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.
Fragmentation of amyloid polymers by the chaperone Hsp104 allows them to propagate as prions in yeast. The factors which determine the frequency of fragmentation are unclear, though it is often presumed to depend on the physical strength of prion polymers. Proteins with long polyglutamine stretches represent a tractable model for revealing sequence elements required for polymer fragmentation in yeast, since they form poorly fragmented amyloids. Here we show that interspersion of polyglutamine stretches with various amino acid residues differentially affects the in vivo formation and fragmentation of the respective amyloids. Aromatic residues tyrosine, tryptophan and phenylalanine strongly stimulated polymer fragmentation, leading to the appearance of oligomers as small as dimers. Alanine, methionine, cysteine, serine, threonine and histidine also enhanced fragmentation, while charged residues, proline, glycine and leucine inhibited polymerization. Our data indicate that fragmentation frequency primarily depends on the recognition of fragmentation-promoting residues by Hsp104 and/or its co-chaperones, rather than on the physical stability of polymers. This suggests that differential exposure of such residues to chaperones defines prion variant-specific differences in polymer fragmentation efficiency.
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.
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.
amyloid; prion; Rnq1; Sup35; Ure2; translation termination; yeast
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.
Polyglutamine expansion causes diseases in humans and other mammals. One example is Huntington's disease. Fragments of human huntingtin protein having an expanded polyglutamine stretch form aggregates and cause cytotoxicity in yeast cells bearing endogenous QN-rich proteins in the aggregated (prion) form. Attachment of the proline(P)-rich region targets polyglutamines to the large perinuclear deposit (aggresome). Aggresome formation ameliorates polyglutamine cytotoxicity in cells containing only the prion form of Rnq1 protein. Here we show that expanded polyglutamines both with (poly-QP) or without (poly-Q) a P-rich stretch remain toxic in the presence of the prion form of translation termination (release) factor Sup35 (eRF3). A Sup35 derivative that lacks the QN-rich domain and is unable to be incorporated into aggregates counteracts cytotoxicity, suggesting that toxicity is due to Sup35 sequestration. Increase in the levels of another release factor, Sup45 (eRF1), due to either disomy by chromosome II containing the SUP45 gene or to introduction of the SUP45-bearing plasmid counteracts poly-Q or poly-QP toxicity in the presence of the Sup35 prion. Protein analysis confirms that polyglutamines alter aggregation patterns of Sup35 and promote aggregation of Sup45, while excess Sup45 counteracts these effects. Our data show that one and the same mode of polyglutamine aggregation could be cytoprotective or cytotoxic, depending on the composition of other aggregates in a eukaryotic cell, and demonstrate that other aggregates expand the range of proteins that are susceptible to sequestration by polyglutamines.
Polyglutamine diseases, including Huntington disease, are associated with expansions of polyglutamine tracts, resulting in aggregation of respective proteins. The severity of Huntington disease is controlled by both DNA and non–DNA factors. Mechanisms of such a control are poorly understood. Polyglutamine may sequester other cellular proteins; however, different experimental models have pointed to different sequestered proteins. By using a yeast model, we demonstrate that the mechanism of polyglutamine toxicity is driven by the composition of other (endogenous) aggregates (for example, yeast prions) present in a eukaryotic cell. Although these aggregates do not necessarily cause significant toxicity on their own, they serve as mediators in protein sequestration and therefore determine which specific proteins are to be sequestered by polyglutamines. We also show that polyglutamine deposition into an aggresome, a perinuclear compartment thought to be cytoprotective, fails to ameliorate cytotoxicity in cells with certain compositions of pre-existing aggregates. Finally, we demonstrate that an increase in the dosage of a sequestered protein due to aneuploidy by a chromosome carrying a respective gene may rescue cytotoxicity. Our data shed light on genetic and epigenetic mechanisms modulating polyglutamine cytotoxicity and establish a new approach for identifying potential therapeutic targets through characterization of the endogenous aggregated proteins.
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.
