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.
Certain proteins can misfold into β-sheet-rich, self-seeding aggregates. Such proteins appear to be associated with neurodegenerative diseases such as prion, Alzheimer's and Parkinson's. Yeast prions also misfold into self-seeding aggregates and provide a good model to study how these rogue polymers first appear. De novo prion appearance can be made very frequent in yeast by transient overexpression of the prion protein in the presence of heterologous prions or prion-like aggregates. Here, we show that the aggregates of one such newly induced prion are initially formed in a dot-like structure near the vacuole. These dots then grow into rings at the periphery of the cell prior to becoming smaller rings surrounding the vacuole and maturing into the characteristic heritable prion tiny dots found throughout the cytoplasm. We found considerable colocalization of two heterologous prion/prion-like aggregates with the newly appearing prion protein aggregates, which is consistent with the prevalent model that existing prion aggregates can cross-seed the de novo aggregation of a heterologous prion protein. However, we failed to find any physical interaction between another heterologous aggregating protein and the newly appearing prion aggregates it stimulated to appear, which is inconsistent with cross-seeding.
Prions are infectious, self-propagating protein conformations. Rnq1 is required for the yeast Saccharomyces cerevisiae prion [PIN+], which is necessary for the de novo induction of a second prion, [PSI+]. Here we isolated a [PSI+]-eliminating mutant, Rnq1Δ100, that deletes the nonprion domain of Rnq1. Rnq1Δ100 inhibits not only [PSI+] prion propagation but also [URE3] prion and huntingtin's polyglutamine aggregate propagation in a [PIN+] background but not in a [pin−] background. Rnq1Δ100, however, does not eliminate [PIN+]. These findings are interpreted as showing a possible involvement of the Rnq1 prion in the maintenance of heterologous prions and polyQ aggregates. Rnq1 and Rnq1Δ100 form a sodium dodecyl sulfate-stable and Sis1 (an Hsp40 chaperone protein)-containing coaggregate in [PIN+] cells. Importantly, Rnq1Δ100 is highly QN-rich and prone to self-aggregate or coaggregate with Rnq1 when coexpressed in [pin−] cells. However, the [pin−] Rnq1-Rnq1Δ100 coaggregate does not represent a prion-like aggregate. These findings suggest that [PIN+] Rnq1-Rnq1Δ100 aggregates interact with other transmissible and nontransmissible amyloids to destabilize them and that the nonprion domain of Rnq1 plays a crucial role in self-regulation of the highly reactive QN-rich prion domain of Rnq1.
Yeast prions are heritable amyloid aggregates of functional yeast proteins; their propagation to subsequent cell generations is dependent upon fragmentation of prion protein aggregates by molecular chaperone proteins. Mounting evidence indicates the J-protein Sis1 may act as an amyloid specificity factor, recognizing prion and other amyloid aggregates and enabling Ssa and Hsp104 to act in prion fragmentation. Chaperone interactions with prions, however, can be affected by variations in amyloid-core structure resulting in distinct prion variants or ‘strains’. Our genetic analysis revealed that Sis1 domain requirements by distinct variants of [PSI+] are strongly dependent upon overall variant stability. Notably, multiple strong [PSI+] variants can be maintained by a minimal construct of Sis1 consisting of only the J-domain and glycine/phenylalanine-rich (G/F) region that was previously shown to be sufficient for cell viability and [RNQ+] prion propagation. In contrast, weak [PSI+] variants are lost under the same conditions but maintained by the expression of an Sis1 construct that lacks only the G/F region and cannot support [RNQ+] propagation, revealing mutually exclusive requirements for Sis1 function between these two prions. Prion loss is not due to [PSI+]-dependent toxicity or dependent upon a particular yeast genetic background. These observations necessitate that Sis1 must have at least two distinct functional roles that individual prions differentially require for propagation and which are localized to the glycine-rich domains of the Sis1. Based on these distinctions, Sis1 plasmid-shuffling in a [PSI+]/[RNQ+] strain permitted J-protein-dependent prion selection for either prion. We also found that, despite an initial report to the contrary, the human homolog of Sis1, Hdj1, is capable of [PSI+] prion propagation in place of Sis1. This conservation of function is also prion-variant dependent, indicating that only one of the two Sis1-prion functions may have been maintained in eukaryotic chaperone evolution.
