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 β‐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.