Molecular chaperones regulate essential steps in the propagation of yeast prions. Yeast prions possess domains enriched in glutamines and asparagines that act as templates to drive the assembly of native proteins into beta-sheet-rich, amyloid-like fibrils. Several recent studies highlight a significant and complex function for Hsp40 co-chaperones in propagation of prion elements in yeast. Hsp40 co-chaperones bind non-native polypeptides and transfer these clients to Hsp70s for refolding or degradation. How Hsp40 co-chaperones bind amyloid-like prion conformers that are enriched in hydrophilic residues such as glutamines and asparagines is a significant question in the field. Interestingly, selective recognition of amyloid-like conformers by distinct Hsp40s appears to confer opposing actions on prion assembly. For example, the Type I Hsp40 Ydj1 and Type II Hsp40 Sis1 bind different regions within the prion protein Rnq1 and function respectively to inhibit or promote [RNQ+] prion assembly. Thus, substrate selectivity enables distinct Hsp40s to act at unique steps in prion propagation.
Hsp40; Ydj1; Sis1; amyloid; prion; Rnq1; J-protein; Hsp70
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.
In eukaryotic cells amyloid aggregates may incorporate various functionally unrelated proteins. In mammalian diseases this may cause amyloid toxicity, while in yeast this could contribute to prion phenotypes. Insolubility of amyloids in the presence of strong ionic detergents, such as SDS or sarcosyl, allows discrimination between amorphous and amyloid aggregates. Here, we used this property of amyloids to study the interdependence of their formation in yeast. We observed that SDS-resistant polymers of proteins with extended polyglutamine domains caused the appearance of SDS or sarcosyl-insoluble polymers of three tested chromosomally-encoded Q/N-rich proteins, Sup35, Rnq1 and Pub1. These polymers were non-heritable, since they could not propagate in the absence of polyglutamine polymers. Sup35 prion polymers caused the appearance of non-heritable sarcosyl-resistant polymers of Pub1. Since eukaryotic genomes encode hundreds of proteins with long Q/N-rich regions, polymer interdependence suggests that conversion of a single protein into polymer form may significantly affect cell physiology by causing partial transfer of other Q/N-rich proteins into a non-functional polymer state.
amyloid; prion; [PSI+]; huntingtin; polyglutamine; Saccharomyces cerevisiae; Sup35/eRF3
The glutamine/asparagine (Q/N)-rich yeast prion protein Sup35 has a low intrinsic propensity to spontaneously self-assemble into ordered, β-sheet-rich amyloid fibrils. In yeast cells, de novo formation of Sup35 aggregates is greatly facilitated by high protein concentrations and the presence of preformed Q/N-rich protein aggregates that template Sup35 polymerization. Here, we have investigated whether aggregation-promoting polyglutamine (polyQ) tracts can stimulate the de novo formation of ordered Sup35 protein aggregates in the absence of Q/N-rich yeast prions. Fusion proteins with polyQ tracts of different lengths were produced and their ability to spontaneously self-assemble into amlyloid structures was analyzed using in vitro and in vivo model systems. We found that Sup35 fusions with pathogenic (≥54 glutamines), as opposed to non-pathogenic (19 glutamines) polyQ tracts efficiently form seeding-competent protein aggregates. Strikingly, polyQ-mediated de novo assembly of Sup35 protein aggregates in yeast cells was independent of pre-existing Q/N-rich protein aggregates. This indicates that increasing the content of aggregation-promoting sequences enhances the tendency of Sup35 to spontaneously self-assemble into insoluble protein aggregates. A similar result was obtained when pathogenic polyQ tracts were linked to the yeast prion protein Rnq1, demonstrating that polyQ sequences are generic inducers of amyloidogenesis. In conclusion, long polyQ sequences are powerful molecular tools that allow the efficient production of seeding-competent amyloid structures.
