Dynamic interactions of Sup35p and PrP prion protein domains modulate aggregate nucleation and seeding

Prions are self-propagating infectious protein aggregates of mammals and fungi. The exact mechanism of prion formation is poorly understood. In a recent study, a comparative analysis of the aggregation propensities of chimeric proteins derived from the yeast Sup35p and mouse PrP prion proteins was performed in neuroblastoma cells. The cytosolic expression of the Sup35p domains NM, PrP and fusion proteins thereof revealed that the carboxyterminal domain of PrP (PrP_90–230) mediated aggregate formation, while Sup35p N and M domains modulated aggregate size and frequency when fused to the globular domain of PrP. Here we further present co-aggregation studies of chimeric proteins with cytosolic PrP or a huntingtin fragment with an extended polyglutamine tract. Our studies demonstrate that cross-seeding by heterologous proteins requires sequence similarity with the aggregated protein domain. Taken together, these results demonstrate that nucleation and seeding of prion protein aggregates is strongly influenced by dynamic interactions between the aggregate core forming domain and its flanking regions.Key words: prion, Sup35, huntingtin, cross-seeding, co-aggregation     

Spatial sequestration and oligomer remodeling during de novo [PSI+] formation

Prions are misfolded, aggregated, infectious proteins found in a range of organisms from mammals to bacteria. In mammals, prion formation is difficult to study because misfolding and aggregation take place prior to symptom presentation. The study of the yeast prion [PSI⁺], which is the misfolded infectious form of Sup35p, provides a tractable system to monitor prion formation in real time. Recently, we showed that the de novo formation of prion aggregates begins with the appearance of highly mobile cytoplasmic foci, called early foci, which assemble into larger ring or dot structures. We also observed SDS-resistant oligomers during formation, and lysates containing newly formed oligomers can convert [psi^?] cells to the [PSI⁺] state, suggesting that these oligomers have infectious potential. Here, we further characterize two aspects of prion formation: spatial sequestration of early foci and oligomerization of endogenous Sup35p. Our data provides important insights into the process of prion formation and explores the minimal oligomer requirement for infectivity.     

Oxidative stress conditions increase the frequency of de novo formation of the yeast [PSI+] prion

Prions are self‐perpetuating amyloid protein aggregates which underlie various neurodegenerative diseases in mammals and heritable traits in yeast. The molecular basis of how yeast and mammalian prions form spontaneously into infectious amyloid‐like structures is poorly understood. We have explored the hypothesis that oxidative stress is a general trigger for prion formation using the yeast [PSI⁺] prion, which is the altered conformation of the Sup35 translation termination factor. We show that the frequency of [PSI⁺] prion formation is elevated under conditions of oxidative stress and in mutants lacking key antioxidants. We detect increased oxidation of Sup35 methionine residues in antioxidant mutants and show that overexpression of methionine sulphoxide reductase abrogates both the oxidation of Sup35 and its conversion to the [PSI⁺] prion. [PSI⁺] prion formation is particularly elevated in a mutant lacking the Sod1 Cu,Zn‐superoxide dismutase. We have used fluorescence microscopy to show that the de novo appearance of [PSI⁺] is both rapid and increased in frequency in this mutant. Finally, electron microscopy analysis of native Sup35 reveals that similar fibrillar structures are formed in both the wild‐type and antioxidant mutants. Together, our data indicate that oxidative stress is a general trigger of [PSI⁺] formation, which can be alleviated by antioxidant defenses.     

Sequestration of essential proteins causes prion associated toxicity in yeast

Prions are infectious, aggregated proteins that cause diseases in mammals but are not normally toxic in fungi. Excess Sup35p, an essential yeast protein that can exist as the [ PSI ⁺] prion, inhibits growth of [ PSI ⁺] but not [ psi ^-] cells. This toxicity is rescued by expressing the Sup35Cp domain of Sup35p, which is sufficient for cell viability but not prion propagation. We now show that rescue requires Sup35Cp levels to be proportional to Sup35p overexpression. Overexpression of Sup35p appeared to cause pre-existing [ PSI ⁺] aggregates to coalesce into larger aggregates, but these were not toxic per se because they formed even when Sup35Cp rescued growth. Overexpression of Sup45p, but not other tested essential Sup35p binding partners, caused rescue. Sup45–GFPp formed puncta that colocalized with large [ PSI ⁺] Sup35-RFPp aggregates in cells overexpressing Sup35p, and the frequency of the Sup45–GFPp puncta was reduced by rescuing levels of Sup35Cp. In contrast, [ PSI ⁺] toxicity caused by a high excess of the Sup35p prion domain (Sup35NMp) was rescued by a single copy of Sup35Cp, was not rescued by Sup45p overexpression and was not associated with the appearance of Sup45–GFPp puncta. This suggests [ PSI ⁺] toxicity caused by excess Sup35p verses Sup35NMp is, respectively, through sequestration/inactivation of Sup45p verses Sup35p.     

Evolutionary conservation of prion-forming abilities of the yeast Sup35 protein

Saccharomyces cerevisiae prion [PSI ] is a self-propagating isoform of the eukaryotic release factor eRF3 (Sup35p). Sup35p consists of the evolutionary conserved release factor domain (Sup35C) and two evolutionary variable regions - Sup35N, which serves as a prion-forming domain in S. cerevisiae, and Sup35M. Here, we demonstrate that the prion form of Sup35p is not observed among industrial and natural strains of yeast. Moreover, the prion ([PSI + ]) state of the endogenous S. cerevisiae Sup35p cannot be transmitted to the next generations via heterologous Sup35p or Sup35NM, originating from the distantly related yeast species Pichia methanolica. This suggests the existence of a 'species barrier' in yeast prion conversion. However, the chimeric Sup35p, containing the Sup35NM region of Pichia, can be turned into a prion in S. cerevisiae by overproduction of the identical Pichia Sup35NM. Therefore, the prion-forming potential of Sup35NM is conserved in evolution. In the heterologous system, overproduction of Pichia Sup35p or Sup35NM induced formation of the prion form of S. cerevisiae Sup35p, albeit less efficiently than overproduction of the endogenous Sup35p. This implies that prion induction by protein overproduction does not require strict correspondence of the 'inducer' and 'inducee' sequences, and can overcome the 'species barrier'.     

A role for the proteasome in the turnover of Sup35p and in [PSI+] prion propagation

Yeast prions are superb models for understanding the mechanisms of self‐perpetuating protein aggregates formation. [PSI⁺] stands among the most documented yeast prions and results from self‐assembly of the translation termination factor Sup35p into protein fibrils. A plethora of cellular factors were shown to affect [PSI⁺] formation and propagation. Clearance of Sup35p prion particles is however poorly understood and documented. Here, we investigated the role of the proteasome in the degradation of Sup35p and in [PSI⁺] prion propagation. We found that cells lacking the RPN4 gene, which have reduced intracellular proteasome pools, accumulated Sup35p and have defects in [PSI⁺] formation and propagation. Sup35p is degraded in vitro by the 26S and 20S proteasomes in a ubiquitin‐independent manner, generating an array of amyloidogenic peptides derived from its prion‐domain. We also demonstrate the formation of a proteasome‐resistant fragment spanning residues 83–685 which is devoid of the prion‐domain that is essential for [PSI⁺] propagation. Most important was the finding that the 26S and 20S proteasomes degrade Sup35p fibrils in vitro and abolish their infectivity. Our results point to an overlooked role of the proteasome in clearing toxic protein aggregates, and have important implications for a better understanding of the life cycle of infectious protein assemblies.     

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.     

