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J. Biol. Chem., Vol. 280, Issue 33, 29470-29478, August 19, 2005

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A High Throughput Screen to Identify Substrates for the Ubiquitin Ligase Rsp5^*

Bart Kus, Aaron Gajadhar, Karen Stanger, Rob Cho, Warren Sun, Nathalie Rouleau¶, Tammy Lee, Donovan Chan¶, Cheryl Wolting||, Aled Edwards, Roger Bosse¶, and Daniela Rotin||**

From the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, ^¶PerkinElmer Life Sciences, Biosignal, Montreal, QuebecH3J 1R4, and ^||The Hospital for Sick Children and Department of Biochemistry, University of Toronto, Toronto, Ontario M5G 1X8, Canada

Ubiquitin-protein ligases (E3s) are implicated in various human disorders and are attractive targets for therapeutic intervention. Although most cellular proteins are ubiquitinated, ubiquitination cannot be linked directly to a specific E3 for a large fraction of these proteins, and the substrates of most E3 enzymes are unknown. We have developed a luminescent assay to detect ubiquitination in vitro, which is more quantitative, effective, and sensitive than conventional ubiquitination assays. By taking advantage of the abundance of purified proteins made available by genomic efforts, we screened hundreds of purified yeast proteins for ubiquitination, and we identified previously reported and novel substrates of the yeast E3 ligase Rsp5. The relevance of these substrates was confirmed in vivo by showing that a number of them interact genetically with Rsp5, and some were ubiquitinated by Rsp5 in vivo. The combination of this sensitive assay and the availability of purified substrates will enable the identification of substrates for any purified E3 enzyme.

Received for publication, February 25, 2005 , and in revised form, June 8, 2005.

^* This work was supported in part by the National Cancer Institute of Canada with funds from the Canadian Cancer Society (to A. E. and D. R.), the Canadian Institute for Health Research (to D. R.), and the Canadian Foundation for Innovation (to D. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains Table S1.

Supported by Ontario Graduate Scholarship.

^** To whom correspondence should be addressed: Program in Cell Biology, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario M5G 1X8, Canada. Tel.: 416-813-5098; Fax: 416-813-5771; E-mail: drotin{at}sickkids.ca.

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