Identification of sumoylated proteins by systematic immunoprecipitation of the budding yeast proteome.
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 ( approximately 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.
Wykoff
Dennis D
DD
Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, University of California, San Francisco, California 94143-2240, USA.
O'Shea
Erin K
EK
D003546:Cysteine Endopeptidases / Q000235:genetics / Q000378:metabolism; D047468:Immunoprecipitation; D011499:Protein Processing, Post-Translational; D020543:Proteome / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics; D025842:SUMO-1 Protein / Q000502:physiology*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D025841:Small Ubiquitin-Related Modifier Proteins
D020543:Proteome; D012097:Repressor Proteins; C099362:SMT3 protein, S cerevisiae; D025842:SUMO-1 Protein; D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease