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 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
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.
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
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 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.
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
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.
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 [URE3] prion of Saccharomyces cerevisiae is a self-propagating amyloid form of Ure2p. The amino-terminal prion domain of Ure2p is necessary and sufficient for prion formation and has a high glutamine (Q) and asparagine (N) content. Such Q/N-rich domains are found in two other yeast prion proteins, Sup35p and Rnq1p, although none of the many other yeast Q/N-rich domain proteins have yet been found to be prions. To examine the role of amino acid sequence composition in prion formation, we used Ure2p as a model system and generated five Ure2p variants in which the order of the amino acids in the prion domain was randomly shuffled while keeping the amino acid composition and C-terminal domain unchanged. Surprisingly, all five formed prions in vivo, with a range of frequencies and stabilities, and the prion domains of all five readily formed amyloid fibers in vitro. Although it is unclear whether other amyloid-forming proteins would be equally resistant to scrambling, this result demonstrates that [URE3] formation is driven primarily by amino acid composition, largely independent of primary sequence.
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.
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
During propagation, yeast prions show a strict sequence preference that confers the specificity of prion assembly. Although propagations of [PSI+] and [RNQ+] are independent of each other, the appearance of [PSI+] is facilitated by the presence of [RNQ+]. To explain the [RNQ+] effect on the appearance of [PSI+], the cross-seeding model was suggested, in which Rnq1 aggregates act as imperfect templates for Sup35 aggregation. If cross-seeding events take place in the cytoplasm of yeast cells, the collision frequency between Rnq1 aggregates and Sup35 will affect the appearance of [PSI+]. In this study, to address whether cross-seeding occurs in vivo, a new [PSI+] induction method was developed that exploits a protein fusion between the prion domain of Sup35 (NM) and Rnq1. This fusion protein successfully joins preexisting Rnq1 aggregates, which should result in the localization of NM around the Rnq1 aggregates and hence in an increased collision frequency between NM and Rnq1 aggregates. The appearance of [PSI+] could be induced very efficiently, even with a low expression level of the fusion protein. This study supports the occurrence of in vivo cross-seeding between Sup35 and Rnq1 and provides a new tool that can be used to dissect the mechanism of the de novo appearance of prions.
Prions 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.
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
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.
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.
Prions are self-propagating, infectious aggregates of misfolded proteins. The mammalian prion, PrPSc, causes fatal neurodegenerative disorders. Fungi also have prions. While yeast prions depend upon glutamine/asparagine(Q/N)-rich regions, the Podospora anserina HET-s and PrP prion proteins, lack such sequences. Nonetheless, we show that the HET-s prion domain fused to GFP propagates as a prion in yeast. Analogously to native yeast prions: transient overexpression of the HET-s fusion induces ring-like aggregates that propagate in daughter cells as cytoplasmically-inherited, detergent-resistant dot aggregates. Efficient dot propagation, but not ring formation, is dependent upon the Hsp104 chaperone. The yeast prion [PIN+] enhances HET-s ring formation, suggesting that prions with and without Q/N-rich regions interact. Finally, HET-s aggregates propagated in yeast are infectious when introduced into Podospora. To our knowledge, this is the first report of prion propagation in a truly foreign host. Since yeast can host non Q/N-rich prions, such native yeast prions may exist.
Onset of proteotoxicity is linked to change in the subcellular location of proteins that cause misfolding diseases. Yet, factors that drive changes in disease protein localization and the impact of residence in new surroundings on proteotoxicity are not entirely clear. To address these issues, we examined aspects of proteotoxicity caused by Rnq1-green fluorescent protein (GFP) and a huntingtin's protein exon-1 fragment with an expanded polyglutamine tract (Htt-103Q), which is dependent upon the intracellular presence of [RNQ+] prions. Increasing heat-shock protein 40 chaperone activity before Rnq1-GFP expression, shifted Rnq1-GFP aggregation from the cytosol to the nucleus. Assembly of Rnq1-GFP into benign amyloid-like aggregates was more efficient in the nucleus than cytosol and nuclear accumulation of Rnq1-GFP correlated with reduced toxicity. [RNQ+] prions were found to form stable complexes with Htt-103Q, and nuclear Rnq1-GFP aggregates were capable of sequestering Htt-103Q in the nucleus. On accumulation in the nucleus, conversion of Htt-103Q into SDS-resistant aggregates was dramatically reduced and Htt-103Q toxicity was exacerbated. Alterations in activity of molecular chaperones, the localization of intracellular interaction partners, or both can impact the cellular location of disease proteins. This, in turn, impacts proteotoxicity because the assembly of proteins to a benign state occurs with different efficiencies in the cytosol and nucleus.
Amyloid protein aggregation is involved in serious neurodegenerative disorders such as Alzheimer's disease and transmissible encephalopathies. The concept of an infectious protein (prion) being the scrapie agent was successfully validated for several yeast and fungi proteins. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically and biochemically identified as prion proteins. Studies on these proteins have revealed critical information on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by rich β sheet content. In a previous work on the HET-s prion protein Podospora, we demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the HET-s prion domain associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review, as are relevant questions about the [Het-s] system of Podospora anserina.
prion; HET-s; Podospora; amyloid; infectious; β sheet; mutagenesis; fold; propagation
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 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.
[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.
Yeast prions, based on self-seeded highly ordered fibrous aggregates (amyloids), serve as a model for human amyloid diseases. Propagation of yeast prions depends on the balance between chaperones of the Hsp100 and Hsp70 families. The yeast prion [PSI+] can be eliminated by an excess of the chaperone Hsp104. This effect is reversed by an excess of the chaperone Hsp70-Ssa. Here we show that the actions of Hsp104 and Ssa on [PSI+] are modulated by the small glutamine-rich tetratricopeptide cochaperone Sgt2. Sgt2 is conserved from yeast to humans, has previously been implicated in the guided entry of tail-anchored proteins (GET) trafficking pathway, and is known to interact with Hsps, cytosolic Get proteins, and tail-anchored proteins. We demonstrate that Sgt2 increases the ability of excess Ssa to counteract [PSI+] curing by excess Hsp104. Deletion of SGT2 also restores trafficking of a tail-anchored protein in cells with a disrupted GET pathway. One region of Sgt2 interacts both with the prion domain of Sup35 and with tail-anchored proteins. Sgt2 levels are increased in response to the presence of a prion when major Hsps are not induced. Our data implicate Sgt2 as an amyloid “sensor” and a regulator of chaperone targeting to different types of aggregation-prone proteins.
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