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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

Fibrillar Ure2p is not degraded by the 26S proteasome.(A) Ure2p fibrils (2 μg) were incubated at 30°C under mild agitation (<300 rpm) in the presence of 2.5 mM ATP, with or without 26S proteasomes (1.6 μg), as indicated. Aliquots were removed at time intervals and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies. (B) Ure2p fibrils were incubated with or without 26S proteasomes and MG132 (100 μM), as indicated in (A). At the indicated time points, aliquots were diluted four-fold in proteasome assay buffer and then filtered through a cellulose acetate membrane (0.2 μm pore size) using a slot-blot vacuum manifold. Each well was then washed twice with 200 μL assay buffer and the membranes were immunostained with anti-Ure2p antibodies. (C) Ure2p fibrils were incubated with or without 26S proteasomes, as indicated in (A). Aliquots were withdrawn at time intervals and analyzed by SDD-AGE followed by immunoblotting with anti-Ure2p antibodies (D) Sup35p fibrils (1 μg) were mixed with purified 26S proteasomes (0.4 μg) in the presence of 2.5 mM ATP. The reactions mixes were incubated at 30°C under mild agitation (<300 rpm), and at the indicated time points, aliquots were removed and analyzed by SDS-PAGE followed by Western blotting using anti-Sup35p antibodies.
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pone.0131789.g005: Fibrillar Ure2p is not degraded by the 26S proteasome.(A) Ure2p fibrils (2 μg) were incubated at 30°C under mild agitation (<300 rpm) in the presence of 2.5 mM ATP, with or without 26S proteasomes (1.6 μg), as indicated. Aliquots were removed at time intervals and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies. (B) Ure2p fibrils were incubated with or without 26S proteasomes and MG132 (100 μM), as indicated in (A). At the indicated time points, aliquots were diluted four-fold in proteasome assay buffer and then filtered through a cellulose acetate membrane (0.2 μm pore size) using a slot-blot vacuum manifold. Each well was then washed twice with 200 μL assay buffer and the membranes were immunostained with anti-Ure2p antibodies. (C) Ure2p fibrils were incubated with or without 26S proteasomes, as indicated in (A). Aliquots were withdrawn at time intervals and analyzed by SDD-AGE followed by immunoblotting with anti-Ure2p antibodies (D) Sup35p fibrils (1 μg) were mixed with purified 26S proteasomes (0.4 μg) in the presence of 2.5 mM ATP. The reactions mixes were incubated at 30°C under mild agitation (<300 rpm), and at the indicated time points, aliquots were removed and analyzed by SDS-PAGE followed by Western blotting using anti-Sup35p antibodies.

Mentions: We previously demonstrated that the 26S proteasome degrades Sup35p fibrils in vitro, thereby abolishing their infectivity in protein transformation experiments [32]. To determine whether Ure2p fibrils are degraded by the proteasome, Ure2p fibrils were incubated at 30°C with or without 26S proteasomes in the presence of ATP under mild agitation and aliquots, withdrawn at the indicated time, were immunoblotted using anti-Ure2p antibodies after SDS-PAGE or trapping on cellulose acetate membranes [41]. The results presented in Fig 5A and 5B, clearly indicate that Ure2p fibrils were not degraded by the 26S proteasomes under our experimental conditions as the intensity of Ure2p band on the SDS-PAGE and the filter trap remained unchanged upon incubation for 6h in the presence of 26S proteasomes. To strengthen this observation and rule out possible proteasome-mediated fibrils remodeling, the integrity of preformed fibrils incubated for up to 6h with the 26S proteasomes and ATP at 30°C was assessed by SDD-AGE [32, 42]. Fig 5C shows that the size distribution of Ure2p fibrils is unaffected by their incubation with the 26S proteasome, and that the intensity of the band remained unchanged. We conclude from these observations that the 26S proteasome neither remodels nor degrades preformed Ure2p fibrils. A control reaction ran in parallel, shows that preformed Sup35p fibrils are degraded by the 26S proteasome (Fig 5D), as described previously [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)

