title: Reconstitution of SUMO-dependent ubiquitylation in vitro.
abstract: In eukaryotic cells, most soluble proteins are degraded via the ubiquitin proteasome system. The recognition signal for the proteasome consists of a lysine 48-linked ubiquitin chain which is posttranslationally conjugated to lysine residues in target proteins. This conjugation reaction is mediated by an enzymatic cascade consisting of specific E1, E2, and E3 enzymes. The small ubiquitin-related modifier (SUMO) is conjugated to target proteins via a similar cascade of SUMO-specific enzymes. Contrary to the long-standing assumption that SUMO does not participate in proteolytic targeting, proteasomal inhibition stabilizes both ubiquitin and SUMO conjugates (SCs). This led to the discovery of ubiquitin ligases for SUMO conjugates (ULS proteins or SUMO-targeted ubiquitin ligases) that target SUMOylated proteins for proteasomal degradation. The so far identified ULS proteins each contains a really interesting new gene domain with ubiquitin-E3 ligase activity and several SUMO interaction motifs that noncovalently bind SUMO. In order to identify ULS proteins and characterize their substrates, it is important to reconstitute this reaction in vitro. In this chapter, we describe step-by-step protocols for the production and purification of recombinant SUMOylated substrates as well as their in vitro ubiquitylation by ULS proteins.
authors.affiliation: Center for Molecular Medicine Cologne (CMMC), Institute for Genetics, University of Cologne, Cologne, Germany.
authors.forename: Kirstin
authors.initials: K
authors.lastname: Keusekotten
authors.forename: Gerrit J K
authors.initials: GJ
authors.lastname: Praefcke
mesh_terms: D006801:Humans; D046988:Proteasome Endopeptidase Complex; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012568:Schizosaccharomyces / Q000378:metabolism; D025841:Small Ubiquitin-Related Modifier Proteins / Q000378:metabolism*; D058207:Sumoylation; D025801:Ubiquitin / Q000378:metabolism*; D043743:Ubiquitin-Protein Ligase Complexes / Q000378:metabolism; D054875:Ubiquitination
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D025801:Ubiquitin; D043743:Ubiquitin-Protein Ligase Complexes; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease