|Ph.D Student||Adi Guterman|
|Subject||The Intrinsic Deubiquitinating Activity of the Yeast 26S|
|Department||Department of Biology||Supervisor||Full Professor Glickman Michael|
Substrates destined for degradation by the 26S proteasome are labeled with polyubiquitin chains. These chains can be dismantled by deubiquitinating enzymes (DUBs). A number of reports have identified different DUBs that can hydrolyze ubiquitin from substrates bound to the proteasome. We measured deubiquitination by both isolated Lid and Base-Core particle subcomplexes ("Lidless"), suggesting that at least two different DUBs are intrinsic components of the 26S proteasome holoenzymes. To the best of our knowledge, this is the first time that direct deubiquitination by purified Lid has been observed. In agreement, we find that highly purified proteasomes contain both Rpn11 and Ubp6, situated within the Lid and Base subcomplexes, respectively. To study their relative contributions, we purified proteasomes from a mutant in the putative metalloprotease domain of Rpn11 and from a ubp6 null. In both preparations we observed slower deubiquitination rates, suggesting that Rpn11 and Ubp6 serve complementary roles. In accord, the double mutant is synthetically lethal. Treatment of proteasomes with ubiquitin-aldehyde and other cysteine modifiers inhibited proteasome-associated deubiquitination but simultaneously promoted degradation of a monoubiquitinated substrate along with the ubiquitin tag. Degradation is unique to intact 26S holoenzyme, whereas the lidless conformation was incompetent for proteolysis despite possessing deubiquitinating activity. The fascinating observation that a single ubiquitin moiety is sufficient for targeting an otherwise stable substrate to proteasomes exposes how rapid deubiquitination of poorly ubiquitinated substrates may counteract degradation. In contrast, one purpose of polyubiquitin chains may be to commit substrates for degradation by slowing down their rate of deubiquitination.