Fibrillar Ure2p is not degraded by the 26S proteasome.(A) Ure2p fibrils (2 μg) were incubated at 30°C under mild agitation (<300 rpm) in the presence of 2.5 mM ATP, with or without 26S proteasomes (1.6 μg), as indicated. Aliquots were removed at time intervals and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies. (B) Ure2p fibrils were incubated with or without 26S proteasomes and MG132 (100 μM), as indicated in (A). At the indicated time points, aliquots were diluted four-fold in proteasome assay buffer and then filtered through a cellulose acetate membrane (0.2 μm pore size) using a slot-blot vacuum manifold. Each well was then washed twice with 200 μL assay buffer and the membranes were immunostained with anti-Ure2p antibodies. (C) Ure2p fibrils were incubated with or without 26S proteasomes, as indicated in (A). Aliquots were withdrawn at time intervals and analyzed by SDD-AGE followed by immunoblotting with anti-Ure2p antibodies (D) Sup35p fibrils (1 μg) were mixed with purified 26S proteasomes (0.4 μg) in the presence of 2.5 mM ATP. The reactions mixes were incubated at 30°C under mild agitation (<300 rpm), and at the indicated time points, aliquots were removed and analyzed by SDS-PAGE followed by Western blotting using anti-Sup35p antibodies.
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pone.0131789.g005: Fibrillar Ure2p is not degraded by the 26S proteasome.(A) Ure2p fibrils (2 μg) were incubated at 30°C under mild agitation (<300 rpm) in the presence of 2.5 mM ATP, with or without 26S proteasomes (1.6 μg), as indicated. Aliquots were removed at time intervals and analyzed by SDS-PAGE followed by Western blotting using anti-Ure2p antibodies. (B) Ure2p fibrils were incubated with or without 26S proteasomes and MG132 (100 μM), as indicated in (A). At the indicated time points, aliquots were diluted four-fold in proteasome assay buffer and then filtered through a cellulose acetate membrane (0.2 μm pore size) using a slot-blot vacuum manifold. Each well was then washed twice with 200 μL assay buffer and the membranes were immunostained with anti-Ure2p antibodies. (C) Ure2p fibrils were incubated with or without 26S proteasomes, as indicated in (A). Aliquots were withdrawn at time intervals and analyzed by SDD-AGE followed by immunoblotting with anti-Ure2p antibodies (D) Sup35p fibrils (1 μg) were mixed with purified 26S proteasomes (0.4 μg) in the presence of 2.5 mM ATP. The reactions mixes were incubated at 30°C under mild agitation (<300 rpm), and at the indicated time points, aliquots were removed and analyzed by SDS-PAGE followed by Western blotting using anti-Sup35p antibodies.
Mentions: We previously demonstrated that the 26S proteasome degrades Sup35p fibrils in vitro, thereby abolishing their infectivity in protein transformation experiments [32]. To determine whether Ure2p fibrils are degraded by the proteasome, Ure2p fibrils were incubated at 30°C with or without 26S proteasomes in the presence of ATP under mild agitation and aliquots, withdrawn at the indicated time, were immunoblotted using anti-Ure2p antibodies after SDS-PAGE or trapping on cellulose acetate membranes [41]. The results presented in Fig 5A and 5B, clearly indicate that Ure2p fibrils were not degraded by the 26S proteasomes under our experimental conditions as the intensity of Ure2p band on the SDS-PAGE and the filter trap remained unchanged upon incubation for 6h in the presence of 26S proteasomes. To strengthen this observation and rule out possible proteasome-mediated fibrils remodeling, the integrity of preformed fibrils incubated for up to 6h with the 26S proteasomes and ATP at 30°C was assessed by SDD-AGE [32, 42]. Fig 5C shows that the size distribution of Ure2p fibrils is unaffected by their incubation with the 26S proteasome, and that the intensity of the band remained unchanged. We conclude from these observations that the 26S proteasome neither remodels nor degrades preformed Ure2p fibrils. A control reaction ran in parallel, shows that preformed Sup35p fibrils are degraded by the 26S proteasome (Fig 5D), as described previously [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