Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase.
The homeostatic abundance of the proteasome in Saccharomyces cerevisiae is controlled by a feedback circuit in which transcriptional activator Rpn4 up-regulates the proteasome genes and is destroyed by the assembled, active proteasome. Remarkably, the degradation of Rpn4 can be mediated by two independent pathways. One pathway is independent of ubiquitin, whereas the other involves ubiquitination on internal lysines. In the present study, we investigated the mechanism underlying the ubiquitin-dependent degradation of Rpn4. We demonstrated, through in vivo and in vitro assays, that Rpn4 is a physiological substrate of the Ubr2 ubiquitin ligase, which was originally identified as a sequence homolog of Ubr1, the E3 component of the N-end rule pathway. The ubiquitin-conjugating enzyme Rad6, which directly interacts with Ubr2, is also required for the ubiquitin-dependent degradation of Rpn4. Furthermore, we showed that deletion of UBR2 exhibited a strong synthetic growth defect with a mutation in the Rpt1 proteasome subunit when Rpn4 was overexpressed. This study not only identified the ubiquitination apparatus for Rpn4 but also unveiled the first physiological substrate of Ubr2. The biological significance of Ubr2-mediated degradation of Rpn4 is also discussed.
Wang
Li
L
Barbara Ann Karmanos Cancer Institute, Department of Pathology, Wayne State University School of Medicine, 110 Warren Avenue, Detroit, MI 48201, USA.
Mao
Xicheng
X
Ju
Donghong
D
Xie
Youming
Y
D000595:Amino Acid Sequence; D000818:Animals; D004268:DNA-Binding Proteins / Q000502:physiology*; D017353:Gene Deletion; D005982:Glutathione Transferase / Q000378:metabolism; D015151:Immunoblotting; D047468:Immunoprecipitation; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D013997:Time Factors; D014157:Transcription Factors / Q000502:physiology*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; C119430:RPN4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D025801:Ubiquitin; C471606:RAD6 protein, S cerevisiae; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; C496536:Ubr2 protein, S cerevisiae; D005982:Glutathione Transferase; D046988:Proteasome Endopeptidase Complex