Identification of Sumoylated Proteins by Systematic Immunoprecipitation of the Budding Yeast Proteome*
- ‡From the Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, California 94143-2240
- ‡To whom correspondence should be addressed: Dept. of Biochemistry and Biophysics, Howard Hughes Medical Inst., University of California, 600 16th St., Genentech Hall, Rm. GH-S472D, San Francisco, CA 94143-2240. Tel.: 415-476-2212; Fax: 415-514-2073;E-mail: oshea{at}biochem.ucsf.edu.
Abstract
The identification of post-translational modifications to proteins is critical for understanding many important aspects of biology. Utilizing a collection of epitope-tagged yeast strains, we developed a novel approach to determine which proteins are modified by the small ubiquitin-related modifier (SUMO). We crossed traits useful for the detection of SUMO conjugation into 4246 tandem affinity purification-tagged strains and successfully immunoprecipitated and screened 2893 of these proteins for association with SUMO (∼70% of the expressed proteome detectable by immunoblot analysis). We found 82 proteins associated with SUMO, including many of low abundance. Because our screen was performed under non-denaturing conditions, we were able to identify multiple members of four complexes that were associated with SUMO: the RSC chromatin remodeling complex, the mediator complex, the TFIID complex, and the septin complex. In addition, we describe five new direct conjugates of SUMO, and we mutated SUMO conjugation sites in four proteins. This is the first attempt to immunoprecipitate a large fraction of the proteome of a eukaryote, and it demonstrates the utility of this method to identify post-translational modifications in the yeast proteome.
Footnotes
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Published, MCP Papers in Press, December 13, 2004, DOI 10.1074/mcp.M400166-MCP200
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1 The abbreviations used are: SUMO, small ubiquitin-related modifier; HA, hemagglutinin; IDA, iminodiacetic acid; ORF, open reading frame; SD, synthetic yeast medium with 2% dextrose; TAP, tandem affinity purification; E1, SUMO-activating enzyme; E2, SUMO carrier protein; E3, SUMO-protein isopeptide ligase; Ura, uracil.
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↵* k was supported by National Institutes of Health Postdoctoral Research Fellowship GM20762 and the Howard Hughes Medical Institute. 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.
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- Received October 22, 2004.
- Revision received December 2, 2004.
- The American Society for Biochemistry and Molecular Biology
