Abstract
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.
Highlights
Evidence accumulated to date has shown that proteins degraded by the ubiquitin (Ub)1-mediated proteolytic system [1, 2] contain structural features that serve as recognition signals for Ub system factors
Portions of the encephalomyocarditis virus (EMCV) 3C protease were first fused with portions of the poliovirus 3C protease, which is known to be a poor substrate for the Ub-mediated proteolytic system [36]
The poliovirus protein does not share extensive sequence homology with the EMCV 3C protease [49, 50], the two do contain the centrally placed common sequence FRD, located at positions 92–94 in the EMCV 3C protease and at positions 83– 85 in the poliovirus 3C protease. This shared sequence was utilized to construct in vitro transcription plasmids carrying either the sequence coding for the N-terminal half of the EMCV 3C protease fused with the C-terminal half of the poliovirus 3C protease (ENPC protein) or the sequence coding for the N-terminal half of the poliovirus 3C protease fused with the C-terminal half of the EMCV 3C protease (PNEC protein)
Summary
Ubiquitin; DTT, dithiothreitol; EMCV, encephalomyocarditis virus; HAV, hepatitis A virus; HIC, hydrophobic interaction chromatography; MeUb, methylated ubiquitin; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase. The 3C proteases are cysteine proteases of approximately 21–24 kDa which exhibit a high degree of substrate specificity [37] These proteins are initially synthesized within large polyproteins encoded by the positive stranded RNA viral genomes. In vivo [41], and it has been directly proven to be quickly degraded in rabbit reticulocyte lysate [36] The destruction of both of these proteins requires both the synthesis of Ub-3C protease conjugates and the action of the proteasome [35, 36]. Neither of these 3C proteases possesses a destabilizing Nterminal amino acid or homology with any reported sequences suspected of being required for recognition by the Ub-mediated proteolytic system. We have identified two E3 Ubprotein ligases, one being E3␣, that recognize the EMCV 3C protease and catalyze the conjugation of Ub to the protein
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