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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

Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also S1 Table). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. (Inset) Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.
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pone.0131789.g003: Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also S1 Table). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. (Inset) Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.

Mentions: We next identified the proteolytic products generated upon soluble Ure2p degradation by the 26S proteasome. Ure2p was incubated alone or in the presence of 26S proteasomes for up to 3 h at 30°C, and the reaction mixtures analyzed by western blot (Fig 3, inset) and nanoLC-LTQ-Orbitrap mass spectrometry. The degradation of Ure2p by the 26S proteasome generated 94 different ~10- to 30-amino-acid-long peptides, spanning the first 103 residues of the protein (Fig 3, S1 Table). None of these peptides was identified in the control reactions without 26S proteasomes, indicating they correspond to specific products of proteasome-dependent Ure2p proteolysis. Most peptides were generated within 1 h of incubation, while others were detected after 2 h or 3 h of incubation (Fig 3, S1 Table). This time-dependent mass-spectrometry analysis suggests that Ure2p degradation by the proteasome proceeds sequentially from the N-terminal end of the protein towards the C-terminus (Fig 3, S1 Table). No peptides spanning residues 103 to 354 were identified in our mass spectrometry analysis within the time frame of the experiment. The proteasomal degradation pattern of Ure2p we observed (Fig 2A, upper panel left and Fig 3, inset) is thus due to processive cleavage of Ure2p N-terminal PrD. The compactly folded globular C-terminal domain of Ure2p [38] appeared to resist proteasomal degradation. Thus, the proteasomal degradation of soluble Ure2p in vitro resembles that of Sup35p [32]. Indeed, both proteins are sequentially degraded via their intrinsically disordered N-terminal PrDs, yielding amyloidogenic peptides and proteasome-resistant fragments encompassing their folded and functional C-terminal domains (Figs 2and 3) [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)

Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also S1 Table). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. (Inset) Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482727&req=5

pone.0131789.g003: Ure2p degradation by the 26S proteasome yields ~10–30 aminoacids-long peptides originating from the N-terminal end of the protein.Purified soluble Ure2p (1 μg) was incubated without or with purified 26S proteasome (0.4 μg) in the presence of 2.5 mM ATP at 30°C under mild agitation (<300 rpm) for 0, 1, 2 or 3 h. Peptides produced during the incubation were identified by nanoLC-LTQ-Orbitrap mass spectrometry (see also S1 Table). These peptides were not produced when Ure2p was incubated alone (data not shown). The color code reflects the time point at which each individual peptide was first detected, as indicated. (Inset) Aliquots from the reaction mixes were analyzed by SDS-PAGE and Western blot using anti-Ure2p antibodies.
Mentions: We next identified the proteolytic products generated upon soluble Ure2p degradation by the 26S proteasome. Ure2p was incubated alone or in the presence of 26S proteasomes for up to 3 h at 30°C, and the reaction mixtures analyzed by western blot (Fig 3, inset) and nanoLC-LTQ-Orbitrap mass spectrometry. The degradation of Ure2p by the 26S proteasome generated 94 different ~10- to 30-amino-acid-long peptides, spanning the first 103 residues of the protein (Fig 3, S1 Table). None of these peptides was identified in the control reactions without 26S proteasomes, indicating they correspond to specific products of proteasome-dependent Ure2p proteolysis. Most peptides were generated within 1 h of incubation, while others were detected after 2 h or 3 h of incubation (Fig 3, S1 Table). This time-dependent mass-spectrometry analysis suggests that Ure2p degradation by the proteasome proceeds sequentially from the N-terminal end of the protein towards the C-terminus (Fig 3, S1 Table). No peptides spanning residues 103 to 354 were identified in our mass spectrometry analysis within the time frame of the experiment. The proteasomal degradation pattern of Ure2p we observed (Fig 2A, upper panel left and Fig 3, inset) is thus due to processive cleavage of Ure2p N-terminal PrD. The compactly folded globular C-terminal domain of Ure2p [38] appeared to resist proteasomal degradation. Thus, the proteasomal degradation of soluble Ure2p in vitro resembles that of Sup35p [32]. Indeed, both proteins are sequentially degraded via their intrinsically disordered N-terminal PrDs, yielding amyloidogenic peptides and proteasome-resistant fragments encompassing their folded and functional C-terminal domains (Figs 2and 3) [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