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 yeast [PSI+], [URE3], and [PIN+] genetic elements are prion forms of Sup35p, Ure2p, and Rnq1p, respectively. Overexpression of Sup35p, Ure2p, or Rnq1p leads to increased de novo appearance of [PSI+], [URE3], and [PIN+], respectively. This inducible appearance of [PSI+] was shown to be dependent on the presence of [PIN+] or [URE3] or overexpression of other yeast proteins that have stretches of polar residues similar to the prion-determining domains of the known prion proteins. In a similar manner, [PSI+] and [URE3] facilitate the appearance of [PIN+]. In contrast to these positive interactions, here we find that in the presence of [PIN+], [PSI+] and [URE3] repressed each other's propagation and de novo appearance. Elevated expression of Hsp104 and Hsp70 (Ssa2p) had little effect on these interactions, ruling out competition between the two prions for limiting amounts of these protein chaperones. In contrast, we find that constitutive overexpression of SSA1 but not SSA2 cured cells of [URE3], uncovering a specific interaction between Ssa1p and [URE3] and a functional distinction between these nearly identical Hsp70 isoforms. We also find that Hsp104 abundance, which critically affects [PSI+] propagation, is elevated when [URE3] is present. Our results are consistent with the notion that proteins that have a propensity to form prions may interact with heterologous prions but, as we now show, in a negative manner. Our data also suggest that differences in how [PSI+] and [URE3] interact with Hsp104 and Hsp70 may contribute to their antagonistic interactions.
A significant body of evidence shows that polyglutamine (polyQ) tracts are important for various biological functions. The characteristic polymorphism of polyQ length is thought to play an important role in the adaptation of organisms to their environment. However, proteins with expanded polyQ are prone to form amyloids, which cause diseases in humans and animals and toxicity in yeast. Saccharomyces cerevisiae contain at least 8 proteins which can form heritable amyloids, called prions, and most of them are proteins with glutamine- and asparagine-enriched domains. Yeast prion amyloids are susceptible to fragmentation by the protein disaggregase Hsp104, which allows them to propagate and be transmitted to daughter cells during cell divisions. We have previously shown that interspersion of polyQ domains with some non-glutamine residues stimulates fragmentation of polyQ amyloids in yeast and that yeast prion domains are often enriched in one of these residues. These findings indicate that yeast prion domains may have derived from polyQ tracts via accumulation and amplification of mutations. The same hypothesis may be applied to polyasparagine (polyN) tracts, since they display similar properties to polyQ, such as length polymorphism, amyloid formation and toxicity. We propose that mutations in polyQ/N may be favored by natural selection thus making prion domains likely by-products of the evolution of polyQ/N.
amyloid; Hsp104; polyglutamine; polyasparagine; polyQ; polyN; prion; yeast
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.
Prions are self-propagating protein conformations. Transmission of the prion state between non-identical proteins, e.g. between homologous proteins from different species, is frequently inefficient. Transmission barriers are attributed to sequence differences in prion proteins, but their underlying mechanisms are not clear. Here we use a yeast Rnq1/[PIN+]-based experimental system to explore the nature of transmission barriers. [PIN+], the prion form of Rnq1, is common in wild and laboratory yeast strains, where it facilitates the appearance of other prions. Rnq1's prion domain carries four discrete QN-rich regions. We start by showing that Rnq1 encompasses multiple prion determinants that can independently drive amyloid formation in vitro and transmit the [PIN+] prion state in vivo. Subsequent analysis of [PIN+] transmission between Rnq1 fragments with different sets of prion determinants established that (i) one common QN-rich region is required and usually sufficient for the transmission; (ii) despite identical sequences of the common QNs, such transmissions are impeded by barriers of different strength. Existence of transmission barriers in the absence of amino acid mismatches in transmitting regions indicates that in complex prion domains multiple prion determinants act cooperatively to attain the final prion conformation, and reveals transmission barriers determined by this cooperative fold.
