The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry Vol. 291; no. 22; pp. 11761 - 11775
• Main Authors: Paci, Alexandr, Liu, Peter X.H., Zhang, Lingjie, Zhao, Rongmin
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 27.05.2016; American Society for Biochemistry and Molecular Biology
• Subjects: Adenosine Triphosphatases - metabolism; HSP90 Heat-Shock Proteins - metabolism; Models, Molecular; molecular chaperone; Nuclear Proteins - metabolism; proteasome; Proteasome Endopeptidase Complex - metabolism; Protein Binding; protein degradation; Protein Folding; Protein Stability; Protein Synthesis and Degradation; protein-protein interaction; Proteolysis; R2TP complex; ribosomal RNA processing (rRNA processing); RNA, Small Nucleolar - metabolism; Saccharomyces cerevisiae - growth & development; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins - metabolism; Ubiquitin - metabolism; ubiquitin-independent degradation; ubiquitylation (ubiquitination); proteasome; ubiquitylation (ubiquitination); molecular chaperone; ribosomal RNA processing (rRNA processing); R2TP complex; protein-protein interaction; ubiquitin-independent degradation; protein degradation
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M115.702043

Pih1 is a scaffold protein of the Rvb1-Rvb2-Tah1-Pih1 (R2TP) protein complex, which is conserved in fungi and animals. The chaperone-like activity of the R2TP complex has been implicated in the assembly of multiple protein complexes, such as the small nucleolar RNA protein complex. However, the mechanism of the R2TP complex activity in vivo and the assembly of the complex itself are still largely unknown. Pih1 is an unstable protein and tends to aggregate when expressed alone. The C-terminal fragment of Pih1 contains multiple destabilization factors and acts as a degron when fused to other proteins. In this study, we investigated Pih1 interactors and identified a specific interaction between Pih1 and the proteasome subunit Rpn8 in yeast Saccharomyces cerevisiae when HSP90 co-chaperone Tah1 is depleted. By analyzing truncation mutants, we identified that the C-terminal 30 amino acids of Rpn8 are sufficient for the binding to Pih1 C terminus. With in vitro and in vivo degradation assays, we showed that the Pih1 C-terminal fragment Pih1(282–344) is able to induce a ubiquitin-independent degradation of GFP. Additionally, we demonstrated that truncation of the Rpn8 C-terminal disordered region does not affect proteasome assembly but specifically inhibits the degradation of the GFP-Pih1(282–344) fusion protein in vivo and Pih1 in vitro. We propose that Pih1 is a ubiquitin-independent proteasome substrate, and the direct interaction with Rpn8 C terminus mediates its proteasomal degradation.