Noncatalytic effect of the ubiquitin ligase Hul5 on proteasome function

Hul5, a major proteasome-associated protein, extends the multiubiquitin chains of proteasome-bound ubiquitin conjugates, and thus promotes their degradation. Active-site mutants in the Hul5 ubiquitin ligase domain recapitulate the phenotype of a full deletion in several respects. Through characterizing mutant phenotypes of Hul5 in a yeast strain with impaired proteasome function, I discovered an unexpected noncatalytic function for Hul5. The noncatalytic effect promotes protein degradation, as judged from several in vivo assays. The strength of the effect is evident from the substantial increase in total cellular ubiquitin conjugate levels when catalytically inactive Hul5 is expressed. The noncatalytic effect of Hul5 was abrogated when a proteasome deubiquitinating enzyme, Ubp6, was rendered catalytically inactive by mutation. This result suggests that the noncatalytic effect of Hul5 may be to inhibit the catalytic activity of Ubp6. The noncatalytic activity has been localized to a small, 53-residue segment at the N terminus of this 910-residue protein. The proteasome binding element of Hul5 was mapped in parallel, and closely overlaps the noncatalytic element. The mapping data suggest that the noncatalytic effect is exerted at the proteasome, consistent with the model that Ubp6 is its target. In summary, Hul5 regulates protein turnover by the proteasome via two distinct mechanisms. The catalytic and noncatalytic modes of Hul5 function are proposed to act coordinately on multiubiquitin chains by promoting their extension and suppressing their disassembly.