RESUMO
Rad23 is a nucleotide-excision repair protein with a previously unknown biochemical function. We determined that yeast and human Rad23 inhibited multi-ubiquitin (Ub) chain formation and the degradation of proteolytic substrates. Significantly, Rad23 could be co-precipitated with a substrate that contained a short multi-Ub chain. The UV sensitivity of rad23Delta was reduced in mutants lacking the E2 enzyme Ubc4, or the multi-Ub chain-promoting factor Ufd2. These studies suggest that the stability of proteolytic substrates is governed by the competing action of multi-Ub chain-promoting and chain-inhibiting factors. The stabilization of DNA repair and stress factors could represent an important biological function of Rad23.
Assuntos
Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Ligases/metabolismo , Proteínas de Saccharomyces cerevisiae , Ubiquitinas/metabolismo , Enzimas Reparadoras do DNA , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Histonas/metabolismo , Humanos , Especificidade por Substrato , Enzimas de Conjugação de UbiquitinaRESUMO
The targeting of proteolytic substrates is accomplished by a family of ubiquitin-conjugating (E2) enzymes and a diverse set of substrate recognition (E3) factors. The ligation of a multiubiquitin chain to a substrate can promote its degradation by the proteasome. However, the mechanism that facilitates the translocation of a substrate to the proteasome in vivo is poorly understood. We have discovered that E2 proteins, including Ubc1, Ubc2, Ubc4, and Ubc5, can interact with the 26S proteasome. Significantly, the interaction between Ubc4 and the proteasome is strongly induced by heat stress, consistent with the requirement for this E2 for efficient stress tolerance. A catalytically inactive derivative of Ubc4 (Ubc4(C86A)), which causes toxicity in yeast cells, can also bind the proteasome. Purified proteasomes can ligate ubiquitin to a test substrate without the addition of exogenous E2 protein, suggesting that the ubiquitylation of some proteolytic substrates might be directly coupled to degradation by the proteasome.
Assuntos
Ligases/metabolismo , Complexos Multienzimáticos , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas de Saccharomyces cerevisiae , Ligação Competitiva , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Temperatura Alta , Ligases/genética , Mutação , Oligopeptídeos , Peptídeo Hidrolases/genética , Peptídeos/genética , Peptídeos/metabolismo , Enzimas de Conjugação de Ubiquitina , Leveduras/genética , Leveduras/metabolismoRESUMO
Rad23 is a member of a novel class of proteins that contain unprocessed ubiquitin-like (UbL) domains. We showed recently that a small fraction of Rad23 can form an interaction with the 26S proteasome. Similarly, a small fraction of Rpn10 is a component of the proteasome. Rpn10 can bind multiubiquitin chains in vitro, but genetic studies have not clarified its role in vivo. We report here that the loss of both Rad23 and Rpn10 results in pleiotropic defects that are not observed in either single mutant. rad23Delta rpn10Delta displays slow growth, cold sensitivity, and a pronounced G2/M phase delay, implicating overlapping roles for Rad23 and Rpn10. Although rad23Delta rpn10Delta displays similar sensitivity to DNA damage as a rad23Delta single mutant, deletion of RAD23 in rpn10Delta significantly increased sensitivity to canavanine, a phenotype associated with an rpn10Delta single mutant. A mutant Rad23 that is unable to bind the proteasome ((DeltaUbL)rad23) does not suppress the canavanine or cold-sensitive defects of rad23Delta rpn10Delta, demonstrating that Rad23/proteasome interaction is related to these effects. Finally, the accumulation of multiubiquitinated proteins and the stabilization of a specific proteolytic substrate in rad23Delta rpn10Delta suggest that proteasome function is altered.
Assuntos
Proteínas de Transporte , Proteínas de Ligação a DNA/metabolismo , Proteínas Fúngicas/metabolismo , Deleção de Genes , Peptídeo Hidrolases/metabolismo , Complexo de Endopeptidases do Proteassoma , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae/metabolismo , Canavanina/farmacologia , Ciclo Celular , Temperatura Baixa , Reparo do DNA/genética , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteínas Fúngicas/genética , Genes Essenciais , Genes Fúngicos/genética , Genes Fúngicos/fisiologia , Teste de Complementação Genética , Peptídeo Hidrolases/genética , Fenótipo , Ligação Proteica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Fatores de Tempo , Ubiquitinas/metabolismo , Raios UltravioletaRESUMO
Rad23 is an evolutionarily conserved protein that is important for nucleotide excision repair. A regulatory role has been proposed for Rad23 because rad23 mutants are sensitive to ultraviolet light but are still capable of incising damaged DNA. Here we show that Rad23 interacts with the 26S proteasome through an amino-terminal ubiquitin-like domain (UbL[R23]). The carboxy terminus of Rad23 binds to the Rad4 DNA repair protein and creates a link between the DNA repair and proteasome pathways. The ultraviolet sensitivity caused by deletion of the UbL(R23) domain may therefore arise from its inability to interact with the proteasome. The fusion proteins glutathione S-transferase (GST)-Rad23 and Rad4-haemagglutinin (HA), and the proteasome subunits Cim3 and Cim5, cofractionate through consecutive chromatography steps. The ubiquitin-like domain of human Rad23 (UbL[HRB]) also interacts with the human proteasome. These results demonstrate that ubiquitin-like domains (UbLs) represent a new class of proteasome-interacting motifs.