Multiple neurodegenerative disorders such as Alzheimer's, Parkinson's and Creutzfeldt-Jakob disease are associated with the accumulation of fibrous protein aggregates collectively termed ‘amyloid.’ In the baker's yeast Saccharomyces cerevisiae, multiple proteins form intracellular amyloid aggregates known as yeast prions. Yeast prions minimally require a core set of chaperone proteins for stable propagation in yeast, including the J-protein Sis1, which appears to be required for the propagation of all yeast prions and functioning similarly in each case. Here we present evidence which challenges the notion of a universal function for Sis1 in prion propagation and asserts instead that Sis1's function in the maintenance of at least two prions, [RNQ+] and [PSI+], is distinct and mutually exclusive for some prion variants. We also find that the human homolog of Sis1, called Hdj1, has retained the ability to support some, but not all yeast prions, indicating a partial conservation of function. Because yeast chaperones have the ability to both bind and fragment amyloids in vivo, further investigations into these prion-specific properties of Sis1 and Hdj1 will likely lead to new insights into the biological management of protein misfolding.
In yeast, fragmentation of amyloid polymers by the Hsp104 chaperone allows them to propagate as prions. The prion-forming domain of the yeast Sup35 protein is rich in glutamine, asparagine, tyrosine, and glycine residues, which may define its prion properties. Long polyglutamine stretches can also drive amyloid polymerization in yeast, but these polymers are unable to propagate because of poor fragmentation and exist through constant seeding with the Rnq1 prion polymers. We proposed that fragmentation of polyglutamine amyloids may be improved by incorporation of hydrophobic amino acid residues into polyglutamine stretches. To investigate this, we constructed sets of polyglutamine with or without tyrosine stretches fused to the non-prion domains of Sup35. Polymerization of these chimeras started rapidly, and its efficiency increased with stretch size. Polymerization of proteins with polyglutamine stretches shorter than 70 residues required Rnq1 prion seeds. Proteins with longer stretches polymerized independently of Rnq1 and thus could propagate. The presence of tyrosines within polyglutamine stretches dramatically enhanced polymer fragmentation and allowed polymer propagation in the absence of Rnq1 and, in some cases, of Hsp104.
[PSI+] is an amyloid-based prion of Sup35p, a subunit of the translation termination factor. Prion “strains” or “variants” are amyloids with different conformations of a single protein sequence, conferring different phenotypes, but each relatively faithfully propagated. Wild Saccharomyces cerevisiae isolates have SUP35 alleles that fall into three groups, called reference, Δ19, and E9, with limited transmissibility of [PSI+] between cells expressing these different polymorphs. Here we show that prion transmission pattern between different Sup35 polymorphs is prion variant-dependent. Passage of one prion variant from one Sup35 polymorph to another need not change the prion variant. Surprisingly, simple mitotic growth of a [PSI+] strain results in a spectrum of variant transmission properties among the progeny clones. Even cells that have grown for >150 generations continue to vary in transmission properties, suggesting that simple variant segregation is insufficient to explain the results. Rather, there appears to be continuous generation of a cloud of prion variants, with one or another becoming stochastically dominant, only to be succeeded by a different mixture. We find that among the rare wild isolates containing [PSI+], all indistinguishably “weak” [PSI+], are several different variants based on their transmission efficiencies to other Sup35 alleles. Most show some limitation of transmission, indicating that the evolved wild Sup35 alleles are effective in limiting the spread of [PSI+]. Notably, a “strong [PSI+]” can have any of several different transmission efficiency patterns, showing that “strong” versus “weak” is insufficient to indicate prion variant uniformity.
