Abstract
The yeast Saccharomyces cerevisiae contains in its proteome at least three prion proteins. These proteins (Ure2p, Sup35p, and Rnq1p) share a set of remarkable properties. In vivo, they form aggregates that self-perpetuate their aggregation. This aggregation is controlled by Hsp104, which plays a major role in the growth and severing of these prions. In vitro, these prion proteins form amyloid fibrils spontaneously. The introduction of such fibrils made from Ure2p or Sup35p into yeast cells leads to the prion phenotypes [URE3] and [PSI], respectively. Previous studies on evolutionary biology of yeast prions have clearly established that [URE3] is not well conserved in the hemiascomycetous yeasts and particularly in S. paradoxus. Here we demonstrated that the S. paradoxus Ure2p is able to form infectious amyloid. These fibrils are more resistant than S. cerevisiae Ure2p fibrils to shear force. The observation, in vivo, of a distinct aggregation pattern for GFP fusions confirms the higher propensity of SpUre2p to form fibrillar structures. Our in vitro and in vivo analysis of aggregation propensity of the S. paradoxus Ure2p provides an explanation for its loss of infective properties and suggests that this protein belongs to the non-prion amyloid world.
Highlights
The yeast prions represent an attractive and valuable model for understanding complex aspects of mammalian prion biology
Purification of the Soluble Form of SpUre2p—After purification of the SpUre2 protein, a single species was observed that migrates with an apparent molecular mass of 42 kDa in SDS-PAGE (Fig. 1A)
We compared the behavior of recombinant same enzymes. The minipRSETa-URE2 (Sp) and ScUre2p by size exclusion chromatography (SEC)
Summary
Materials—GSH, -NADPH, CHP, and glutathione reductase were from Sigma. Construction of Ure2p Expression—The URE2 open reading frame was PCR-amplified from pUHE-URE2 and cloned into pET3a resulting in pET3a-URE2. Digestion was stopped by the addition of electrophoresis sample buffer pH 6.8, containing 4% SDS, 2% mercaptoethanol (v/v), 12% glycerol (w/v), 0.01% Serva Blue G, and phenylmethylsulfonyl fluoride to a final concentration of 1 mM and immediately incubated at 100 °C for 5 min. Assay of Ure GPx Activity during the Time Course of Amyloid-like Fibril Formation—The initial sample was centrifuged at 10,000 ϫ g for 30 min at 4 °C to remove any preexisting aggregates, and 300 l of the supernatant was transferred into each of a series of tubes, one for each time point. The reaction mixture contained 46 M full-length S. paradoxus Ure in 50 mM Tris-HCl buffer, pH 8.4, containing 0.2 M NaCl. The samples were incubated in parallel at a constant temperature of 25 °C with shaking as described previously [16]. The values of GPx activity are mean values of two independent assays
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