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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

The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.(A) Cartoon representation of the Ure2p variants used in this study. (B) Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE (C) Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. (D) Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.
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pone.0131789.g004: The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.(A) Cartoon representation of the Ure2p variants used in this study. (B) Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE (C) Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. (D) Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.

Mentions: We previously showed that a truncated form of Sup35p lacking the N-terminal 82 amino acid residues (Sup35Δ1–82) resists proteasomal degradation suggesting a prominent role for Sup35p PrD in recognition and degradation by the 26S proteasome [32]. The results presented in Figs 2 and 3 suggest that Ure2p PrD behaves as a degron that is recognized and engaged by the 26S proteasome. This is supported by the finding that a truncated form of Ure2p lacking its N-terminal domain (Ure2Δ1–93, Fig 4A), used to solve the structure of the compactly folded domain of Ure2p [39], and that is unable to assemble into fibrils (Fig 4B and 4C), fully resists proteasomal degradation (Fig 4D). It should be noted here that a Ure2p variant lacking its prion domain (Ure2Δ2–94) was shown to be unstable in vivo [37]. Nor the reasons behind this instability, nor the protease(s) responsible for Ure2Δ2–94 degradation in vivo were identified [37]. Therefore, Ure2Δ1–93 (or UreΔ2–94) may very well be highly resistant to proteasomal degradation, as shown in Fig 4D, but not to other proteolytic machineries.


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)

The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.(A) Cartoon representation of the Ure2p variants used in this study. (B) Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE (C) Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. (D) Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4482727&req=5

pone.0131789.g004: The deletion of residues 3–25 prevents the proteasomal degradation of Ure2p.(A) Cartoon representation of the Ure2p variants used in this study. (B) Time-courses of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assembly at 6°C, monitored by thioflavin T binding (a.u., arbitrary units). Data represent the mean of three independent experiments ± SE (C) Negative-stained electron micrographs of Ure2p, Ure2Δ3–25 and Ure2Δ1–93 (25 μM) assemblies after 30 days of incubation at 6°C (scale bar: 500 nm). Only amorphous aggregates were detected for Ure2Δ1–93. (D) Purified Ure2p, Ure2Δ3–25 or Ure2Δ1–93 (250 nM) were incubated with or without purified 26S proteasomes (2 nM), as indicated, and in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed from the reaction mix and analyzed by SDS-PAGE and Western blotting using anti- Ure2p antibodies.
Mentions: We previously showed that a truncated form of Sup35p lacking the N-terminal 82 amino acid residues (Sup35Δ1–82) resists proteasomal degradation suggesting a prominent role for Sup35p PrD in recognition and degradation by the 26S proteasome [32]. The results presented in Figs 2 and 3 suggest that Ure2p PrD behaves as a degron that is recognized and engaged by the 26S proteasome. This is supported by the finding that a truncated form of Ure2p lacking its N-terminal domain (Ure2Δ1–93, Fig 4A), used to solve the structure of the compactly folded domain of Ure2p [39], and that is unable to assemble into fibrils (Fig 4B and 4C), fully resists proteasomal degradation (Fig 4D). It should be noted here that a Ure2p variant lacking its prion domain (Ure2Δ2–94) was shown to be unstable in vivo [37]. Nor the reasons behind this instability, nor the protease(s) responsible for Ure2Δ2–94 degradation in vivo were identified [37]. Therefore, Ure2Δ1–93 (or UreΔ2–94) may very well be highly resistant to proteasomal degradation, as shown in Fig 4D, but not to other proteolytic machineries.

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