Localization of prion-destabilizing mutations in the N-terminal non-prion domain of Rnq1 in Saccharomyces cerevisiae

[PIN⁺] is the prion form of Rnq1 in Saccharomyces cerevisiae and is necessary for the de novo induction of a second prion, [PSI⁺]. The function of Rnq1, however, is little understood. The limited availability of defective rnq1 alleles impedes the study of its structure-function relationship by genetic analysis. In this study, we isolated rnq1 mutants that are defective in the stable maintenance of the [PIN⁺] prion. Since there is no rnq1 phenotype available that is applicable to a direct selection or screening for loss-of-function rnq1 mutants, we took advantage of a prion inhibitory agent, Rnq1Δ100, to develop a color-based genetic screen. Rnq1Δ100 eliminates the [PSI⁺] prion in the [PIN⁺] state but not in the [pin⁻] state. This allows us to find loss-of-[PIN⁺] rnq1 mutants as white [PSI⁺] colonies. Nine rnq1 mutants with single-amino-acid substitutions were defined. These mutations impaired the stable maintenance of [PIN⁺] and, as a consequence, were also partially defective in the de novo induction of [PSI⁺]. Interestingly, eight of the nine alleles were mapped to the N-terminal region of Rnq1, which is known as the non-prion domain preceding the asparagine and glutamine rich prion domain of Rnq1. Notably, overexpression of these rnq1 mutant proteins restored [PIN⁺] prion activity, suggesting that each of the rnq1 mutants was not completely inactive. These findings indicate that the N-terminal non-prion domain of Rnq1 harbors a potent activity to regulate the maintenance of the [PIN⁺] prion.Key words: Rnq1, [PIN⁺], Sup35, [PSI⁺], yeast prion     

Chaperone Proteins Select and Maintain [PIN+] Prion Conformations in Saccharomyces cerevisiae

Prions are proteins that can adopt different infectious conformations known as “strains” or “variants,” each with a distinct, epigenetically inheritable phenotype. Mechanisms by which prion variants are determined remain unclear. Here we use the Saccharomyces cerevisiae prion Rnq1p/[PIN⁺] as a model to investigate the effects of chaperone proteins upon prion variant determination. We show that deletion of specific chaperone genes alters [PIN⁺] variant phenotypes, including [PSI⁺] induction efficiency, Rnq1p aggregate morphology/size and variant dominance. Mating assays demonstrate that gene deletion-induced phenotypic changes are stably inherited in a non-Mendelian manner even after restoration of the deleted gene, confirming that they are due to a bona fide change in the [PIN⁺] variant. Together, our results demonstrate a role for chaperones in regulating the prion variant complement of a cell.     

Increased [PSI+] Appearance by Fusion of Rnq1 with the Prion Domain of Sup35 in Saccharomyces cerevisiae