Yeast Prions: Evolution of the Prion Concept

Prions (infectious proteins) analogous to the scrapie agent have been identified in Saccharomyces cerevisiae and Podospora anserina based on their special genetic characteristics. Each is a protein acting as a gene, much like nucleic acids have been shown to act as enzymes. The [URE3], [PSI⁺], [PIN⁺] and [Het-s] prions are self-propagating amyloids of Ure2p, Sup35p, Rnq1p and the HET-s protein, respectively. The [β] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI⁺] are clearly diseases, while [Het-s] and [β] carry out normal cell functions. Surprisingly, the prion domains of Ure2p and Sup35p can be randomized without loss of ability to become a prion. Thus amino acid content and not sequence determine these prions. Shuffleability also suggests amyloids with a parallel in-register β-sheet structure.Key Words: Ure2, Sup35, Rnq1, HETs, PrP, prion, amyloid     

Prion and Nonprion Amyloids: A Comparison Inspired by the Yeast Sup35 Protein

Yeast prion determinants are related to polymerization of some proteins into amyloid-like fibers. The [PSI⁺] determinant reflects polymerization of the Sup35 protein. Fragmentation of prion polymers by the Hsp104 chaperone represents a key step of the prion replication cycle. The frequency of fragmentation varies depending on the structure of the prion polymers and defines variation in the prion phenotypes, e.g., the suppressor strength of [PSI⁺] and stability of its inheritance. Besides [PSI⁺], overproduction of Sup35 can produce nonheritable phenotypically silent Sup35 amyloid-like polymers. These polymers are fragmented poorly and are present due to efficient seeding with the Rnq1 prion polymers, which occurs by several orders of magnitude more frequently than seeding of [PSI⁺] appearance. Such Sup35 polymers resemble human nonprion amyloids by their nonheritability, mode of appearance and increased size. Thus, a single protein, Sup35, can model both prion and nonprion amyloids. In yeast, these phenomena are distinguished by the frequency of polymer fragmentation. We argue that in mammals the fragmentation frequency also represents a key factor defining differing properties of prion and nonprion amyloids, including infectivity. By analogy with the Rnq1 seeding of nonheritable Sup35 polymers, the “species barrier” in prion transmission may be due to seeding by heterologous prion of nontransmissible type of amyloid, rather than due to the lack of seeding.Key Words: amyloid, prion, Rnq1, Sup35, Ure2, translation termination, yeast     

Growth phase‐dependent changes in the size and infectivity of SDS‐resistant Sup35p assemblies associated with the [PSI+] prion in yeast

The translation termination factor Sup35p can form self‐replicating fibrillar aggregates responsible for the [PSI⁺] prion state. Sup35p aggregation yields detergent‐resistant assemblies detectable on agarose gels under semi‐denaturant conditions and fluorescent puncta within the yeast cytosol when the protein is fused to GFP. It is still unclear whether any of these manifestations of [PSI⁺] truly correspond to the Sup35p assemblies that faithfully transmit the [PSI⁺] prion from mother to daughter cells. The infectious titer of prions in cells can be indirectly assessed by the ability of [PSI⁺] cells lysates to induce the prion state when introduced into naïve cells. Here, we report that the dramatic changes in the size and amounts of SDS‐resistant Sup35p that occur during growth do not correlate with the infectious titer. Our results suggest that fluorescent Sup35‐GFP puncta and detergent‐resistant Sup35p assemblies are good indicators of Sup35p conversion to the prion state but not of infectious particles number.     

Autophagy protects against de novo formation of the [PSI+] prion in yeast

Prions are self-propagating, infectious proteins that underlie several neurodegenerative diseases. The molecular basis underlying their sporadic formation is poorly understood. We show that autophagy protects against de novo formation of [PSI⁺], which is the prion form of the yeast Sup35 translation termination factor. Autophagy is a cellular degradation system, and preventing autophagy by mutating its core components elevates the frequency of spontaneous [PSI⁺] formation. Conversely, increasing autophagic flux by treating cells with the polyamine spermidine suppresses prion formation in mutants that normally show a high frequency of de novo prion formation. Autophagy also protects against the de novo formation of another prion, namely the Rnq1/[PIN⁺] prion, which is not related in sequence to the Sup35/[PSI⁺] prion. We show that growth under anaerobic conditions in the absence of molecular oxygen abrogates Sup35 protein damage and suppresses the high frequency of [PSI⁺] formation in an autophagy mutant. Autophagy therefore normally functions to remove oxidatively damaged Sup35, which accumulates in cells grown under aerobic conditions, but in the absence of autophagy, damaged/misfolded Sup35 undergoes structural transitions favoring its conversion to the propagatable [PSI⁺] form.     

