Genome-wide analysis identifies MYND-domain protein Mub1 as an essential factor for Rpn4 ubiquitylation.
The proteasome homeostasis in Saccharomyces cerevisiae is regulated by a negative feedback circuit in which the Rpn4 transcription factor upregulates the proteasome genes and is rapidly degraded by the proteasome. Previous work has identified Ubr2 and Rad6 as the cognate E3 and E2 enzymes for Rpn4 ubiquitylation. However, our recent attempts to ubiquitylate Rpn4 using purified Ubr2 and Rad6 proteins in a reconstitution system have been unsuccessful, suggesting that an additional factor is required for Rpn4 ubiquitylation. Here, we screened the entire collection of the single-gene-deletion yeast mutants generated by the Saccharomyces Genome Deletion Project and identified the mub1Delta mutant defective in ubiquitin-dependent degradation of Rpn4. An in vitro reconstitution ubiquitylation assay confirms that Mub1 is the missing factor for Rpn4 ubiquitylation. We further show that Mub1 directly interacts with Ubr2 and Rpn4. The MYND domain of Mub1 may play an important role in Rpn4 ubiquitylation. Interestingly, Mub1 itself is a short-lived protein and its degradation is dependent on the Ubr2/Rad6 ubiquitin ligase. Together, these data suggest that Mub1 and Ubr2 cooperate to transfer ubiquitin to Rpn4 from Rad6 and that Mub1 may switch from a partner to a substrate of the Ubr2/Rad6 ubiquitin ligase.
Ju
Donghong
D
Barbara Ann Karmanos Cancer Institute, Wayne State University School of Medicine, 110 E. Warren Ave., Detroit, MI 48201, USA.
Wang
Xiaogang
X
Xu
Haiming
H
Xie
Youming
Y
D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D016681:Genome, Fungal / Q000235:genetics*; D008954:Models, Biological; D009154:Mutation / Q000235:genetics; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases / Q000378:metabolism; D054875:Ubiquitination
D002352:Carrier Proteins; D004268:DNA-Binding Proteins; C526592:Mub1 protein, S cerevisiae; 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