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The importance of the ubiquitin system largely depends on ubiquitin ligases, E3s, as they determine the specificity of the system. Rbx1/ROC1/Hrt1, a RING finger protein, functions as an important component of the cullin-containing SCF and VBC-Cul2 ligases. Modification of cullins by NEDD8 (NEDDylation), has been shown to be essential for the E3 activity of both SCF and VBC-Cul2, and it was suggested that Rbx1 acts as the E3 for cullin NEDDylation. RING finger is composed of eight cysteine and histidine residues that bind to zinc ions. Rbx1 is a highly evolutionarily conserved protein; however, the eighth coordination residue in its RING finger is aspartate (D97) rather than cysteine. Substitution of D97 with each of the other 19 amino acids demonstrates that aspartate is superior to cysteine in cullin NEDDylation. Interestingly, however, different D97 mutants demonstrate different activities towards 6 cullins tested. Importantly, we were able to discriminate between the NEDDylating activity of Rbx1 and its involvement in the ubiquitylation reaction within the context of VBC-Cul2. Moreover, while Rbx1 is not involved in governing the stability of SCF, Rbx1 mutants destabilize VBC-Cul2. Taken together, these results indicate that various mechanisms regulate both the activities and the stability of cullin-based ligases.