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 cyt osol of sensitive cells where it inhibits protein synthesis by inactiv ating elongation factor 2 (EF-2). We have constructed a number of diph theria toxin mutants that are degraded by the N-end rule pathway in Ve ro cells, and that display a wide range of intracellular stabilities. The degradation could be inhibited by the proteasome inhibitor lactacy stin, indicating that the proteasome is responsible for N-end rule-med iated degradation in mammalian cells. Previously, the N-end rule has b een investigated by studying the co-translational degradation of intra cellularly 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 intracell ular stability of the mutants and their toxic effect on cells, thus de monstrating a novel manner of modulating the toxicity of a protein tox in. The data also indicate that the inactivation of EF-2 is the rate-l imiting step in the intoxication process.