Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

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.     

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 ⁺].     

The sensitive [SWI+] prion

Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these “nouveau prions” has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI⁺], differs from the best studied, archetypal prion [PSI⁺] in several significant ways.¹ Notably, [SWI⁺] is highly sensitive to alterations in Hsp70 system chaperone activity and is lost upon growth at elevated temperatures. In that report we briefly noted a correlation amongst prions regarding amino acid composition, seed number and sensitivity to the activity of the Hsp70 chaperone system. Here we extend that analysis and put forth the idea that [SWI⁺] may be representative of a class of asparagine-rich yeast prions which also includes [URE3], [MOT3⁺] and [ISP⁺], distinct from the glutamine-rich prions such as [PSI⁺] and [RNQ⁺]. While much work remains, it is apparent that our understanding of the extent of the diversity of prion characteristics is in its infancy.     

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.     

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.     

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     

The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI⁺] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI⁺] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI⁺] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI⁺] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI⁺] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI⁺] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI⁺] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.     

Forms and abundance of chaperone proteins influence yeast prion variant competition

[PSI⁺] variants are different infectious conformations of the same Sup35 protein. We show that when [PSI⁺] variants VK and VL co‐infect a dividing host, only one prevails in the end and the host genetic background is involved in winner selection. In the 5V‐H19 background, the VK variant dominates over the VL variant. The order of dominance is reversed in the 74‐D694 background, where VL can coexists with VK for a short period, but will eventually take over. Differential interaction of chaperone proteins with distinct prion variant conformations can influence the outcome of competition. Expanding the Glycine/Methionine‐rich domain of Sis1, an Hsp40 protein, helps the propagation of VL. Over‐expression of the Hsp70 protein Ssa2 lowers the number of prion particles (propagons) in the cell. There is more reduction for VK than VL, causing the latter to dominate in some of the 5V‐H19 and all of the 74‐D694 cells tested. Consistently, depleting Ssa1 in 74‐D694 strengthens VK. Swapping chromosomal alleles of SSA1/2 and SIS1 between 5V‐H19 and 74‐D694, including cognate promoters, is not sufficient to change the native dominance order of each background, suggesting there exist additional polymorphic factors that modulate [PSI⁺] competition.     

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN⁺], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.     

An insight into the complex prion-prion interaction network in the budding yeast Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a valuable model system for studying prion-prion interactions as it contains multiple prion proteins. A recent study from our laboratory showed that the existence of Swi1 prion ([SWI⁺]) and overproduction of Swi1 can have strong impacts on the formation of 2 other extensively studied yeast prions, [PSI⁺] and [PIN⁺] ([RNQ⁺]) (Genetics, Vol. 197, 685–700). We showed that a single yeast cell is capable of harboring at least 3 heterologous prion elements and these prions can influence each other's appearance positively and/or negatively. We also showed that during the de novo [PSI⁺] formation process upon Sup35 overproduction, the aggregation patterns of a preexisting inducer ([RNQ⁺] or [SWI⁺]) can undergo significant remodeling from stably transmitted dot-shaped aggregates to aggregates that co-localize with the newly formed Sup35 aggregates that are ring/ribbon/rod- shaped. Such co-localization disappears once the newly formed [PSI⁺] prion stabilizes. Our finding provides strong evidence supporting the “cross-seeding” model for prion-prion interactions and confirms earlier reports that the interactions among different prions and their prion proteins mostly occur at the initiation stages of prionogenesis. Our results also highlight a complex prion interaction network in yeast. We believe that elucidating the mechanism underlying the yeast prion-prion interaction network will not only provide insight into the process of prion de novo generation and propagation in yeast but also shed light on the mechanisms that govern protein misfolding, aggregation, and amyloidogenesis in higher eukaryotes.     

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.     

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.     

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI⁺] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates.     

The sensitive [SWI+] prion: New perspectives on yeast prion diversity

Yeast prions are heritable protein-based genetic elements which rely on molecular chaperone proteins for stable transmission to cell progeny. Within the past few years, five new prions have been validated and 18 additional putative prions identified in Saccharomyces cerevisiae. The exploration of the physical and biological properties of these “nouveau prions” has begun to reveal the extent of prion diversity in yeast. We recently reported that one such prion, [SWI⁺], differs from the best studied, archetypal prion [PSI⁺] in several significant ways.¹ Notably, [SWI⁺] is highly sensitive to alterations in Hsp70 system chaperone activity and is lost upon growth at elevated temperatures. In that report we briefly noted a correlation amongst prions regarding amino acid composition, seed number and sensitivity to the activity of the Hsp70 chaperone system. Here we extend that analysis and put forth the idea that [SWI⁺] may be representative of a class of asparagine-rich yeast prions which also includes [URE3], [MOT3⁺] and [ISP⁺], distinct from the glutamine-rich prions such as [PSI⁺] and [RNQ⁺]. While much work remains, it is apparent that our understanding of the extent of the diversity of prion characteristics is in its infancy.Key words: Sis1, Hsp40, chromatin remodeling, Swi1, Ssa, heat-shock, protein misfolding, cell stress, Hsp 104, PINYeast prions are heritable elements, most of which are amyloid aggregates of single proteins. The three best studied yeast prions [PSI⁺], [RNQ⁺] (also called [PIN⁺]), and [URE3] are formed from amyloid aggregates of the cytosolic yeast proteins Sup35, Rnq1 and Ure2, respectively.² Yeast prions can spontaneously arise in an otherwise clonal cell population, a process referred to as prion formation or nucleation, but once formed their continued propagation is intimately related to molecular chaperone activity. Chaperone function is needed to fragment prion amyloids to create heritable seeds which can then be passed on to cell progeny, thus maintaining the prion in the cell line.³ Yeast prions vary in the steady-state number of heritable seeds per cell; having more seeds increases the chances of passing the prion to progeny and hence prions with higher seed numbers are more mitotically stable.^4–6The currently accepted model of prion fragmentation posits that components of the Hsp70 chaperone system work in congress with the disaggregase Hsp104.^1,7–9 Hsp70-type chaperones function by repeatedly binding and releasing client polypeptides in an ATP-dependent manner, a cycle that is tightly regulated by co-chaperone proteins (Fig. 1). J-proteins (Hsp40s) stimulate Hsp70 ATP hydrolysis and peptide binding via a conserved J-domain whereas nucleotide exchange factors (NEFs) stimulate ADP/ATP exchange, restoring the ATP-bound (peptide unbound) state. In prion fragmentation, the J-protein Sis1, the Hsp70 Ssa, and nucleotide exchange factors (NEFs) of the Sse family are co-chaperones required as partners for the Hsp70 Ssa. While chaperone proteins may have additional functions in prion biology, e.g., prion formation, these additional functions are still poorly understood.⁹Open in a separate windowFigure 1The Cyclic Hsp70 Chaperone System. Ssa (purple), the yeast cytosolic Hsp70, binds and releases client polypeptides (blue) in a regulated and ATP-dependent manner. J-proteins (aquamarine) including Sis1, Ydj1 and others, stimulate Ssa ATP hydrolysis by virtue of a conserved J-domain and thereby catalyze the “forward” direction of the cycle as indicated above. ADP•Ssa more stably associates with client polypeptides than the ATP-bound form and hence J-proteins favor the ADP•Ssa•Peptide complex. In some cases, J-proteins can also bind and deliver client polypeptides to Hsp70s via C-terminal domains (also shown above). Nucleotide exchange factors (NEFs), including the Sse proteins (dark blue) which share some structural homology with Ssa, catalyze the “reverse” direction of the cycle by facilitating ADP release and subsequent ATP binding, and thus favor an ATP•Ssa state with a dissociated peptide.In the past few years, the number of known yeast prions has rapidly grown such that, to date, a total of eight yeast prions have been identified and an additional 18 proteins have been annotated as putative prions.¹⁰ The biological and physical properties of these newly discovered prions are only beginning to be explored. We recently reported the results of an investigation into the biological properties of the prion [SWI⁺], which is formed from the chromatin-remodeling factor Swi1.¹ Swi1 is part of the SWI/SNF chromatin-remodeling complex that regulates the expression of approximately 6% of all yeast genes.¹¹ The presence of [SWI⁺] causes partial loss of SWI/SNF chromatin-remodeling function, resulting in the impaired ability to uptake certain sugars, among other phenotypes.¹¹ [SWI⁺] is a prion of particular interest because of its potential to alter global gene expression. Below we describe its intriguing interactions with molecular chaperone proteins and environmental stress, and the implications of these properties on yeast prion biology.     

Expanding the yeast prion world

Mammalian and fungal prion proteins form self-perpetuating β-sheet-rich fibrillar aggregates called amyloid. Prion inheritance is based on propagation of the regularly oriented amyloid structures of the prion proteins. All yeast prion proteins identified thus far contain aggregation-prone glutamine/asparagine (Gln/Asn)-rich domains, although the mammalian prion protein and fungal prion protein HET-s do not contain such sequences. In order to fill this gap, we searched for novel yeast prion proteins lacking Gln/Asn-rich domains via a genome-wide screen based on cross-seeding between two heterologous proteins and identified Mod5, a yeast tRNA isopentenyltransferase, as a novel non-Gln/Asn-rich yeast prion protein. Mod5 formed self-propagating amyloid fibers in vitro and the introduction of Mod5 amyloids into non-prion yeast induced dominantly and cytoplasmically heritable prion state [MOD⁺], which harbors aggregates of endogenous Mod5. [MOD⁺] yeast showed an increased level of membrane lipid ergosterol and acquired resistance to antifungal agents. Importantly, enhanced de novo formation of [MOD⁺] was observed when non-prion yeast was grown under selective pressures from antifungal drugs. Our findings expand the family of yeast prions to non-Gln/Asn-rich proteins and reveal the acquisition of a fitness advantage for cell survival through active prion conversion.     

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.     

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.     

The story of stolen chaperones

Prions are self-seeding alternate protein conformations. Most yeast prions contain glutamine/asparagine (Q/N)-rich domains that promote the formation of amyloid-like prion aggregates. Chaperones, including Hsp104 and Sis1, are required to continually break these aggregates into smaller “seeds.” Decreasing aggregate size and increasing the number of growing aggregate ends facilitates both aggregate transmission and growth. Our previous work showed that overexpression of 11 proteins with Q/N-rich domains facilitates the de novo aggregation of Sup35 into the [PSI⁺] prion, presumably by a cross-seeding mechanism. We now discuss our recent paper, in which we showed that overexpression of most of these same 11 Q/N-rich proteins, including Pin4C and Cyc8, destabilized pre-existing Q/N rich prions. Overexpression of both Pin4C and Cyc8 caused [PSI⁺] aggregates to enlarge. This is incompatible with a previously proposed “capping” model where the overexpressed Q/N-rich protein poisons, or “caps,” the growing aggregate ends. Rather the data match what is expected of a reduction in prion severing by chaperones. Indeed, while Pin4C overexpression does not alter chaperone levels, Pin4C aggregates sequester chaperones away from the prion aggregates. Cyc8 overexpression cures [PSI⁺] by inducing an increase in Hsp104 levels, as excess Hsp104 binds to [PSI⁺] aggregates in a way that blocks their shearing.