title: The Proteasome Subunit Rpn8 Interacts with the Small Nucleolar RNA Protein (snoRNP) Assembly Protein Pih1 and Mediates Its Ubiquitin-independent Degradation in Saccharomyces cerevisiae.
abstract: 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.
authors.affiliation: From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada.
authors.forename: Alexandr
authors.initials: A
authors.lastname: Paci
authors.affiliation: From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada.
authors.forename: Peter X H
authors.initials: PX
authors.lastname: Liu
authors.affiliation: From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada.
authors.forename: Lingjie
authors.initials: L
authors.lastname: Zhang
authors.affiliation: From the Department of Biological Sciences, University of Toronto, Toronto, Ontario M1C 1A4, Canada rzhao@utsc.utoronto.ca.
authors.forename: Rongmin
authors.initials: R
authors.lastname: Zhao
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D008958:Models, Molecular; D009687:Nuclear Proteins / Q000378:metabolism*; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism*; D011485:Protein Binding; D017510:Protein Folding; D055550:Protein Stability; D059748:Proteolysis; D020537:RNA, Small Nucleolar / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D025801:Ubiquitin / Q000378:metabolism*
chemical_list: D018841:HSP90 Heat-Shock Proteins; D009687:Nuclear Proteins; C497702:PIH1 protein, S cerevisiae; D020537:RNA, Small Nucleolar; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D046988:Proteasome Endopeptidase Complex; C573321:Rpn8 protein, S cerevisiae; D000251:Adenosine Triphosphatases
keywords: R2TP complex; molecular chaperone; proteasome; protein degradation; protein-protein interaction; ribosomal RNA processing (rRNA processing); ubiquitin-independent degradation; ubiquitylation (ubiquitination)