Characterization of the Non‐catalytic Inhibitory Effect of Ubp6 on Proteasome Function

Selective protein degradation in eukaryotes is mediated primarily by the ubiquitin‐proteasome system (UPS) in which ubiquitin is attached to a target protein to signal its degradation by the proteasome. Therefore, the proteasome, a multisubunit protease, is a central enzyme in the system and controls the levels of hundreds of proteins. However, little attention has been given to how the proteasome is regulated. One important mechanism of proteasome regulation involves reversibly‐associated deubiquitinating enzymes (DUBs). One of these DUBs is the yeast protein Ubp6 (whose mammalian ortholog is Usp14). Interestingly, in addition to its catalytic activity of deubiquitination, Ubp6, has a second, highly unexpected function: a capacity to delay the degradation of ubiquitinated proteins (termed the non‐catalytic inhibitory effect). The degradation delay caused by Ubp6 might serve to ensure that the deubiquitinating activity of Ubp6 has adequate time to regulate substrate commitment to degradation. However, the mechanism of this inhibitory effect is unknown. In search of this mechanism we have characterized noncatalytic proteasome inhibition by Ubp6 with the benefit of site‐directed mutagenesis, quantitative biochemical assays, and yeast genetics.