Prions, self-propagating protein conformations and causative agents of lethal neurodegenerative diseases, present a serious public health threat: they can arise sporadically and then spread by transmission to the same, as well as other, species. The risk of infecting humans with prions originating in wild and domestic animals is determined by the so-called transmission barriers. These barriers are attributed to differences in prion proteins from different species, but their underlying mechanisms are not clear. Recent findings that the prion state is transmitted through the interaction between short transmitting regions within prion domains revealed one type of transmission barrier, where productive templating is impeded by non-matching amino acids within transmitting regions. Here we present studies of the prion domain of the [PIN+]-forming protein, Rnq1, and describe a distinct type of transmission barrier not involving individual amino acid mismatches in the transmitting regions. Rnq1's prion domain is complex and encompasses four regions that can independently transmit the prion state. Our data suggest that multiple prion determinants of a complex prion domain act cooperatively to attain the prion conformation, and transmission barriers occur between protein variants that cannot form the same higher order structure, despite the identity of the region(s) driving the transmission.
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.
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.
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 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
Prions are self-propagating protein aggregates that are characteristically transmissible. In mammals, the PrP protein can form a prion that causes the fatal transmissible spongiform encephalopathies. Prions have also been uncovered in fungi, where they act as heritable, protein-based genetic elements. We previously showed that the yeast prion protein Sup35 can access the prion conformation in Escherichia coli. Here, we demonstrate that E. coli can propagate the Sup35 prion under conditions that do not permit its de novo formation. Furthermore, we show that propagation requires the disaggregase activity of the ClpB chaperone. Prion propagation in yeast requires Hsp104 (a ClpB ortholog), and prior studies have come to conflicting conclusions about ClpB's ability to participate in this process. Our demonstration of ClpB-dependent prion propagation in E. coli suggests that the cytoplasmic milieu in general and a molecular machine in particular are poised to support protein-based heredity in the bacterial domain of life.
Unlike most infectious agents—such as viruses or bacteria—that contain genetic material in the form of DNA or RNA, a prion is simply an aggregate of misfolded proteins. Although they are not living organisms, these prion aggregates can self-propagate; when they enter a healthy organism, they cause existing, correctly folded proteins to adopt the prion fold. Within the aggregate, the prion proteins have a corrugated structure that allows them to stack together tightly, which in turn makes the aggregates very stable. As more prions are formed, they then trigger other protein molecules to misfold and join the aggregates, and the aggregates continue to grow and spread within the infected organism causing tissue damage and cell death.
Prion diseases are well known in mammals, where the prion aggregates typically destroy tissue within the brain or nervous system. Bovine spongiform encephalopathy (also commonly known as BSE or ‘mad cow disease’) is an example of a prion disease that affects cattle and can be transmitted to humans by eating infected meat. Prions also form in yeast and other fungi. These prions, however, do not cause disease or cell death; instead, yeast prions act as protein-based elements that can be inherited over multiple generations and which provide the yeast with new traits or characteristics. Although prions can form spontaneously in yeast cells, their stable propagation depends on so-called chaperone proteins that help to remodel the prion aggregates. Previous work has shown that bacterial cells can also support the formation of prion-like aggregates. The bacteria were engineered to produce two yeast prion proteins—one of which spontaneously formed aggregates that were needed to trigger the conversion of the other to its prion form. However, it was not known if bacterial cells could support the stable propagation of prions if the initial trigger for prion conversion was removed.
Yuan et al. now reveal that the bacterium Escherichia coli can propagate a yeast prion for over a hundred generations, even when the cells can no longer make the protein that serves as the trigger for the initial conversion. This propagation depends on a bacterial chaperone protein called ClpB, which is related to another chaperone protein that is required for stable prion propagation in yeast. As such, the findings of Yuan et al. raise the possibility that, even though a prion specific to bacteria has yet to be identified, prions or prion-like proteins might also contribute to the diversity of traits found in bacteria. Furthermore, since both yeast and bacteria form and propagate prions in similar ways, such protein-based inheritance might have evolved in these organisms' common ancestor over two billion years ago.
