Identification and characterization of a protein destruction signal in the encephalomyocarditis virus 3C protease

• Published in: The Journal of biological chemistry 1999-04, Vol.274 (14), p.9904-9980
• Main Authors: Lawson, TG, Gronros, DL, Evans, PE, Bastien, MC, Michalewich, KM, Clark, JK, Edmonds, JH, Graber, KH, Werner, JA, Lurvey, BA, Cate, JM
• Format: Article
• Language: English
• ISSN: 1083-351X

The amino acid sequence LLVRGRTLVV, which is probably located in a strand-turn-strand structure, has been identified as a protein destruction signal in the rapidly degraded encephalomyocarditis virus 3C protease. Mutations within this sequence reduced the susceptibility of the 3C protease toward ubiquitination and degradation in rabbit reticulocyte lysate. This signal is transferable, since poliovirus 3C protease, which is a poor ubiquitin-mediated proteolytic system substrate, was found to be ubiquitinated and degraded when the signal sequence was either generated at an internal location in the protein or fused to the N terminus. An evaluation of the behavior of 3C protease proteins containing mutations in the signal region indicates that considerable variability in the primary structure is tolerated, although the conservation of certain features appears to be required for signal function. Two E3 ubiquitin-protein ligases that recognize the encephalomyocarditis virus 3C protease as a substrate were also partially purified. One of these was identified as the previously described E3alpha, and this was shown to require the destruction signal sequence to catalyze efficiently the ubiquitination of the 3C protease. The other is a Ubc5-dependent E3 that appears to recognize a different, unidentified feature of the 3C protease.