During propagation, yeast prions show a strict sequence preference that confers the specificity of prion assembly. Although propagations of [PSI⁺] and [RNQ⁺] are independent of each other, the appearance of [PSI⁺] is facilitated by the presence of [RNQ⁺]. To explain the [RNQ⁺] effect on the appearance of [PSI⁺], the cross-seeding model was suggested, in which Rnq1 aggregates act as imperfect templates for Sup35 aggregation. If cross-seeding events take place in the cytoplasm of yeast cells, the collision frequency between Rnq1 aggregates and Sup35 will affect the appearance of [PSI⁺]. In this study, to address whether cross-seeding occurs in vivo, a new [PSI⁺] induction method was developed that exploits a protein fusion between the prion domain of Sup35 (NM) and Rnq1. This fusion protein successfully joins preexisting Rnq1 aggregates, which should result in the localization of NM around the Rnq1 aggregates and hence in an increased collision frequency between NM and Rnq1 aggregates. The appearance of [PSI⁺] could be induced very efficiently, even with a low expression level of the fusion protein. This study supports the occurrence of in vivo cross-seeding between Sup35 and Rnq1 and provides a new tool that can be used to dissect the mechanism of the de novo appearance of prions.Prions were originally defined as proteinaceous infectious particles responsible for transmissible spongiform encephalopathies in mammals (reviewed in reference 23). Since a non-Mendelian genetic element, [URE3], was identified as a yeast prion (37), however, this concept has been expanded to include protein-based genetic elements. In addition to [URE3], there are at least two more proteinaceous genetic elements in Saccharomyces cerevisiae, namely, [PSI⁺] and [RNQ⁺] (20, 22, 28). [Het-s] was also identified as a prion in the filamentous fungus Podospora anserina (2).Despite the absence of any structural and functional homologies between various prion proteins from different organisms, they share a common feature, i.e., prion proteins can adopt two distinct conformational states. One of these, the aggregated prion state, can stimulate the soluble, nonprion conformation to convert into the prion form. Gaining intermolecular β-sheet structures, purified yeast prion proteins aggregate and form amyloid fibers in vitro (8, 12, 28, 32). Protein extract from yeast cells in the prion state can facilitate the in vitro polymerization of soluble prion protein from nonprion cells (21), and amyloid fibers of purified yeast prion proteins can convert the cells into the prion state when introduced into yeast cells, demonstrating the protein-only hypothesis (15, 31). Thus, intracellular prion aggregates are thought to have the same structural basis as amyloid fibers formed in vitro.Yeast prion biology has provided invaluable insights into the prion concept at the molecular level. Because of its experimental convenience, [PSI⁺] has been investigated most intensively among various yeast prions. [PSI⁺] results from the aggregation of Sup35 protein, which is essential for terminating the translation at stop codons. When Sup35 is in the [PSI⁺] aggregated state, ribosomes often fail to release polypeptides at stop codons, causing a non-Mendelian trait which is easily detected by nonsense suppression. ade1 or ade2 nonsense mutants are used as marker genes to determine the [PSI⁺] state. These mutants cannot grow on adenine-deficient medium and form red colonies on medium supplemented with a limiting amount of adenine, such as yeast extract-peptone-dextrose (YPD). ade mutants in the [PSI⁺] state, however, can grow on adenine-deficient medium and form white colonies, as they produce functional Ade1 or Ade2 by virtue of a nonsense mutation readthrough. To sustain propagation, all yeast prions need the disaggregation activity of Hsp104, which can be inhibited by guanidine hydrochloride (GuHCl) (9). Since yeast prions are cured by growth on guanidine-containing medium, prion phenotypes can easily be distinguished from chromosomal suppressor mutants.Sup35 (eRF3) of S. cerevisiae has a prion-determining N-terminal domain (N), a highly charged middle domain (M) that confers solubility on the molecule, and an essential C-terminal domain that binds guanine nucleotides and stimulates the polypeptide release reaction catalyzed by Sup45 (eRF1) (17, 29, 33). The de novo appearance of [PSI⁺] can be induced by overexpression of SUP35 or its prion domain-containing fragments (NM) (6). [PSI⁺] induction, however, can be achieved only in [RNQ⁺] cells that harbor the prion state of the Rnq1 protein (4, 19). Two hypotheses about how [RNQ⁺] can affect the appearance of [PSI⁺] have been suggested. One is an inhibitor titration model that postulates the molecules preventing the aggregation of Sup35 and the recruitment of these inhibitors to Rnq1 aggregates in [RNQ⁺] cells. The other is a cross-seeding model in which Rnq1 aggregates directly catalyze the polymerization of Sup35. In vitro cross-seeding between different amyloidogenic proteins was reported, and Rnq1 amyloid fiber can also act as a seed for Sup35 polymerization in vitro (7, 13). These in vitro data support the possibility of cross-seeding between Rnq1 and Sup35. However, because the milieu of cytoplasm is very different from that of a test tube, whether this cross-seeding really occurs in vivo is still obscure. For this study, we developed a new, robust [PSI⁺] induction method that confirms the cross-seeding events in the cytoplasmic environment.     

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.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.Key words: prion, Sup35p, Ure2p, Rnq1p, [PSI⁺], [URE3], [PIN⁺], amyloid fibrils     

Prion formation and polyglutamine aggregation are controlled by two classes of genes

Prions are self-perpetuating aggregated proteins that are not limited to mammalian systems but also exist in lower eukaryotes including yeast. While much work has focused around chaperones involved in prion maintenance, including Hsp104, little is known about factors involved in the appearance of prions. De novo appearance of the [PSI ⁺] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by transient overexpression of SUP35 in the presence of the prion form of the Rnq1 protein, [PIN ⁺]. When fused to GFP and overexpressed in [ps⁻] [PIN ⁺] cells, Sup35 forms fluorescent rings, and cells with these rings bud off [PSI ⁺] daughters. We investigated the effects of over 400 gene deletions on this de novo induction of [PSI ⁺]. Two classes of gene deletions were identified. Class I deletions (bug1Δ, bem1Δ, arf1Δ, and hog1Δ) reduced the efficiency of [PSI ⁺] induction, but formed rings normally. Class II deletions (las17Δ, vps5Δ, and sac6Δ) inhibited both [PSI⁺] induction and ring formation. Furthermore, class II deletions reduced, while class I deletions enhanced, toxicity associated with the expanded glutamine repeats of the huntingtin protein exon 1 that causes Huntington''s disease. This suggests that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.     