prions; chaperones; Sup35; ClpB; protein-based heredity; E. coli; S. cerevisiae
The cause of Huntington's disease is expansion of polyglutamine (polyQ) domain in huntingtin, which makes this protein both neurotoxic and aggregation prone. Here we developed the first yeast model, which establishes a direct link between aggregation of expanded polyQ domain and its cytotoxicity. Our data indicated that deficiencies in molecular chaperones Sis1 and Hsp104 inhibited seeding of polyQ aggregates, whereas ssa1, ssa2, and ydj1–151 mutations inhibited expansion of aggregates. The latter three mutants strongly suppressed the polyQ toxicity. Spontaneous mutants with suppressed aggregation appeared with high frequency, and in all of them the toxicity was relieved. Aggregation defects in these mutants and in sis1–85 were not complemented in the cross to the hsp104 mutant, demonstrating an unusual type of inheritance. Since Hsp104 is required for prion maintenance in yeast, this suggested a role for prions in polyQ aggregation and toxicity. We screened a set of deletions of nonessential genes coding for known prions and related proteins and found that deletion of the RNQ1 gene specifically suppressed aggregation and toxicity of polyQ. Curing of the prion form of Rnq1 from wild-type cells dramatically suppressed both aggregation and toxicity of polyQ. We concluded that aggregation of polyQ is critical for its toxicity and that Rnq1 in its prion conformation plays an essential role in polyQ aggregation leading to the toxicity.
aggregation; polyglutamine; toxicity; prions; yeast
Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.
Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another
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.
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.
Molecular chaperones play a significant role in preventing protein misfolding and aggregation. Indeed, some protein conformational disorders have been linked to changes in the chaperone network. Curiously, in yeast, chaperones also play a role in promoting prion maintenance and propagation. While many amyloidogenic proteins are associated with disease in mammals, yeast prion proteins, and their ability to undergo conformational conversion into a prion state, are proposed to play a functional role in yeast biology. The chaperone Hsp104, a AAA+ ATPase, is essential for yeast prion propagation. Hsp104 fragments large prion aggregates to generate a population of smaller oligomers that can more readily convert soluble monomer and be transmitted to daughter cells. Here, we show that the middle (M) domain of Hsp104, and its mobility, plays an integral part in prion propagation. We generated and characterized mutations in the M-domain of Hsp104 that are predicted to stabilize either a repressed or de-repressed conformation of the M-domain (by analogy to ClpB in bacteria). We show that the predicted stabilization of the repressed conformation inhibits general chaperone activity. Mutation to the de-repressed conformation, however, has differential effects on ATP hydrolysis and disaggregation, suggesting that the M-domain is involved in coupling these two activities. Interestingly, we show that changes in the M-domain differentially affect the propagation of different variants of the [PSI+] and [RNQ+] prions, which indicates that some prion variants are more sensitive to changes in the M-domain mobility than others. Thus, we provide evidence that regulation of the M-domain of Hsp104 is critical for efficient prion propagation. This shows the importance of elucidating the function of the M-domain in order to understand the role of Hsp104 in the propagation of different prions and prion variants.
Sis1 and Ydj1, functionally distinct heat shock protein (Hsp)40 molecular chaperones of the yeast cytosol, are homologs of Hdj1 and Hdj2 of mammalian cells, respectively. Sis1 is necessary for propagation of the Saccharomyces cerevisiae prion [RNQ+]; Ydj1 is not. The ability to function in [RNQ+] maintenance has been conserved, because Hdj1 can function to maintain Rnq1 in an aggregated form in place of Sis1, but Hdj2 cannot. An extended glycine-rich region of Sis1, composed of a region rich in phenylalanine residues (G/F) and another rich in methionine residues (G/M), is critical for prion maintenance. Single amino acid alterations in a short stretch of amino acids of the G/F region of Sis1 that are absent in the otherwise highly conserved G/F region of Ydj1 cause defects in prion maintenance. However, there is some functional redundancy within the glycine-rich regions of Sis1, because a deletion of the adjacent glycine/methionine (G/M) region was somewhat defective in propagation of [RNQ+] as well. These results are consistent with a model in which the glycine-rich regions of Hsp40s contain specific determinants of function manifested through interaction with Hsp70s.