Comparative assay of amyloid and prion contents in yeast cells

Amyloid contents were quantitatively assayed in crude yeast lysates treated with thioflavin T that specifically stained amyloid fibrils. We demonstrated that guanidine hydrochloride (GuHCl) treatment and overexpression of Hsp104p chaperone resulted in the elimination of the [PSI ⁺] factor and that the stable decline in amyloid contents followed from the reduced fluorescence intensity (IF) of thioflavin T. Overexpression of the SUP35 gene coding the protein prionizable to [PSI ⁺] results in the generation of [PSI ⁺] clones with increased thioflavin T IF. Transmission of [PSI ⁺] factor by cytoduction in crossings of recipients with low IF was also accompanied by stable IF enhancement in cytoductants, indicating enriched amyloid contents. Thus, in model experiments, modifying the quantity of [PSI ⁺] factor, a yeast prion amyloid, the change in thioflavin T IF corresponds to the expected shift in amyloid contents, the IF shift behaving as a cytoplasm hereditary determinant. It is concluded that thioflavin T IF allows for the quantitative estimation of amyloid contents in cells. The stable mitotic IF shift induced by agents affecting the prion composition permits the quantitative evaluation of prion contribution into amyloid pool. It is possible to assume that the monitoring of thiophlavin T IF shifts under the exposure of agents affecting prion pattern may be helpful to disclose previously unknown prions in yeast strains.     

Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion "strains," [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.     

Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans

How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.     

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.     

Destabilizing interactions among [PSI(+)] and [PIN(+)] yeast prion variants

The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi-] or [pin-], respectively, or the multiple infectious amyloid-like forms called [PSI+] or [PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.     

New aspects of research upon the yeast Saccharomyces cerevisiae [PSI+] prion

One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.     

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.     

Yeast prions form infectious amyloid inclusion bodies in bacteria

ABSTRACT: BACKGROUND: Prions were first identified as infectious proteins associated with fatal brain diseases in mammals. However, fungal prions behave as epigenetic regulators that can alter a range of cellular processes. These proteins propagate as self-perpetuating amyloid aggregates being an example of structural inheritance. The best-characterized examples are the Sup35 and Ure2 yeast proteins, corresponding to [PSI+] and [URE3] phenotypes, respectively. RESULTS: Here we show that both the prion domain of Sup35 (Sup35-NM) and the Ure2 protein (Ure2p) form inclusion bodies (IBs) displaying amyloid-like properties when expressed in bacteria. These intracellular aggregates template the conformational change and promote the aggregation of homologous, but not heterologous, soluble prionogenic molecules. Moreover, in the case of Sup35-NM, purified IBs are able to induce different [PSI+] phenotypes in yeast, indicating that at least a fraction of the protein embedded in these deposits adopts an infectious prion fold. CONCLUSIONS: An important feature of prion inheritance is the existence of strains, which are phenotypic variants encoded by different conformations of the same polypeptide. We show here that the proportion of infected yeast cells displaying strong and weak [PSI+] phenotypes depends on the conditions under which the prionogenic aggregates are formed in E. coli, suggesting that bacterial systems might become useful tools to generate prion strain diversity.     

The Genetic Control of the Formation and Propagation of the [PSI+] Prion of Yeast

It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI+] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learned much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI+] prion.     

Prion diseases of yeast: amyloid structure and biology

Prion "variants" or "strains" are prions with the identical protein sequence, but different characteristics of the prion infection: e.g. different incubation periods for scrapie strains or different phenotype intensities for yeast prion variants. We have shown that infectious amyloids of the yeast prions [PSI+], [URE3] and [PIN+] each have an in-register parallel β-sheet architecture. Moreover, we have pointed out that this amyloid architecture can explain how one protein can faithfully transmit any of several conformations to new protein monomers. This explains how proteins can be genes.     

Nonsense suppression in yeast cells overproducing Sup35 (eRF3) is caused by its non-heritable amyloids