The [PSI+] prion (infectious protein) of yeast is a self-propagating amyloid (filamentous protein polymer) of the Sup35 protein, a subunit of the translation termination factor. A single protein can form many biologically distinct prions, called prion variants. Wild yeast strains have three groups of Sup35 sequences (polymorphs), which partially block transmission of the [PSI+] prion from cell to cell. We find that [PSI+] variants (including the rare [PSI+] from wild yeasts) show different transmission patterns from one Sup35 sequence to another. Moreover, we find segregation of different prion variants on mitotic growth and evidence for generation of new variants with growth under non-selective conditions. This data supports the “prion cloud” model, that prions are not uniform structures but have an array of related self-propagating amyloid structures.
De novo formation of two structurally and compositionally distinct yeast prions, [PSI+] and [Het-s]y, is similar starting with formation of a peripheral ring and then internal ring, and then daughters propagate the prions as dots. The ring formation, but not dot propagation, requires an endocytic protein Sla2.
Various proteins, like the infectious yeast prions and the noninfectious human Huntingtin protein (with expanded polyQ), depend on a Gln or Asn (QN)-rich region for amyloid formation. Other prions, e.g., mammalian PrP and the [Het-s] prion of Podospora anserina, although still able to form infectious amyloid aggregates, do not have QN-rich regions. Furthermore, [Het-s] and yeast prions appear to differ dramatically in their amyloid conformation. Despite these differences, a fusion of the Het-s prion domain to GFP (Het-sPrD-GFP) can propagate in yeast as a prion called [Het-s]y. We analyzed the properties of two divergent prions in yeast: [Het-s]y and the native yeast prion [PSI+] (prion form of translational termination factor Sup35). Curiously, the induced appearance and transmission of [PSI+] and [Het-s]y aggregates is remarkably similar. Overexpression of tagged prion protein (Sup35-GFP or Het-sPrD-GFP) in nonprion cells gives rise to peripheral, and later internal, ring/mesh-like aggregates. The cells with these ring-like aggregates give rise to daughters with one (perivacuolar) or two (perivacuolar and juxtanuclear) dot-like aggregates per cell. These line, ring, mesh, and dot aggregates are not really the transmissible prion species and should only be regarded as phenotypic markers of the presence of the prions. Both [PSI+] and [Het-s]y first appear in daughters as numerous tiny dot-like aggregates, and both require the endocytic protein, Sla2, for ring formation, but not propagation.
Replicating amyloids, called prions, are responsible for transmissible
neurodegenerative diseases in mammals and some heritable phenotypes in fungi.
The transmission of prions between species is usually inhibited, being highly
sensitive to small differences in amino acid sequence of the prion-forming
proteins. To understand the molecular basis of this prion interspecies barrier,
we studied the transmission of the
[PSI+] prion state from
Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with
prion-forming domains from four other closely related
Saccharomyces species. Whereas all the hybrid Sup35
proteins could adopt a prion form in S. cerevisiae, they could
not readily acquire the prion form from the
[PSI+] prion of S.
cerevisiae. Expression of the hybrid Sup35 proteins in S.
cells often resulted in frequent loss of the native
[PSI+] prion. Furthermore,
all hybrid Sup35 proteins showed different patterns of interaction with the
native [PSI+] prion in terms of
co-polymerization, acquisition of the prion state, and induced prion loss, all
of which were also dependent on the
[PSI+] variant. The
observed loss of S. cerevisiae
[PSI+] can be related to
inhibition of prion polymerization of S. cerevisiae Sup35 and
formation of a non-heritable form of amyloid. We have therefore identified two
distinct molecular origins of prion transmission barriers between closely
sequence-related prion proteins: first, the inability of heterologous proteins
to co-aggregate with host prion polymers, and second, acquisition by these
proteins of a non-heritable amyloid fold.
Amyloid; Prions; Protein Folding; Translation Release Factors; Yeast; Saccharomyces; Sup35; Prion Interference; Prion Species Barrier
The [PSI+] prion is the aggregated self-propagating form of the Sup35 protein from the yeast Saccharomyces cerevisiae. Aggregates of Sup35 in [PSI+] cells exist in different heritable conformations, called “variants,” and they are composed of detergent-resistant Sup35 polymers, which may be closely associated with themselves, other proteins, or both. Here, we report that disassembly of the aggregates into individual Sup35 polymers and non-Sup35 components increases their infectivity while retaining their variant specificity, showing that variant-specific [PSI+] infection can be transmitted by Sup35 polymers alone. Morphological analysis revealed that Sup35 isolated from [PSI+] yeast has the appearance of short barrels, and bundles, which seem to be composed of barrels. We show that the major components of two different variants of [PSI+] are interacting infectious Sup35 polymers and Ssa1/2. Using a candidate approach, we detected Hsp104, Ssb1/2, Sis1, Sse1, Ydj1, and Sla2 among minor components of the aggregates. We demonstrate that Ssa1/2 efficiently binds to the prion domain of Sup35 in [PSI+] cells, but that it interacts poorly with the nonaggregated Sup35 found in [psi−] cells. Hsp104, Sis1, and Sse1 interact preferentially with the prion versus nonprion form of Sup35, whereas Sla2 and Ssb1/2 interact with both forms of Sup35 with similar efficiency.
Inheritance of phenotypic traits depends on two key events: replication of the determinant of that trait and partitioning of these copies between mother and daughter cells. Although these processes are well understood for nucleic acid–based genes, the mechanisms by which protein-only or prion-based genetic elements direct phenotypic inheritance are poorly understood. Here, we report a process crucial for inheritance of the Saccharomyces cerevisiae prion [PSI+], a self-replicating conformer of the Sup35 protein. By tightly controlling expression of a Sup35-GFP fusion, we directly observe remodeling of existing Sup35[PSI+] complexes in vivo. This dynamic change in Sup35[PSI+] is lost when the molecular chaperone Hsp104, a factor essential for propagation of all yeast prions, is functionally impaired. The loss of Sup35[PSI+] remodeling by Hsp104 decreases the mobility of these complexes in the cytosol, creates a segregation bias that limits their transmission to daughter cells, and consequently diminishes the efficiency of conversion of newly made Sup35 to the prion form. Our observations resolve several seemingly conflicting reports on the mechanism of Hsp104 action and point to a single Hsp104-dependent event in prion propagation.
The inheritance of phenotypic traits (the observable characteristics of the organism) is a fundamental process in biology. Most phenotypes are controlled by a cell's genes, and a particular phenotype becomes heritable when this underlying genetic information is copied and transmitted to progeny. In contrast, another group of phenotypes appears to be inherited through a protein-only, or prion, mechanism in which the structure of a protein rather than its sequence is the molecular determinant of the phenotype. It is thought that the presence of a prion in a cell forces conversion of a normal cellular protein into a differently folded shape (the prion form), which simultaneously deprives the cell of the protein's normal function and causes the prion-folded protein to aggregate within the cell. However, prion inheritance (how prions are passed down to daughter cells) remains poorly understood.
Using the yeast prion [PSI+] as a model system, we have elucidated a process necessary for protein-only inheritance. Here we show that the molecular chaperone Hsp104, a factor necessary for the inheritance of all known yeast prions, plays a single primary role in generating additional templates for protein-state replication. In the absence of this activity, existing prion templates are inefficiently transferred to daughter cells. As a consequence, the rate of protein-state replication is greatly decreased, and the protein-based phenotype is progressively lost.
The authors examine the role of the molecular chaperone Hsp104 in controlling inheritance of the prion form of Sup35[PSI+].
Amyloidogenic proteins associated with a variety of unrelated diseases are typically capable of forming several distinct self-templating conformers. In prion diseases, these different structures, called prion strains (or variants), confer dramatic variation in disease pathology and transmission. Aggregate stability has been found to be a key determinant of the diverse pathological consequences of different prion strains. Yet, it remains largely unclear what other factors might account for the widespread phenotypic variation seen with aggregation-prone proteins. Here, we examined a set of yeast prion variants of the [RNQ+] prion that differ in their ability to induce the formation of another yeast prion called [PSI+]. Remarkably, we found that the [RNQ+] variants require different, non-contiguous regions of the Rnq1 protein for both prion propagation and [PSI+] induction. This included regions outside of the canonical prion-forming domain of Rnq1. Remarkably, such differences did not result in variation in aggregate stability. Our analysis also revealed a striking difference in the ability of these [RNQ+] variants to interact with the chaperone Sis1. Thus, our work shows that the differential influence of various amyloidogenic regions and interactions with host cofactors are critical determinants of the phenotypic consequences of distinct aggregate structures. This helps reveal the complex interdependent factors that influence how a particular amyloid structure may dictate disease pathology and progression.
Protein conformational disorders, including many neurodegenerative diseases, result when a protein misfolds and undergoes a conformational change to form self-templating aggregates, called amyloid. Interestingly, the proteins that misfold in these diseases tend to form a wide variety of distinct aggregate structures. In prion diseases, these different amyloid conformations are called prion strains. The different conformations of prion strains are responsible for modulating disease progression, pathology, and transmission. Previous work with yeast prions has provided tremendous insight into how distinct prion conformers can cause such phenotypic variability. Here, we used a set of [RNQ+] prion variants to show the complex web of interactions involved in the propagation of distinct aggregate structures. We found that several different non-adjacent regions of Rnq1, even outside the prion-forming domain, make varying contributions to the propagation of distinct variants of the [RNQ+] prion. Moreover, our data support the hypothesis that the [RNQ+] variants differentially interact with the molecular chaperone Sis1. These data strongly suggest that the variable phenotypic manifestations of different aggregate conformations depend upon a unique set of primary structural elements and differential interactions with host cofactors.
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.
Background: Yeast have been used to study hungtingtin toxicity.
Results: Both HttQ103 and HttQP103 are toxic in yeast with [PSI+] prion. This toxicity is markedly rescued by a Sup35 fragment.
Conclusion: Sequestration of the essential protein, Sup35, contributes to Htt toxicity in yeast.
Significance: This research demonstrates the complex nature of Htt toxicity.
Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN+] prion, which is the amyloid form of Rnq1, or [PSI+] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI+] yeast than in [PIN+], even though HttQP103 formed multiple aggregates in both [PSI+] and [PIN+] yeast. This toxicity was only observed in the strong [PSI+] variant, not the weak [PSI+] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI+] prion is primarily due to sequestration of the essential protein, Sup35.
Huntington Disease; Prions; Protein Folding; Yeast; Yeast Physiology
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.
Certain proteins that exist in functional unaggregated conformers can also form self-perpetuating infectious aggregates called prions. Here we investigate factors involved in the initial switch to the prion form. De novo appearance of the [PSI+] prion, which is the aggregated form of the Sup35 protein, is dramatically enhanced by overexpression of the SUP35 gene in the presence of the prion form of the Rnq1 protein, [PIN+]. When tagged with green fluorescent protein and transiently overexpressed in [psi−] [PIN+] cells, Sup35 forms fluorescent rings, and cells with these rings give rise to daughter cells that are [PSI+]. Here, we investigate factors required for this induction of [PSI+]. Analyses of over 400 gene deletions revealed two classes that reduce [PSI+] induction: one class forms fluorescent rings normally, and the other does not. Interestingly, the former class enhanced, while the latter class reduced, toxicity associated with the expanded polyglutamine repeats of the huntingtin protein exon 1 that causes Huntington's disease. These results suggest that prion formation and polyglutamine aggregation involve a multi-phase process that can be inhibited at different steps.
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 prion; [PSI+]; Pin+; [RNQ+]; [RNQ1Δ100+]; [URE3]; Lsm4; New1
Prions are self-propagating conformations of proteins that can cause heritable phenotypic traits. Most yeast prions contain glutamine (Q)/asparagine (N)-rich domains that facilitate the accumulation of the protein into amyloid-like aggregates. Efficient transmission of these infectious aggregates to daughter cells requires that chaperones, including Hsp104 and Sis1, continually sever the aggregates into smaller “seeds.” We previously identified 11 proteins with Q/N-rich domains that, when overproduced, facilitate the de novo aggregation of the Sup35 protein into the [PSI
+] prion state. Here, we show that overexpression of many of the same 11 Q/N-rich proteins can also destabilize pre-existing [PSI+] or [URE3] prions. We explore in detail the events leading to the loss (curing) of [PSI+] by the overexpression of one of these proteins, the Q/N-rich domain of Pin4, which causes Sup35 aggregates to increase in size and decrease in transmissibility to daughter cells. We show that the Pin4 Q/N-rich domain sequesters Hsp104 and Sis1 chaperones away from the diffuse cytoplasmic pool. Thus, a mechanism by which heterologous Q/N-rich proteins impair prion propagation appears to be the loss of cytoplasmic Hsp104 and Sis1 available to sever [PSI+].
Certain proteins can occasionally misfold into infectious aggregates called prions. Once formed, these aggregates grow by attracting the soluble form of that protein to join them. The presence of these aggregates can cause profound effects on cells and, in humans, can cause diseases such as transmissible spongiform encephalopathies (TSEs). In yeast, the aggregates are efficiently transmitted to daughter cells because they are cut into small pieces by molecular scissors (chaperones). Here we show that heritable prion aggregates are frequently lost when we overproduce certain other proteins with curing activity. We analyzed one such protein in detail and found that when it is overproduced it forms aggregates that sequester chaperones. This sequestration appears to block the ability of the chaperones to cut the prion aggregates. The result is that the prions get too large to be transmitted to daughter cells. Such sequestration of molecular scissors provides a potential approach to thwart the propagation of disease-causing infectious protein aggregates.
Prions are self-propagating infectious protein conformations. The mammalian prion, PrPSc, responsible for neurodegenerative diseases like mad-cow and Creutzfeldt Jacob’s diseases, appears to be a β-sheet rich amyloid conformation of PrPc that converts PrPc into PrPSc. However, an unequivocal demonstration of ‘protein-only’ infection by PrPSc is still lacking. So far, “protein only” infection has been proven for three prions, [PSI+], [URE3] and [Het-s], all of fungal origin. Considerable evidence also supports the hypothesis that another protein, the yeast Rnq1p, can form a prion, [PIN+]. While Rnq1p does not lose any known function upon prionization, [PIN+] has interesting positive phenotypes: facilitating the appearance and destabilization of other prions as well as the aggregation of poly-Gln extensions of the Huntingtin protein. Here, we polymerize a Gln/Asn-rich recombinant fragment of Rnq1p into β-sheet-rich amyloid-like aggregates. While the method used for [PSI+] and [URE3] infectivity assays did not yield ‘protein-only’ infection for the Rnq1p aggregates, we did successfully obtain ‘protein-only’ infection by modifying the protocol. The work proves that [PIN+] is a prion mediated by amyloid-like aggregates of Rnq1p, and supports the hypothesis that heterologous prions affect each others appearance and propagation through interaction of their amyloid-like regions.
Amyloid-like; [PIN+]; Prion; Protein-Only; Rnq1p
The yeast and fungal prions determine heritable and infectious traits, and are thus genes composed of protein. Most prions are inactive forms of a normal protein as it forms a self-propagating filamentous β – sheet - rich polymer structure called amyloid. Remarkably, a single prion protein sequence can form two or more faithfully inherited prion variants, in effect alleles of these genes. What protein structure explains this protein-based inheritance? Using solid-state NMR, we showed that the infectious amyloids of the prion domains of Ure2p, Sup35p and Rnq1p have an in-register parallel architecture. This structure explains how the amyloid filament ends can template the structure of a new protein as it joins the filament.
The yeast prions [PSI+] and [URE3] are not found in wild strains, indicating they are a disadvantage to the cell. Moreover, the prion domains of Ure2p and Sup35p have functions unrelated to prion formation, indicating that these domains are not present for the purpose of forming prions. Indeed, prion forming ability is not conserved, even within S. cerevisiae, suggesting that the rare formation of prions is a disease. The prion domain sequences generally vary more rapidly in evolution than does the remainder of the molecule, producing a barrier to prion transmission, perhaps selected in evolution by this protection.
prion; amyloid; in-register parallel structure
In vivo propagation of [PSI+], an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104. Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation. Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele. In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of [PSI+] and another candidate yeast prion, [PIN+]. Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation. The [PSI+] response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased. A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35PSI+ aggregates and an increase in their size. This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle. Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).
Prions in the yeast Saccharomyces cerevisiae show a surprising degree of interdependence. Specifically, the rate of appearance of the [PSI+] prion, which is thought to be an important mechanism to respond to changing environmental conditions, is greatly increased by another prion, [RNQ+]. While the domains of the Rnq1 protein important for formation of the [RNQ+] prion have been defined, the specific residues required remain unknown. Furthermore, residues in Rnq1p that mediate the interaction between [PSI+] and [RNQ+] are unknown. To identify residues important for prion protein interactions, we created a mutant library of Rnq1p clones in the context of a chimera that serves as proxy for [RNQ+] aggregates. Several of the mutant Rnq1p proteins showed structural differences in the aggregates they formed, as revealed by SDD-AGE. Additionally, several of the mutants showed a striking defect in the ability to promote [PSI+] induction. These data indicate that the mutants formed strain variants of [RNQ+]. By dissecting the mutations in the isolated clones, we found five single mutations that caused [PSI+] induction defects, S223P, F184S, Q239R, N297S, and Q298R. These are the first specific mutations characterized in Rnq1p that alter [PSI+] induction. Additionally, we have identified a region important for the propagation of certain strain variants of [RNQ+]. Deletion of this region (amino acids 284–317) affected propagation of the high variant but not medium or low [RNQ+] strain variants. Furthermore, when the low [RNQ+] strain variant was propagated by Δ284–317, [PSI+] induction was greatly increased. These data suggest that this region is important in defining the structure of the [RNQ+] strain variants. These data are consistent with a model of [PSI+] induction caused by physical interactions between Rnq1p and Sup35p.
The frequency with which the yeast [PSI+] prion form of Sup35 arises de novo is controlled by a number of genetic and environmental factors. We have previously shown that in cells lacking the antioxidant peroxiredoxin proteins Tsa1 and Tsa2, the frequency of de novo formation of [PSI+] is greatly elevated. We show here that Tsa1/Tsa2 also function to suppress the formation of the [PIN+] prion form of Rnq1. However, although oxidative stress increases the de novo formation of both [PIN+] and [PSI+], it does not overcome the requirement of cells being [PIN+] to form the [PSI+] prion. We use an anti-methionine sulfoxide antibody to show that methionine oxidation is elevated in Sup35 during oxidative stress conditions. Abrogating Sup35 methionine oxidation by overexpressing methionine sulfoxide reductase (MSRA) prevents [PSI+] formation, indicating that Sup35 oxidation may underlie the switch from a soluble to an aggregated form of Sup35. In contrast, we were unable to detect methionine oxidation of Rnq1, and MSRA overexpression did not affect [PIN+] formation in a tsa1 tsa2 mutant. The molecular basis of how yeast and mammalian prions form infectious amyloid-like structures de novo is poorly understood. Our data suggest a causal link between Sup35 protein oxidation and de novo [PSI+] prion formation.
Methionine; Oxidative Stress; Peroxiredoxin; Prions; Protein Aggregation; Translation; Translation Release Factors; Yeast
The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions. The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ+] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ+] status of cells to perform a screen for mutants that are unable to maintain [RNQ+]. We found eight separate mutations in Hsp104p that caused [RNQ+] cells to become [rnq−]. These mutations also caused the loss of the [PSI+] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ+] and [PSI+] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ+] and was unable to maintain [PSI+]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ+] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI+].
[RNQ+]; [PSI+]; Hsp104p; Sis1p; mutagenesis
The authors define how small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial prion, and then they exploit this knowledge to identify the human amyloid depolymerase.
How small heat shock proteins (sHsps) might empower proteostasis networks to control beneficial prions or disassemble pathological amyloid is unknown. Here, we establish that yeast sHsps, Hsp26 and Hsp42, inhibit prionogenesis by the [PSI+] prion protein, Sup35, via distinct and synergistic mechanisms. Hsp42 prevents conformational rearrangements within molten oligomers that enable de novo prionogenesis and collaborates with Hsp70 to attenuate self-templating. By contrast, Hsp26 inhibits self-templating upon binding assembled prions. sHsp binding destabilizes Sup35 prions and promotes their disaggregation by Hsp104, Hsp70, and Hsp40. In yeast, Hsp26 or Hsp42 overexpression prevents [PSI+] induction, cures [PSI+], and potentiates [PSI+]-curing by Hsp104 overexpression. In vitro, sHsps enhance Hsp104-catalyzed disaggregation of pathological amyloid forms of α-synuclein and polyglutamine. Unexpectedly, in the absence of Hsp104, sHsps promote an unprecedented, gradual depolymerization of Sup35 prions by Hsp110, Hsp70, and Hsp40. This unanticipated amyloid-depolymerase activity is conserved from yeast to humans, which lack Hsp104 orthologues. A human sHsp, HspB5, stimulates depolymerization of α-synuclein amyloid by human Hsp110, Hsp70, and Hsp40. Thus, we elucidate a heretofore-unrecognized human amyloid-depolymerase system that could have applications in various neurodegenerative disorders.
Amyloid fibers are protein aggregates that are associated with numerous neurodegenerative diseases, including Parkinson's disease, for which there are no effective treatments. They can also play beneficial roles; in yeast, for example, they are associated with increased survival and the evolution of new traits. Amyloid fibers are also central to many revolutionary concepts and important questions in biology and nanotechnology, including long-term memory formation and versatile self-organizing nanostructures. Thus, there is an urgent need to understand how we can promote beneficial amyloid assembly, or reverse pathogenic assembly, at will. In this study, we define the mechanisms by which small heat-shock proteins synergize to regulate the assembly and disassembly of a beneficial yeast prion. We then exploit this knowledge to discover an amyloid depolymerase machinery that is conserved from yeast to humans. Remarkably, the human small heat shock protein, HspB5, stimulates Hsp110, Hsp70, and Hsp40 chaperones to gradually depolymerize amyloid fibers formed by α-synuclein (which are implicated in Parkinson's disease) from their ends on a biologically relevant timescale. This newly identified and highly conserved amyloid-depolymerase system could have important therapeutic applications for various neurodegenerative disorders.
[PSI+], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI+] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI+] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI+] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI+]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI+] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.
Mutations in the PRNP gene account for ~15% of all prion disease cases. Little is understood about the mechanism of how some of these mutations in PRNP cause the protein to aggregate into amyloid fibers or cause disease. We have taken advantage of a chimeric protein system to study the oligopeptide repeat domain (ORD) expansions of the prion protein, PrP, and their effect on protein aggregation and amyloid fiber formation. We replaced the ORD of the yeast prion protein Sup35p with that from wild type and expanded ORDs of PrP and compared their biochemical properties in vitro. We previously determined that these chimeric proteins maintain the [PSI+] yeast prion phenotype in vivo. Interestingly, we noted that the repeat expanded chimeric prions seemed to be able to maintain a stronger strain of [PSI+] and convert from [psi-] to [PSI+] with a much higher frequency. In this study we have attempted to understand the biochemical properties of these chimeric proteins and to establish a system to study the properties of the ORD of PrP both in vivo and in vitro.
Investigation of the chimeric proteins in vitro reveals that repeat-expansions increase aggregation propensity and that the kinetics of fiber formation depends on the number of repeats. The fiber formation reactions are promiscuous in that the chimeric protein containing 14 repeats can readily cross-seed fiber formation of proteins that have the wild type number of repeats. Morphologically, the amyloid fibers formed by repeat-expanded proteins associate with each other to form large clumps that were not as prevalent in fibers formed by proteins containing the wild type number of repeats. Despite the increased aggregation propensity and lateral association of the repeat expanded proteins, there was no corresponding increase in the stability of the fibers formed. Therefore, we predict that the differences in fibers formed with different repeat lengths may not be due to gross changes in the amyloid core.
The biochemical observations presented here explain the properties of these chimeric proteins previously observed in yeast. More importantly, they suggest a mechanism for the observed correlation between age of onset and disease severity with respect to the length of the ORD in humans.
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.
prion; Sup35p; Ure2p; Rnq1p; [PSI+]; [URE3]; [PIN+]; amyloid fibrils