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

Site-specific structural analysis of a yeast prion strain with species-specific seeding activity

Prion proteins misfold and aggregate into multiple infectious strain variants that possess unique abilities to overcome prion species barriers, yet the structural basis for the species-specific infectivities of prion strains is poorly understood. Therefore, we have investigated the site-specific structural properties of a promiscuous chimeric form of the yeast prion Sup35 from Saccharomyces cerevisiae and Candida albicans. The Sup35 chimera forms two strain variants, each of which selectively infect one species but not the other. Importantly, the N-terminal and middle domains of the Sup35 chimera (collectively referred to as Sup35NM) contain two prion recognition elements (one from each species) that regulate the nucleation of each strain. Mutations in either prion recognition element significantly bias nucleation of one strain conformation relative to the other. Herein, we have investigated the folding of each prion recognition element for the serine-to-arginine mutant at residue 17 of Sup35NM chimera known to promote nucleation of C. albicans strain conformation. Using cysteine-specific labeling analysis, we find that residues in the C. albicans prion recognition element are solvent-shielded, while those outside the recognition sequence (including most of those in the S. cerevisiae recognition element) are solvent-exposed. Moreover, we find that proline mutations in the C. albicans recognition sequence disrupt the prion templating activity of this strain conformation. Our structural findings reveal that differential folding of complementary and non-complementary prion recognition elements within the prion amyloid core of the Sup35NM chimera is the structural basis for its species-specific templating activity.     

Glucose availability dictates the export of the soluble and prion forms of Sup35p via periplasmic or extracellular vesicles

The yeast [PSI⁺] prion originates from the self-perpetuating transmissible aggregates of the translation termination factor Sup35p. We previously showed that infectious Sup35p particles are exported outside the cells via extracellular vesicles (EV). This finding suggested a function for EV in the vertical and horizontal transmission of yeast prions. Here we report a significant export of Sup35p within periplasmic vesicles (PV) upon glucose starvation. We show that PV are up to three orders of magnitude more abundant than EV. However, PV and EV are different in terms of size and protein content, and their export is oppositely regulated by glucose availability in the growth medium. Overall, our work suggests that the export of prion particles to both the periplasm and the extracellular space needs to be considered to address the physiological consequences of vesicle-mediated yeast prions trafficking.     

A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.     

A bipolar personality of yeast prion proteins

Prions are infectious, self-propagating protein conformations. [PSI⁺], [RNQ⁺] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1, and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI⁺] prion in [RNQ⁺] or [URE3] strain, but not in non-prion strain. However, [RNQ⁺] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1Δ100, and Lsm4, hamper the maintenance of [PSI⁺]. These findings point to a bipolar activity of [RNQ⁺], [URE3], Rnq1Δ100, and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI⁺] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed.     

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.     

Amyloid-mediated sequestration of essential proteins contributes to mutant huntingtin toxicity in yeast

Background

Polyglutamine expansion is responsible for several neurodegenerative disorders, among which Huntington disease is the most well-known. Studies in the yeast model demonstrated that both aggregation and toxicity of a huntingtin (htt) protein with an expanded polyglutamine region strictly depend on the presence of the prion form of Rnq1 protein ([PIN ⁺]), which has a glutamine/asparagine-rich domain.

Principal Findings

Here, we showed that aggregation and toxicity of mutant htt depended on [PIN ⁺] only quantitatively: the presence of [PIN ⁺] elevated the toxicity and the levels of htt detergent-insoluble polymers. In cells lacking [PIN ⁺], toxicity of mutant htt was due to the polymerization and inactivation of the essential glutamine/asparagine-rich Sup35 protein and related inactivation of another essential protein, Sup45, most probably via its sequestration into Sup35 aggregates. However, inhibition of growth of [PIN ⁺] cells depended on Sup35/Sup45 depletion only partially, suggesting that there are other sources of mutant htt toxicity in yeast.

Conclusions

The obtained data suggest that induced polymerization of essential glutamine/asparagine-rich proteins and related sequestration of other proteins which interact with these polymers represent an essential source of htt toxicity.     

Chimeric Yeast Prions with Unstable Inheritance

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressor effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.