The Stability of the Small Nucleolar Ribonucleoprotein (snoRNP) Assembly Protein Pih1 in Saccharomyces cerevisiae Is Modulated by Its C Terminus

Paci, Alexandr; Liu, Xiao Hu; Huang, Hao; Lim, Abelyn; Houry, Walid; Zhao, Rongmin

doi:10.1074/jbc.m112.408849

Cited by 27 publications

(44 citation statements)

References 37 publications

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“…1G, GFP-Pih1(282-344) is degraded quickly in cells treated with DMSO only, whereas its degradation is significantly impaired with the RPN8 gene being repressed. These data suggest that in vivo degradation of both GFP-Pih1(231-344) and GFP-Pih1(282-344) is dependent on the 26S protea- some, and Pih1(282-344) induces a much faster degradation of GFP, also in agreement with our previous observations (8).…”

Section: Pih1 C-terminal Fragment Triggers Proteasome-dependentsupporting

confidence: 82%

“…To construct the plasmid expressing DHFR-CytB-His 6 , the DHFRCytB-His 6 coding sequence in pET22b-UB4DHFR-CytB-His 6 was cleaved with NcoI and HindIII and inserted into pProEXHTb. The plasmids expressing GFP-Pih1(231-344) and GFP-Pih1(282-344) in E. coli and yeast were described previously (8). GFP-Pih1(282-344⌬K) expressed from p415GPD vector was constructed by replacing the Pih1(282-344) coding sequencing (EcoRI/HindIII) with a new EcoRI/HindIII fragment that was synthesized as oligonucleotides and annealed together, in which the codons for lysine were replaced by arginine codons.…”

Section: Methodsmentioning

confidence: 99%

“…Intrinsically disordered regions are important features of a protein, and they often mediate protein-protein interaction or are involved in posttranslational regulation of the protein activities (25). Additionally, we previously showed that the Pih1 extreme C-terminal fragment, Pih1(282-344), contains multiple destabilization elements sufficient to induce fast degradation of well folded proteins, such as GFP, in vivo (8,12). Therefore, Pih1 is a multidomain scaffold protein responsible for binding different interacting partners, whereas the stability and turnover of Pih1 in vivo is probably controlled by a complex mechanism.…”

Section: The R2tpmentioning

confidence: 99%

“…R2TP is a conserved protein complex and has also been identified in mammalian cells (3)(4)(5), fruit fly (6), plasmodium (7), and many other fungi species (8), whereas it is absent in higher plants (9). There are more than 70 different snoRNP complexes that are directly involved in ribosomal RNA processing, a critical step for ribosome assembly that requires a total of more than 200 assembly factors (10).…”

Section: The R2tpmentioning

confidence: 99%

“…In addition to a Pih1 domain and a CS domain, we previously showed that Pih1 also contains two intrinsically disordered regions that are essential for the binding to the Rvb1-Rvb2 AAAϩ family DNA helicases (8). Intrinsically disordered regions are important features of a protein, and they often mediate protein-protein interaction or are involved in posttranslational regulation of the protein activities (25).…”

Section: The R2tpmentioning

confidence: 99%

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

Paci

Liu

Zhang

et al. 2016

Journal of Biological Chemistry

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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. The R2TP2 complex, which is composed of Rvb1, Rvb2, Tah1, and Pih1, was first discovered in baking yeast (1) and is required for box C/D small nucleolar RNA protein (snoRNP) complex assembly and thus for the ribosomal RNA biogenesis (2). R2TP is a conserved protein complex and has also been identified in mammalian cells (3-5), fruit fly (6), plasmodium (7), and many other fungi species (8), whereas it is absent in higher plants (9). There are more than 70 different snoRNP complexes that are directly involved in ribosomal RNA processing, a critical step for ribosome assembly that requires a total of more than 200 assembly factors (10). The assembly of R2TP complex in vivo is also a highly dynamic process and is regulated by the molecular chaperone HSP90 and nutrition status (11). However, the mechanism by which R2TP controls box C/D snoRNP complex assembly is still largely unknown.Pih1, the scaffold protein within the R2TP complex, mediates the interaction between Tah1 and Rvb1-Rvb2 and subsequently controls the biogenesis of box C/D snoRNP (2, 3). Tah1 recruits HSP90 and protects Pih1 from degradation in vivo (12, 13). Pih1 interacts directly with Nop58, one of the core protein subunits in box C/D snoRNP (11) and affects its stability (14). Pih1 also interacts with Rsa1, another snoRNP assembly factor through which the R2TP complex may interact with the snoRNP core protein Snu13 (15). Similar to the role of Tah1 in protecting Pih1, a small protein, Hit1, was also identified to interact with and protect Rsa1, thus revealing a complex...

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Section: Pih1 C-terminal Fragment Triggers Proteasome-dependentsupporting

confidence: 82%

Section: Methodsmentioning

confidence: 99%

Section: The R2tpmentioning

confidence: 99%

Section: The R2tpmentioning

confidence: 99%

Section: The R2tpmentioning

confidence: 99%

See 3 more Smart Citations

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

Paci

Liu

Zhang

et al. 2016

Journal of Biological Chemistry

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The Multiple Functions of the PAQosome: An R2TP- and URI1 Prefoldin-Based Chaperone Complex

Lynham

Houry

2018

Advances in Experimental Medicine and Biology

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The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response

et al. 2021

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Cells respond to protein-damaging (proteotoxic) stress by activation of the Heat Shock Response (HSR). The HSR provides cells with an enhanced ability to endure proteotoxic insults and plays a crucial role in determining subsequent cell death or survival. The HSR is, therefore, a critical factor that influences the toxicity of protein stress. While named for its vital role in the cellular response to heat stress, various components of the HSR system and the molecular chaperone network execute essential physiological functions as well as responses to other diverse toxic insults. The effector molecules of the HSR, the Heat Shock Factors (HSFs) and Heat Shock Proteins (HSPs), are also important regulatory targets in the progression of neurodegenerative diseases and cancers. Modulation of the HSR and/or its extended network have, therefore, become attractive treatment strategies for these diseases. Development of effective therapies will, however, require a detailed understanding of the HSR, important features of which continue to be uncovered and are yet to be completely understood. We review recently described and hallmark mechanistic principles of the HSR, the regulation and functions of HSPs, and contexts in which the HSR is activated and influences cell fate in response to various toxic conditions.

show abstract

The Stability of the Small Nucleolar Ribonucleoprotein (snoRNP) Assembly Protein Pih1 in Saccharomyces cerevisiae Is Modulated by Its C Terminus

Cited by 27 publications

References 37 publications

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

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

The Multiple Functions of the PAQosome: An R2TP- and URI1 Prefoldin-Based Chaperone Complex

The functions and regulation of heat shock proteins; key orchestrators of proteostasis and the heat shock response

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