The [PSI+] prion determinant of Saccharomyces cerevisiae causes nonsense suppressor phenotype due to a reduced function of the translation termination factor Sup35 (eRF3) polymerized into amyloid fibrils. Prion state of the Rnq1 protein, [PIN+], is required for the [PSI+] de novo generation but not propagation. Yeast [psi-] [PIN+] cells overproducing Sup35 can exhibit nonsense suppression without generation of a stable [PSI+]. Here, we show that in such cells, most of Sup35 represents amyloid polymers, although the remaining Sup35 monomer is sufficient for normal translation termination. The presence of these polymers strictly depends on [PIN+], suggesting that their maintenance relies on efficient generation de novo rather than inheritance. Sup35 polymers contain Rnq1, confirming a hypothesis that Rnq1 polymers seed Sup35 polymerization. About 10% of cells overproducing Sup35 form colonies on medium selective for suppression, which suggests that the proportion of Sup35 monomers to polymers varies between cells of transformants, allowing selection of cells deficient for soluble Sup35. A hybrid Sup35 with the N-terminal domain replaced for 66 glutamine residues also polymerizes and can cause nonsense suppression when overproduced. The described polymers of these proteins differ from the [PSI+] polymers by poor heritability and very high frequency of the de novo appearance, thus being more similar to amyloids than to prions.     

Cross-talk between RNA and prions

As concepts evolve in mammalian and yeast prion biology, rather preliminary research investigating the interplay between prion and RNA processes are gaining momentum. The yeast prion [PSI+] represents an aggregated state of the translation termination factor Sup35 resulting in the tendency of ribosomes to readthrough stop codons. This "nonsense suppression" activity is investigated for its possible physiological role to engender on Saccharomyces cerevisiae the ability to respond to stress or variable growth conditions and thereby act as a capacitor to evolve. The interaction between prion and RNA is a two way street--the cell may have adopted RNA processes in translation to govern the presence of prions and the [PSI+] prion's nonsense suppressor phenotype may exhibit different growth phenotypes by its control of translation termination. RNA processes in the mammalian cell also effect and are affected by prions.     

Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism

[PSI+] is a protein-based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion-like behaviour of the endogenous Sup35p protein release factor. [PSI+] strains exhibit a marked decrease in translation termination efficiency, which permits decoding of translation termination signals and, presumably, the production of abnormally extended polypeptides. We have examined whether the [PSI+]-induced expression of such an altered proteome might confer some selective growth advantage over [psi-] strains. Although otherwise isogenic [PSI+] and [psi-] strains show no difference in growth rates under normal laboratory conditions, we demonstrate that [PSI+] strains do exhibit enhanced tolerance to heat and chemical stress, compared with [psi-] strains. Moreover, we also show that the prion-like determinant [PSI+] is able to regulate translation termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of translation termination and a loss of the [PSI+] phenotype. We present a model to describe the prion-mediated regulation of translation termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi.     

Molecular basis of a yeast prion species barrier

The yeast [PSI+] factor is inherited by a prion mechanism involving self-propagating Sup35p aggregates. We find that Sup35p prion function is conserved among distantly related yeasts. As with mammalian prions, a species barrier inhibits prion induction between Sup35p from different yeast species. This barrier is faithfully reproduced in vitro where, remarkably, ongoing polymerization of one Sup35p species does not affect conversion of another. Chimeric analysis identifies a short domain sufficient to allow foreign Sup35p to cross this barrier. These observations argue that the species barrier results from specificity in the growing aggregate, mediated by a well-defined epitope on the amyloid surface and, together with our identification of a novel yeast prion domain, show that multiple prion-based heritable states can propagate independently within one cell.     

Self-perpetuating changes in Sup35 protein conformation as a mechanism of heredity in yeast

Recently, a novel mode of inheritance has been described in the yeast Saccharomyces cerevisiae. The mechanism is based on the prion hypothesis, which posits that self-perpetuating changes in the conformation of single protein, PrP, underlie the severe neurodegeneration associated with the transmissible spongiform enchephalopathies in mammals. In yeast, two prions, [URE3] and [PSI+], have been identified, but these factors confer unique phenotypes rather than disease to the organism. In each case, the prion-associated phenotype has been linked to alternative conformations of the Ure2 and Sup35 proteins. Remarkably, Ure2 and Sup35 proteins existing in the alternative conformations have the unique capacity to transmit this physical state to the newly synthesized protein in vivo. Thus, a mechanism exists to ensure replication of the conformational information that underlies protein-only inheritance. We have characterized the mechanism by which Sup35 conformational information is replicated in vitro. The assembly of amyloid fibres by a region of Sup35 encompassing the N-terminal 254 amino acids faithfully recapitulates the in vivo propagation of [PSI+]. Mutations that alter [PSI+] inheritance in vivo change the kinetics of amyloid assembly in vitro in a complementary fashion, and lysates from [PSI+] cells, but not [psi-] cells, accelerate assembly in vitro. Using this system we propose a mechanism by which the alternative conformation of Sup35 is adopted by an unstructured oilgomeric intermediate at the time of assembly.     

