Peptide Fragments; D011994:Recombinant Proteins; C029795:diphtheria toxin fragment A; D009243:NAD; D000246:Adenosine Diphosphate Ribose; D003545:Cysteine