Characterization of deubiquitylating enzymes in yeast

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Protein ubiquitylation is an essential post-translational modification responsible for targeted protein degradation, cellular signaling activation, DNA damage response, and protein trafficking. Deubiquitylating enzymes (DUBs) functions as an eraser of the ubiquitin signals by removing ubiquitin(s) from the ubiquitylated proteins. Despite of their importance, biological significance of DUBs poorly understood. To test enzymatic properties of DUBs, we expressed and purified yeast 20 DUBs by a wheat germ expression system. Although most DUBs did not have chain-type specificities, we found that four DUBs can cleave a ubiquitin precursor Ubi4. Among the proteasomal DUBs, consistently with previous studies, Rpn11 is essential for degradation of the ubiquitinated substrate whereas Ubp6 has an inhibitory effect for the substrate degradation in vitro. However, both DUBs can cleave both K48- and K63-linked ubiquitin chains, suggesting that other factors might specify the proteasomal degradation signal. We also found that Ubp6 facilitates proteasomal assembly by clearing ubiquitylated substrates from assembly precursors by its deubiquitylating activity. Furthermore, we applied parallel reaction monitoring (PRM) method to quantify the ubiquitin chains and analyzed DUB mutants.