Assembly of the Yeast Prion Ure2p into Protein Fibrils

Abstract

The [URE3] phenotype in Saccharomyces cerevisiaepropagates by a prion mechanism, involving the aggregation of the normally soluble and highly helical protein Ure2. Previous data have shown that the protein spontaneously forms in vitro long, straight, insoluble fibrils at neutral pH that are similar to amyloids in that they bind Congo red and show green-yellow birefringence and have an increased resistance to proteolysis. These fibrils are not amyloids as they are devoid of a cross-␤ core. Here we further document the mechanism of assembly of Ure2p into fibrils. The critical concentration for Ure2p assembly is measured, and the minimal size of the nuclei that are the precursors of Ure2p fibrils is determined. Our data indicate that the assembly process is irreversible. As a consequence, the critical concentration is very low. By analyzing the elongation rates of preformed fibrils and combining the results with singlefiber imaging experiments of a variant Ure2p labeled by fluorescent dyes, we reveal the polarity of the fibrils and differences in the elongation rates at their ends. These results bring novel insight in the process of Ure2p assembly into fibrils and the mechanism of propagation of yeast prions.Creutzfeldt-Jacob disease, bovine spongiform encephalopathy, and sheep scrapie are transmissible spongiform encephalopathies. They are believed to be caused by a constitutive protein called prion protein (PrP) in an altered conformation (1). This rare form of the protein is thought to convert the normal form to the infectious conformation in a catalytic manner (for review, see Ref.2). The molecular events at the origin of propagation are not yet elucidated. In the lower eukaryotes Saccharomyces cerevisiae and Podospora anserina prion proteins have been identified taking advantage of traits they confer (3-7). These proteins are harmless for humans and constitute valuable model systems to characterize prion propagation.The [URE3] phenotype in S. cerevisiae is caused by a disorder in a signal transduction cascade that regulates nitrogen catabolism (8). Yeast cells exhibiting the [URE3] phenotype have the ability to take up ureidosuccinate, the structural analogue of allantoate, a poor nitrogen source in the presence of a good nitrogen source such as ammonium ions. This is believed to be because of the loss of the function of the protein determinant of [URE3], Ure2p, whose function is unknown although reported to be involved in yeast heavy metal and strong oxidant detoxification (9). Importantly, Ure2p [URE3] promotes the conversion of the newly synthesized active Ure2p into an inactive form. Thus, the Ure2p [URE3] state is heritable, independent of any nucleic acid (10).Native soluble Ure2p assembles in vitro into fibrils that exhibit the characteristics of amyloids in that they are about 20 nm wide and more than 1 m long, bind thioflavin T and Congo Red, show yellow-green birefringence in polarized light upon Congo Red binding, and exhibit an increased resistance to proteolysis (11-13). The assembly reaction fo...

Molecular Chaperones and the Assembly of the Prion Ure2p in Vitro

“…This indicates that five Ure2p molecules are sequestered in an assembly-incompetent state by each molecule of Ssa1p. We previously showed that hexameric Ure2p is the precursor of the fibrillar form of the protein (27,33). Thus, our results strongly suggest that Ssa1p binds to the hexameric form of Ure2p.…”

“…Because Ure2p assembly into protein fibrils is irreversible (27) if Ure2p fibrils were indeed severed by Hsp104p, one would expect to generate shorter fibrils, but the overall amount of fibrils should not change. Fibrillar material should be, therefore, detected trapped to the cellulose acetate filters.…”

“…Expression and Purification of Ure2p-Recombinant fulllength Ure2p, Ure2p-(94 -354), and the variants Ure2pC355 and Ure2p-(94 -354)C355 were produced as previously described (4,6,27). Protein concentrations were determined spectrophotometrically (HP 8453 diode array spectrophotometer, Hewlett-Packard) using an extinction coefficient of 0.67 mg⅐cm Ϫ2 at 280 nm and molecular masses of 40.2 and 30 kDa for full-length Ure2p and Ure2p-(94 -354), respectively.…”

The yeast prion protein Ure2: Structure, function and folding

“…Another commonly described feature is that the length of the lag time decreases with increasing protein concentration in an approximately exponential relationship. In fact, for Ure2 [36,102] and also for Sup35 [103], the relationship between increasing protein concentration and decreasing lag time is linear. This may reflect a variation in mechanism, the oligomeric nature of the native state, or simply the choice of reaction conditions [36,101,103].…”

Formation and participation of nano-amyloids in pathogenesis of Alzheimer's disease and other amyloidogenic diseases

“…На основании этого можно предположить, что размер критического ядра для образования наноамилоидной формы Аb будет больше четырёх или семи. Для белка Ure2p было найдено, что размер критического ядра состоит из шести цепей, варьируя лаг-период за счет изменения концентрации белка [69]. Из-за сложности задачи эти данные не были подтверждены теоретическими расчётами.…”