Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity

Saeki, Y., Isono, E., Toh-E, A. (2005)

Methods in Enzymology, 399, 215-227

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.

Subject headings: Electrophoresis, Polyacrylamide Gel; Endosomal Sorting Complexes Required for Transport; Proteasome Endopeptidase Complex/metabolism; Saccharomyces cerevisiae Proteins/metabolism; Substrate Specificity; Ubiquitin/chemistry/metabolism; Ubiquitin-Protein Ligase Complexes/metabolism

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Type: Journal Article

Serial number: 2937