Rnq1 protein protects [<Emphasis Type="Italic">PSI</Emphasis><Superscript>+</Superscript>] prion from effect of the <Emphasis Type="Italic">PNM</Emphasis> mutation

The interaction of [PSI ⁺] and [PIN ⁺] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [PIN ⁺] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [PSI ⁺] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [PSI ⁺] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [PSI ⁺].     

Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI⁺], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN⁺], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN⁺] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI ⁺][PIN ⁺] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI ⁺], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN⁺], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI ⁺]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI⁺] induction in the absence of [PIN ⁺]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI ⁺], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI⁺] aggregates in a [PIN ⁺]-independent pathway.     

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     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.     

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     

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.     

The actin cytoskeletal network plays a role in yeast prion transmission and contributes to prion stability

Chaperone networks are required for the shearing and generation of transmissible propagons from pre-existing prion aggregates. However, other cellular networks needed for maintaining yeast prions are largely uncharacterized. Here, we establish a novel role for actin networks in prion maintenance. The [PIN⁺] prion, also known as [RNQ⁺], exists as stable variants dependent upon the chaperone machinery for the transmission of propagons to daughter cells during cell division and cytoplasmic transfer. Loss of the Hsp104 molecular chaperone leads to the growth of prion particles until they are too large to be transmitted. Here, we isolated a unique [PIN⁺] variant, which is unstable in actin mutants. This prion loss is observed over many generations, and coincides with the detection of both high molecular weight species of Rnq1 and large visible aggregates that are asymmetrically retained during cell division. Our data suggest that the irregular actin networks found in these mutants may influence propagon number by slowly permitting aggregate growth over time, resulting in the generation of nontransmissible large aggregates. Thus, we show the potential contribution of cytoskeletal networks in the transmission of prion propagons, which parallels models that have been proposed for cell-to-cell transmission of small amyloids in neurodegenerative protein aggregation diseases.     

Analysis of [SWI+] formation and propagation events

The budding yeast, Saccharomyces cerevisiae, harbors several prions that are transmitted as altered, heritable protein conformations. [SWI⁺] is one such prion whose determinant is Swi1, a subunit of the evolutionarily conserved chromatin‐remodeling complex SWI/SNF. Despite the importance of Swi1, the molecular events that lead to [SWI⁺] prionogenesis remain poorly understood. In this study, we have constructed floccullin‐promoter‐based URA3 reporters for [SWI⁺] identification. Using these reporters, we show that the spontaneous formation frequency of [SWI⁺] is significantly higher than that of [PSI⁺] (prion form of Sup35). We also show that preexisting [PSI⁺] or [PIN⁺] (prion form of Rnq1), or overproduction of Swi1 prion‐domain (PrD) can considerably promote Swi1 prionogenesis. Moreover, our data suggest a strain‐specific effect of overproduction of Sse1 – a nucleotide exchange factor of the molecular chaperone Hsp70, and its interaction with another molecular chaperone Hsp104 on [SWI⁺] maintenance. Additionally, we show that Swi1 aggregates are initially ring/ribbon‐like then become dot‐like in mature [SWI⁺] cells. In the presence of [PSI⁺] or [PIN⁺], Swi1 ring/ribbon‐like aggregates predominantly colocalize with the Sup35 or Rnq1 aggregates; without a preexisting prion, however, such colocalizations are rarely seen during Swi1‐PrD overproduction‐promoted Swi1 prionogenesis. We have thus demonstrated a complex interacting mechanism of yeast prionogenesis.     

The Schizosaccharomyces pombe Hsp104 Disaggregase Is Unable to Propagate the [PSI +] Prion

The molecular chaperone Hsp104 is a crucial factor in the acquisition of thermotolerance in yeast. Under stress conditions, the disaggregase activity of Hsp104 facilitates the reactivation of misfolded proteins. Hsp104 is also involved in the propagation of fungal prions. For instance, the well-characterized [PSI⁺] prion of Saccharomyces cerevisiae does not propagate in Δhsp104 cells or in cells overexpressing Hsp104. In this study, we characterized the functional homolog of Hsp104 from Schizosaccharomyces pombe (Sp_Hsp104). As its S. cerevisiae counterpart, Sp_hsp104⁺ is heat-inducible and required for thermotolerance in S. pombe. Sp_Hsp104 displays low disaggregase activity and cannot propagate the [PSI⁺] prion in S. cerevisiae. When overexpressed in S. cerevisiae, Sp_Hsp104 confers thermotolerance to Δhsp104 cells and reactivates heat-aggregated proteins. However, overexpression of Sp_Hsp104 does not propagate nor eliminate [PSI⁺]. Strikingly, [PSI⁺] was cured by overexpression of a chimeric chaperone bearing the C-terminal domain (CTD) of the S. cerevisiae Hsp104 protein. Our study demonstrates that the ability to untangle aggregated proteins is conserved between the S. pombe and S. cerevisiae Hsp104 homologs, and points to a role of the CTD in the propagation of the S. cerevisiae [PSI⁺] prion.     

