Isolation, characterization, and partial purification of a novel ubiquitin-protein ligase, E3 - Targeting of protein substrates via multiple and distinct recognition signals and conjugating enzymes

Degradation of a protein via the ubiquitin system involves two discrete steps, conjugation of ubiquitin to the substrate and degradation of the adduct, Conjugation follows a three-step mechanism. First, ubiquitin is activated by the ubiquitin-activating enzyme, E1, Following activation, one of several E2 enzymes (ubiquitin-carrier proteins or ubiquitin-conjugating enzymes, UBCs) transfers ubiquitin from E1 to the protein substrate that is bound to one of several ubiquitin-protein ligases, E3s. These enzymes catalyze the last step in the process, covalent attachment of ubiquitin to the protein substrate. The binding of the substrate to E3 is specific and implies that E3s play a major role in recognition and selection of proteins for conjugation and subsequent degradation, So far, only a few ligases have been identified, and it is clear that many more have not been discovered yet, Here, we describe a novel Ligase that is involved in the conjugation and degradation of non ''N-end rule'' protein substrates such as actin, troponin T, and MyoD. This substrate specificity suggests that the enzyme may be involved in degradation of muscle proteins, The ligase acts in concert with E2-F1, a previously described non N-end rule UBC, Interestingly, it is also involved in targeting lysozyme, a bona fide N-end rule substrate that is recognized by E3 alpha and E2-14 kDa. The novel ligase recognizes lysozyme via a signal(s) that is distinct from the N-terminal residue of the protein, Thus, it appears that certain proteins can be targeted via multiple recognition motifs and distinct pairs of conjugating enzymes, We have purified the ligase similar to 200-fold and demonstrated that it is different from other known E3s, including E3 alpha/UBR1, E3 beta, and EG-AP. The native enzyme has an apparent molecular mass of similar to 550 kDa and appears to be a homodimer. Because of its unusual size, we designated this novel ligase E3L (large).