title: Two-step affinity purification of multiubiquitylated proteins from Saccharomyces cerevisiae.
abstract: In budding yeast and higher eukaryotic genomes, there are, respectively, 50 and up to 400 or more distinct genes that encode for ubiquitin-ligases, and approximately 15-90 genes that encode for ubiquitin isopeptidases (TM and RJD, Semple et al., 2003). This puts ubiquitylation on par with phosphorylation as the most common reversible posttranslational modifications in eukaryotic cells. A key challenge that has met with limited success to date is to identify the proteins that are the substrates for this large collection of enzymes. To begin to address this daunting challenge, we sought to identify ubiquitylated proteins that are potential substrates of the 26S proteasome. Here, we describe a two-step affinity purification protocol that uses a budding yeast strain that expresses hexahistidine-tagged ubiquitin. In the first step, native cell lysate was chromatographed on a UBA domain-containing matrix that binds preferentially to K48-linked multiubiquitin chains. Free ubiquitin and presumably monoubiquitylated proteins did not bind this column, whereas proteins that are potential substrates of the proteasome were enriched. In the second step, UBA domain-binding proteins were subjected to immobilized metal ion affinity chromatography (IMAC) under denaturing conditions on magnetic nickel beads, resulting in >3000-fold enrichment of ubiquitin conjugates relative to crude cell extract.
authors.affiliation: Division of Biology, California Institute of Technology, Pasadena, California, USA.
authors.forename: Thibault
authors.initials: T
authors.lastname: Mayor
authors.forename: Raymond J
authors.initials: RJ
authors.lastname: Deshaies
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D029701:Saccharomyces cerevisiae Proteins / Q000302:isolation & purification* / Q000378:metabolism; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin