title: Rad23 promotes the targeting of proteolytic substrates to the proteasome.
abstract: Rad23 contains a ubiquitin-like domain (UbL(R23)) that interacts with catalytically active proteasomes and two ubiquitin (Ub)-associated (UBA) sequences that bind Ub. The UBA domains can bind Ub in vitro, although the significance of this interaction in vivo is poorly understood. Rad23 can interfere with the assembly of multi-Ub chains in vitro, and high-level expression caused stabilization of proteolytic substrates in vivo. We report here that Rad23 interacts with ubiquitinated cellular proteins through the synergistic action of its UBA domains. Rad23 plays an overlapping role with Rpn10, a proteasome-associated multi-Ub chain binding protein. Mutations in the UBA domains prevent efficient interaction with ubiquitinated proteins and result in poor suppression of the growth and proteolytic defects of a rad23 Delta rpn10 Delta mutant. High-level expression of Rad23 revealed, for the first time, an interaction between ubiquitinated proteins and the proteasome. This increase was not observed in rpn10 Delta mutants, suggesting that Rpn10 participates in the recognition of proteolytic substrates that are delivered by Rad23. Overexpression of UbL(R23) caused stabilization of a model substrate, indicating that an unregulated UbL(R23)-proteasome interaction can interfere with the efficient delivery of proteolytic substrates by Rad23. Because the suppression of a rad23 Delta rpn10 Delta mutant phenotype required both UbL(R23) and UBA domains, our findings support the hypothesis that Rad23 encodes a novel regulatory factor that translocates ubiquitinated substrates to the proteasome.
authors.affiliation: Department of Biochemistry, Robert Wood Johnson Medical School, Piscataway, New Jersey 08854, USA.
authors.forename: Li
authors.initials: L
authors.lastname: Chen
authors.forename: Kiran
authors.initials: K
authors.lastname: Madura
mesh_terms: D001483:Base Sequence; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000235:genetics; D064447:Checkpoint Kinase 2; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004249:DNA Damage; D005656:Fungal Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D009154:Mutation; D046988:Proteasome Endopeptidase Complex; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin / Q000378:metabolism; D015003:Yeasts / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D005656:Fungal Proteins; D009097:Multienzyme Complexes; C122019:RPN10 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D064447:Checkpoint Kinase 2; D017346:Protein Serine-Threonine Kinases; C067022:RAD53 protein, S cerevisiae; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex