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The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro.

Wang K, Redeker V, Madiona K, Melki R, Kabani M - PLoS ONE (2015)

Bottom Line: Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates.In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation.Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

View Article: PubMed Central - PubMed

Affiliation: Paris-Saclay Institute of Neuroscience, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.

ABSTRACT
Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI+] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

No MeSH data available.


Related in: MedlinePlus

Soluble Ure2p is a proteasomal substrate in vivo.(A) Yeast cells overexpressing Ure2p (see Materials and Methods) were grown to mid-log phase and then treated with DMSO or MG132 (50 μM) for 30 min at 30°C. Protein expression was shut off by the addition of cycloheximide (100 μg.ml-1), and aliquots were withdrawn at the times indicated (in hours). Cell extracts were prepared and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies (upper panel) or Ponceau staining (lower panel). (B) Quantification of western blots such as those shown in (A) was performed using ImageJ. The amount of Ure2p at time zero was set to 100% (data points represent the mean ± SE of independent experiments performed in triplicate).
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pone.0131789.g001: Soluble Ure2p is a proteasomal substrate in vivo.(A) Yeast cells overexpressing Ure2p (see Materials and Methods) were grown to mid-log phase and then treated with DMSO or MG132 (50 μM) for 30 min at 30°C. Protein expression was shut off by the addition of cycloheximide (100 μg.ml-1), and aliquots were withdrawn at the times indicated (in hours). Cell extracts were prepared and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies (upper panel) or Ponceau staining (lower panel). (B) Quantification of western blots such as those shown in (A) was performed using ImageJ. The amount of Ure2p at time zero was set to 100% (data points represent the mean ± SE of independent experiments performed in triplicate).

Mentions: To determine whether Ure2p is a proteasomal substrate in vivo, we assessed its stability in the presence or absence of proteasome inhibitors using cycloheximide-chase experiments [35]. In order to allow the detection of Ure2p in cell extracts by Western blot, these experiments were carried out in a prion-free [ure-0] wild-type yeast strain bearing a plasmid that drives URE2 overexpression under the control of a tet-inducible promoter (see Materials and Methods) [36]. As described previously [37], we found that Ure2p is slowly degraded over time (Fig 1A and 1B). In contrast with what we observed with DMSO, Ure2p degradation was abolished for over 4h in the presence of the proteasome inhibitor MG132 (Fig 1A and 1B). This suggests that the proteasome contributes to soluble Ure2p degradation. We were not able to make similar observations in cells harboring the [URE3] phenotype. Nonetheless, the experiments depicted in Fig 1A and 1B prompted us to investigate the proteasomal degradation of Ure2p in well-defined in vitro assays.


The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro.

Wang K, Redeker V, Madiona K, Melki R, Kabani M - PLoS ONE (2015)

Soluble Ure2p is a proteasomal substrate in vivo.(A) Yeast cells overexpressing Ure2p (see Materials and Methods) were grown to mid-log phase and then treated with DMSO or MG132 (50 μM) for 30 min at 30°C. Protein expression was shut off by the addition of cycloheximide (100 μg.ml-1), and aliquots were withdrawn at the times indicated (in hours). Cell extracts were prepared and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies (upper panel) or Ponceau staining (lower panel). (B) Quantification of western blots such as those shown in (A) was performed using ImageJ. The amount of Ure2p at time zero was set to 100% (data points represent the mean ± SE of independent experiments performed in triplicate).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482727&req=5

pone.0131789.g001: Soluble Ure2p is a proteasomal substrate in vivo.(A) Yeast cells overexpressing Ure2p (see Materials and Methods) were grown to mid-log phase and then treated with DMSO or MG132 (50 μM) for 30 min at 30°C. Protein expression was shut off by the addition of cycloheximide (100 μg.ml-1), and aliquots were withdrawn at the times indicated (in hours). Cell extracts were prepared and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies (upper panel) or Ponceau staining (lower panel). (B) Quantification of western blots such as those shown in (A) was performed using ImageJ. The amount of Ure2p at time zero was set to 100% (data points represent the mean ± SE of independent experiments performed in triplicate).
Mentions: To determine whether Ure2p is a proteasomal substrate in vivo, we assessed its stability in the presence or absence of proteasome inhibitors using cycloheximide-chase experiments [35]. In order to allow the detection of Ure2p in cell extracts by Western blot, these experiments were carried out in a prion-free [ure-0] wild-type yeast strain bearing a plasmid that drives URE2 overexpression under the control of a tet-inducible promoter (see Materials and Methods) [36]. As described previously [37], we found that Ure2p is slowly degraded over time (Fig 1A and 1B). In contrast with what we observed with DMSO, Ure2p degradation was abolished for over 4h in the presence of the proteasome inhibitor MG132 (Fig 1A and 1B). This suggests that the proteasome contributes to soluble Ure2p degradation. We were not able to make similar observations in cells harboring the [URE3] phenotype. Nonetheless, the experiments depicted in Fig 1A and 1B prompted us to investigate the proteasomal degradation of Ure2p in well-defined in vitro assays.

Bottom Line: Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates.In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation.Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

View Article: PubMed Central - PubMed

Affiliation: Paris-Saclay Institute of Neuroscience, Centre National de la Recherche Scientifique, Gif-sur-Yvette, France.

ABSTRACT
Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI+] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI+] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.

No MeSH data available.


Related in: MedlinePlus