Modulation of the intracellular stability and toxicity of diphtheria toxin through degradation by the N-end rule pathway.
The enzymatically active A-fragment of diphtheria toxin enters the cytosol of sensitive cells where it inhibits protein synthesis by inactivating elongation factor 2 (EF-2). We have constructed a number of diphtheria toxin mutants that are degraded by the N-end rule pathway in Vero cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacystin, indicating that the proteasome is responsible for N-end rule-mediated degradation in mammalian cells. Previously, the N-end rule has been investigated by studying the co-translational degradation of intracellularly expressed beta-galactosidase. Our work shows that a mature protein entering the cytosol from the exterior can also be degraded by the N-end rule pathway with a similar, but not identical specificity to that previously found. We found a correlation between the intracellular stability of the mutants and their toxic effect on cells, thus demonstrating a novel manner of modulating the toxicity of a protein toxin. The data also indicate that the inactivation of EF-2 is the rate-limiting step in the intoxication process.
Falnes
P O
PO
Institute for Cancer Research, The Norwegian Radium Hospital, Montebello, Oslo. pfalnes@ulrik.uio.no
Olsnes
S
S
D000595:Amino Acid Sequence; D000818:Animals; D002458:Cell Fractionation; D002522:Chlorocebus aethiops; D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004151:Dipeptides / Q000494:pharmacology; D004167:Diphtheria Toxin / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D007424:Intracellular Fluid / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism; D016296:Mutagenesis; D009842:Oligopeptides; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism* / Q000633:toxicity*; D010455:Peptides / Q000235:genetics; D046988:Proteasome Endopeptidase Complex; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism; D012156:Reticulocytes; D014709:Vero Cells
D015853:Cysteine Proteinase Inhibitors; D004151:Dipeptides; D004167:Diphtheria Toxin; D009097:Multienzyme Complexes; D009842:Oligopeptides; D010446:Peptide Fragments; D010455:Peptides; D011993:Recombinant Fusion Proteins; C029795:diphtheria toxin fragment A; C085706:FLAG peptide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex