Preparation of Ubiquitinated Substrates by the PY Motif‐Insertion Method for Monitoring 26S Proteasome Activity

For analysis of the mechanism of the 26S proteasome–mediated protein degradation in vitro, the preparation of well‐defined substrate, the ubiquitinated proteins, of the 26S proteasome is inevitable. However, no method has been available to ubiquitinate a given protein. Here, we propose a relatively simple method for preparation of the ubiquitinated substrates using HECT‐type ubiquitin ligase Rsp5, termed the PY motif‐insertion method. The principle of this method is that the PY motif, known as the Rsp5‐binding motif, is inserted into protein to be ubiquitinated by Rsp5. In this communication, we describe that Sic1 was successfully ubiquitinated by the PY motif‐insertion method and demonstrate that Sic1 thus ubiquitinated was degraded by the purified yeast 26S proteasome.