Constitutively active Cullin-RING-Ligases fail to rescue loss of NEDD8 conjugation in Schizosaccharomyces pombe

In fission yeast, the only known essential function of Ned8p is the modification of the cullin, Pcu1p, and subsequent Cullin-RING-Ligase (CRL) activation and substrate ubiquitination. We show here that a functional Pcu1p mutant, deleted for its C-terminal autoinhibitory domain, which negates the requirement of neddylation for ligase activity, is unable to rescue the loss of neddylation. These findings suggest that the neddylation of non-cullin substrate(s) are required for Schizosaccharomyces pombe viability.

► Generation of uba3 and pcu1 temperature-sensitive mutants.
► Deletion of NEDD8 specific ligase, dcn1.
► The activity of Cullin-RING-Ligase activity and varying levels of neddylation were studied.
► Levels of Cullin neddylation did not directly correlate with activity.
► Constitutively active Pcu1p mutants could not rescue lethality from loss of neddylation.