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.
Saeki
Y
Y
Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan.
Isono
E
E
Toh-E
A
A
D004591:Electrophoresis, Polyacrylamide Gel; D056827:Endosomal Sorting Complexes Required for Transport; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D013379:Substrate Specificity; D025801:Ubiquitin / Q000737:chemistry / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism
D056827:Endosomal Sorting Complexes Required for Transport; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; C469287:RSP5 protein, S cerevisiae