Newly identified prions in budding yeast, and their possible functions

Yeast prions are atypical genetic elements that are transmitted as heritable protein conformations. [PSI+], [URE3], and [PIN+] are three well-studied prions in the budding yeast, Saccharomyces cerevisiae. In the last three years, several additional prions have been reported in yeast, including [SWI+], [OCT+], [MCA], [GAR+], [MOT3+], [ISP+], and [NSI+]. The growing number of yeast prions suggests that protein-based inheritance might be a widespread biological phenomenon. In this review, we summarize the characteristics of each prion element, and discuss their potential functional roles in yeast biology.     

Protein inheritance (prions) based on parallel in-register beta-sheet amyloid structures

Most prions (infectious proteins) are self-propagating amyloids (filamentous protein multimers), and have been found in both mammals and fungal species. The prions [URE3] and [PSI+] of yeast are disease agents of Saccharomyces cerevisiae while [Het-s] of Podospora anserina may serve a normal cellular function. The parallel in-register beta-sheet structure shown by prion amyloids makes possible a templating action at the end of filaments which explains the faithful transmission of variant differences in these molecules. This property of self-reproduction, in turn, allows these proteins to act as de facto genes, encoding heritable information.     

PSI+] Prion transmission barriers protect Saccharomyces cerevisiae from infection: intraspecies 'species barriers'

[PSI+] is a prion of Sup35p, an essential translation termination and mRNA turnover factor. The existence of lethal [PSI+] variants, the absence of [PSI+] in wild strains, the mRNA turnover function of the Sup35p prion domain, and the stress reaction to prion infection suggest that [PSI+] is a disease. Nonetheless, others have proposed that [PSI+] and other yeast prions benefit their hosts. We find that wild Saccharomyces cerevisiae strains are polymorphic for the sequence of the prion domain and particularly in the adjacent M domain. Here we establish that these variations within the species produce barriers to prion transmission. The barriers are partially asymmetric in some cases, and evidence for variant specificity in barriers is presented. We propose that, as the PrP 129M/V polymorphism protects people from Creutzfeldt-Jakob disease, the Sup35p polymorphisms were selected to protect yeast cells from prion infection. In one prion incompatibility group, the barrier is due to N109S in the Sup35 prion domain and several changes in the middle (M) domain, with either the single N109S mutation or the group of M changes (without the N109S) producing a barrier. In another, the barrier is due to a large deletion in the repeat domain. All are outside the region previously believed to determine transmission compatibility. [SWI+], a prion of the chromatin remodeling factor Swi1p, was also proposed to benefit its host. We find that none of 70 wild strains carry this prion, suggesting that it is not beneficial.     

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases

The yeast prions [URE3] and [PSI] are not found in wild strains, suggesting they are not an advantage. Prion-forming ability is not conserved, even within Saccharomyces, suggesting it is a disease. Prion domains have non-prion functions, explaining some conservation of sequence. However, in spite of the sequence being constrained in evolution by these non-prion functions, the prion domains vary more rapidly than the remainder of the molecule, and these changes produce a transmission barrier, suggesting that these changes were selected to block prion infection. Yeast prions [PSI] and [URE3] induce a cellular stress response (Hsp104 and Hsp70 induction), suggesting the cells are not happy about being infected. Recently, we showed that the array of [PSI] and [URE3] prions includes a majority of lethal or very toxic variants, a result not expected if either prion were an adaptive cellular response to stress.Key words: [URE3], [PSI⁺], prion, Sup35p, Ure2pfMammalian prions are uniformly fatal, but a lethal yeast prion would not be detected by the usual procedure, which requires growth of a colony under some selective condition. As a result, the prion variants commonly studied are quite mild in their effects. This circumstance has led to the suggestion that yeast prions actually benefit their host. Sup35p, the translation termination subunit whose amyloid becomes the [PSI⁺] prion, is essential for growth and Ure2p, the nitrogen regulation protein whose amyloid constitutes the [URE3] prion, is important for growth, with ure2 mutants showing noticeably slowed growth.When yeast prions were discovered,¹ we assumed they were diseases, by analogy with the mammalian diseases and the many non-prion amyloid diseases. Inactivating the essential Sup35p or the desireable Ure2p did not seem like a useful strategy. While control of either protein''s activity might be advantageous, and Ure2p activity control is the key to regulation of nitrogen catabolism, prion formation is a stochastic process, so it makes control of activity of these proteins random instead of appropriate to the circumstances. The [Het-s] prion changed that picture.² Here was a prion necessary for a normal function, heterokaryon incompatibility, and we suggested that it was the first beneficial prion.³