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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 vitro.(A) Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. (B) Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). (C) Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).
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pone.0131789.g002: Soluble Ure2p is a proteasomal substrate in vitro.(A) Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. (B) Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). (C) Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).

Mentions: We previously showed that purified yeast 26S proteasomes were able to degrade soluble native Sup35p in vitro [32]. Proteasomal degradation of Sup35p proceeded sequentially from the N-terminal PrD, generating an array of overlapping amyloidogenic peptides and a C-terminal proteasome-resistant Sup35p fragment spanning residues 83–685 [32]. To determine whether Ure2p is degraded by yeast 26S proteasomes in vitro and compare its proteolytic processing to that of Sup35p, we incubated soluble Ure2p in the presence of purified yeast 26S proteasomes and ATP and in the presence or absence of the proteasome inhibitor MG132 at 30°C. Aliquots were withdrawn at time intervals and analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies. A control reaction where Sup35p was incubated under exactly the same conditions was run in parallel. Fig 2A (upper panel, left) shows that full-length Ure2p is degraded in a time-dependent manner by the 26S proteasome. The proteasome inhibitor MG132 abolished degradation (Fig 2A, upper panel, right). Beside a major proteolytic fragment, denoted Ure2p*, two lower molecular-weight Ure2p fragments persisted several hours after the onset of the reaction (Fig 2A, upper panel, left). Overall, Ure2p degradation is reminiscent of what we observe for Sup35p in the control reaction (Fig 2A, lower panels and as we previously reported, [32]).


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 vitro.(A) Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. (B) Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). (C) Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).
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pone.0131789.g002: Soluble Ure2p is a proteasomal substrate in vitro.(A) Purified 26S proteasomes (2 nM) were mixed with purified soluble Ure2p (250 nM) (upper panel) or Sup35p (125 nM) (lower panel) in the presence of 2.5 mM ATP, with or without MG132 (100 μM), as indicated. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). At the indicated time points, aliquots were removed and analyzed by SDS-PAGE and Western blotting using anti-Ure2p or anti-Sup35p antibodies. Ure2p* and Sup35p* indicate proteasome-resistant fragments. (B) Purified 26S proteasomes (2 nM) were mixed with purified Ure2p (250 nM) and without or with increasing concentrations of purified Sup35p (250 nM to 1 μM), in the presence of 2.5 mM ATP. Reaction mixes were incubated and analyzed as described in (A). (C) Purified 26S proteasomes (2 nM) were mixed with purified Sup35p (125 nM) and without or with increasing concentrations of purified Ure2p (250 nM to 500 nM), in the presence of 2.5 mM ATP. The reaction mixes were incubated at 30°C under mild agitation (<300 rpm). Reaction mixes were incubated and analyzed as described in (A).
Mentions: We previously showed that purified yeast 26S proteasomes were able to degrade soluble native Sup35p in vitro [32]. Proteasomal degradation of Sup35p proceeded sequentially from the N-terminal PrD, generating an array of overlapping amyloidogenic peptides and a C-terminal proteasome-resistant Sup35p fragment spanning residues 83–685 [32]. To determine whether Ure2p is degraded by yeast 26S proteasomes in vitro and compare its proteolytic processing to that of Sup35p, we incubated soluble Ure2p in the presence of purified yeast 26S proteasomes and ATP and in the presence or absence of the proteasome inhibitor MG132 at 30°C. Aliquots were withdrawn at time intervals and analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies. A control reaction where Sup35p was incubated under exactly the same conditions was run in parallel. Fig 2A (upper panel, left) shows that full-length Ure2p is degraded in a time-dependent manner by the 26S proteasome. The proteasome inhibitor MG132 abolished degradation (Fig 2A, upper panel, right). Beside a major proteolytic fragment, denoted Ure2p*, two lower molecular-weight Ure2p fragments persisted several hours after the onset of the reaction (Fig 2A, upper panel, left). Overall, Ure2p degradation is reminiscent of what we observe for Sup35p in the control reaction (Fig 2A, lower panels and as we previously reported, [32]).

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