| Preparation of ubiquitinated substrates by the PY motif-insertion method for monitoring 26S proteasome activity. | |
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MedLine Citation:
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PMID: 16338358 Owner: NLM Status: MEDLINE |
Abstract/OtherAbstract:
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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. |
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Authors:
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Y Saeki; E Isono; A Toh-E |
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Publication Detail:
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Type: Journal Article; Research Support, Non-U.S. Gov't |
Journal Detail:
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Title: Methods in enzymology Volume: 399 ISSN: 0076-6879 ISO Abbreviation: Meth. Enzymol. Publication Date: 2005 |
Date Detail:
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Created Date: 2005-12-12 Completed Date: 2007-05-31 Revised Date: 2009-11-19 |
Medline Journal Info:
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Nlm Unique ID: 0212271 Medline TA: Methods Enzymol Country: United States |
Other Details:
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Languages: eng Pagination: 215-27 Citation Subset: IM |
Affiliation:
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Department of Biological Sciences, Graduate School of Science, The University of Tokyo, Tokyo, Japan. |
Export Citation:
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| MeSH Terms | |
Descriptor/Qualifier:
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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 |
| Chemical | |
Reg. No./Substance:
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0/Endosomal Sorting Complexes Required for Transport; 0/Saccharomyces cerevisiae Proteins; 0/Ubiquitin; EC 3.4.25.1/Proteasome Endopeptidase Complex; EC 3.4.99.-/ATP dependent 26S protease; EC 6.3.2.19/RSP5 protein, S cerevisiae; EC 6.3.2.19/Ubiquitin-Protein Ligase Complexes |
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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