Allosteric regulation and substrate specificity of the proteasome deubiquitinase Rpn11 (936.2)

The 26S proteasome is a multi‐protein complex responsible for ubiquitin‐mediated protein degradation in eukaryotic cells. Rpn11, the only essential proteasome deubiquitinase, removes ubiquitin chains from protein substrates during degradation, however very little is known about the regulation and intrinsic ubiquitin cleavage specificity of the enzyme. We utilized a heterologous expression system for the proteasome lid subcomplex, mutational analyses and in‐vitro deubiquitination assays to study the activity of Rpn11 in different assembly intermediates of the proteasome. We found that Rpn11 shows no preference for K48 or K63 linked di‐ubiquitin, indicating that it is promiscuous in substrate cleavage specificity. Furthermore, our studies have shown that Rpn5 inhibits Rpn11 in the lid complex and that this inhibition is relieved by incorporation of the lid into the proteasome holoenzyme, or by truncation of the Rpn5 N‐terminus. These observations support a model where the N‐terminus of Rpn5 blocks the Rpn11 active site in the isolated lid, but swings away from the enzyme to make contacts with the base and core particle upon formation of the proteasome. This allosteric regulation by Rpn5 may prevent spurious dubiquitination by Rpn11 in the isolated lid and thus ensure that the enzyme only acts on substrates committed to proteasomal degradation. Grant Funding Source : Supported by National Science Foundation Graduate Research Student Fellowship