Complex Adaptations Can Drive the Evolution of the Capacitor [PSI +], Even with Realistic Rates of Yeast Sex

The [PSI⁺] prion may enhance evolvability by revealing previously cryptic genetic variation, but it is unclear whether such evolvability properties could be favored by natural selection. Sex inhibits the evolution of other putative evolvability mechanisms, such as mutator alleles. This paper explores whether sex also prevents natural selection from favoring modifier alleles that facilitate [PSI⁺] formation. Sex may permit the spread of “cheater” alleles that acquire the benefits of [PSI⁺] through mating without incurring the cost of producing [PSI⁺] at times when it is not adaptive. Using recent quantitative estimates of the frequency of sex in Saccharomyces paradoxus, we calculate that natural selection for evolvability can drive the evolution of the [PSI⁺] system, so long as yeast populations occasionally require complex adaptations involving synergistic epistasis between two loci. If adaptations are always simple and require substitution at only a single locus, then the [PSI⁺] system is not favored by natural selection. Obligate sex might inhibit the evolution of [PSI⁺]-like systems in other species.     

Prion‐promoted phosphorylation of heterologous amyloid is coupled with ubiquitin‐proteasome system inhibition and toxicity

Many neurodegenerative diseases are associated with conversion of a soluble protein into amyloid deposits, but how this is connected to toxicity remains largely unknown. Here, we explore mechanisms of amyloid associated toxicity using yeast. [PIN⁺], the prion form of the Q/N‐rich Rnq1 protein, was known to enhance aggregation of heterologous proteins, including the overexpressed Q/N‐rich amyloid forming domain of Pin4 (Pin4C), and Pin4C aggregates were known to attract chaperones, including Sis1. Here we show that in [PIN⁺] but not [pin^?] cells, overexpression of Pin4C is deadly and linked to hyperphosphorylation of aggregated Pin4C. Furthermore, Pin4C aggregation, hyperphosphorylation and toxicity are simultaneously reversed by Sis1 overexpression. Toxicity may result from proteasome overload because hyperphosphorylated Pin4C aggregation is associated with reduced degradation of a ubiquitin‐protein degradation reporter. Finally, hyperphosphorylation of endogenous full‐length Pin4 was also facilitated by [PIN⁺], revealing that a prion can regulate post‐translational modification of another protein.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.     

A Promiscuous Prion: Efficient Induction of [URE3] Prion Formation by Heterologous Prion Domains

The [URE3] and [PSI⁺] prions are the infections amyloid forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively. Randomizing the order of the amino acids in the Ure2 and Sup35 prion domains while retaining amino acid composition does not block prion formation, indicating that amino acid composition, not primary sequence, is the predominant feature driving [URE3] and [PSI⁺] formation. Here we show that Ure2p promiscuously interacts with various compositionally similar proteins to influence [URE3] levels. Overexpression of scrambled Ure2p prion domains efficiently increases de novo formation of wild-type [URE3] in vivo. In vitro, amyloid aggregates of the scrambled prion domains efficiently seed wild-type Ure2p amyloid formation, suggesting that the wild-type and scrambled prion domains can directly interact to seed prion formation. To test whether interactions between Ure2p and naturally occurring yeast proteins could similarly affect [URE3] formation, we identified yeast proteins with domains that are compositionally similar to the Ure2p prion domain. Remarkably, all but one of these domains were also able to efficiently increase [URE3] formation. These results suggest that a wide variety of proteins could potentially affect [URE3] formation.AMYLOID fibril formation is associated with numerous human diseases, including Alzheimer''s disease, type II diabetes, and the transmissible spongiform encephalopathies. Yeast prions provide a powerful model system for examining amyloid fibril formation in vivo. [URE3] and [PSI⁺] are the prion forms of the Saccharomyces cerevisiae proteins Ure2p and Sup35p, respectively (Wickner 1994). In both cases, prion formation is thought to result from conversion of the native protein into an inactive amyloid form (Glover et al. 1997; King et al. 1997; Taylor et al. 1999). Both proteins contain an N-terminal glutamine/asparagine (Q/N)-rich prion-forming domain (PFD) and a C-terminal functional domain (Ter-Avanesyan et al. 1993; Ter-Avanesyan et al. 1994; Masison and Wickner 1995; Liebman and Derkatch 1999; Maddelein and Wickner 1999). Sup35p contains an additional highly charged middle domain (M) that is not required either for prion formation or for normal protein function, but stabilizes [PSI⁺] aggregates (Liu et al. 2002).Amyloid fibril formation is thought to occur through a seeded polymerization mechanism. In vitro, amyloid fibril formation from native proteins is generally characterized by a significant lag time, thought to result from the slow rate of formation of amyloid nuclei; addition of a small amount of preformed amyloid aggregates (seeds) eliminates the lag time, resulting in rapid polymerization (Glover et al. 1997; Taylor et al. 1999; Serio et al. 2000).Despite considerable study, the mechanism by which amyloid seeds initially form is unclear. At least some of the amyloid proteins involved in human disease can interact with unrelated amyloidogenic proteins, resulting in cross-seeding and modulation of toxicity. Injecting mice with amyloid-like fibrils formed by a variety of short synthetic peptides promotes amyloid formation by amyloid protein A, a protein whose deposition is found in systemic AA amyloidosis (Johan et al. 1998). In yeast, [PSI⁺] and [PIN⁺], the prion form of the protein Rnq1p (Sondheimer and Lindquist 2000; Derkatch et al. 2001), both promote the aggregation of and increase toxicity of expanded polyglutamine tracts, like those seen in Huntington''s disease (Osherovich and Weissman 2001; Meriin et al. 2002; Derkatch et al. 2004; Gokhale et al. 2005; Duennwald et al. 2006); however, in Drosophila, [PSI⁺] aggregates reduce polyglutamine toxicity (Li et al. 2007). Thus, interactions between heterologous amyloidogenic proteins can influence amyloid formation both positively and negatively in vivo.A variety of interactions have been observed among the yeast prions. Under normal cellular conditions, efficient formation, but not maintenance, of [PSI⁺] requires the presence of [PIN⁺] (Derkatch et al. 2000). Overexpression of various Q/N-rich proteins can effectively substitute for [PIN⁺], allowing [PSI⁺] formation in cells lacking [PIN⁺] (Derkatch et al. 2001; Osherovich and Weissman 2001). In vitro and in vivo evidence suggest that the ability of [PIN⁺] to facilitate [PSI⁺] formation is the result of a direct interaction between Rnq1p aggregates and Sup35p (Derkatch et al. 2004; Bardill and True 2009; Choe et al. 2009). [PIN⁺] also increases the frequency of [URE3] formation, while [PSI⁺] inhibits [URE3] formation (Bradley et al. 2002; Schwimmer and Masison 2002).It is unclear whether the ability of Ure2p, Sup35p, and Rnq1p to cross-react is an intrinsic feature of all similar amyloidogenic proteins, or whether it has specifically evolved to regulate prion formation. There is debate as to whether yeast prion formation is a beneficial phenomenon, allowing for regulation of the activity of the prion protein (True and Lindquist 2000; True et al. 2004), or a deleterious event analogous to human amyloid disease (Nakayashiki et al. 2005). Either way, it is likely that interactions between the yeast prion proteins have specifically evolved, either to minimize the detrimental effects of amyloid formation or to regulate beneficial amyloid formation.For both Ure2p and Sup35p, the amino acid composition of the PFD is the predominant feature that drives prion formation. Scrambled versions of Ure2p and Sup35p (in which the order of the amino acids in the PFD was randomized while maintaining amino acid composition) are able to form prions when expressed in yeast as the sole copy Ure2p or Sup35p (Ross et al. 2004, 2005). To examine whether amino acid composition can similarly drive interactions between heterologous proteins, we tested whether the scrambled PFDs can interact with their wild-type counterparts to stimulate prion formation. When overexpressed, scrambled Ure2 PFDs promoted de novo prion formation by wild-type Ure2p, suggesting that the Ure2p PFD can promiscuously interact with compositionally similar PFDs during prion formation. When we searched the yeast proteome for proteins with regions of high compositional similarity to Ure2p, four of the top five proteins were able to efficiently stimulate [URE3] formation. However, there were limits to this promiscuity; overexpression of wild-type or scrambled Sup35 PFDs did not increase [URE3] levels. We propose that this ability to promiscuously interact may have evolved as a mechanism to regulate Ure2p activity and/or prion formation.