Destabilizing interactions among [PSI(+)] and [PIN(+)] yeast prion variants

The yeast Sup35 and Rnq1 proteins can exist in either the noninfectious soluble forms, [psi-] or [pin-], respectively, or the multiple infectious amyloid-like forms called [PSI+] or [PIN+] prion variants (or prion strains). It was previously shown that [PSI+] and [PIN+] prions enhance one another's de novo appearance. Here we show that specific prion variants of [PSI+] and [PIN+] disrupt each other's stable inheritance. Acquiring [PSI+] often impedes the inheritance of particular [PIN+] variants. Conversely, the presence of some [PIN+] variants impairs the inheritance of weak [PSI+] but not strong [PSI+] variants. These same [PIN+] variants generate a single-dot fluorescence pattern when a fusion of Rnq1 and green fluorescent protein is expressed. Another [PIN+] variant, which forms a distinctly different multiple-dot fluorescence pattern, does not impair [PSI+] inheritance. Thus, destabilization of prions by heterologous prions depends upon the variants involved. These findings may have implications for understanding interactions among other amyloid-forming proteins, including those associated with certain human diseases.     

Specificity of prion assembly in vivo. [PSI+] and [PIN+] form separate structures in yeast

The yeast prions [PSI+] and [PIN+] are self-propagating amyloid aggregates of the Gln/Asn-rich proteins Sup35p and Rnq1p, respectively. Like the mammalian PrP prion "strains," [PSI+] and [PIN+] exist in different conformations called variants. Here, [PSI+] and [PIN+] variants were used to model in vivo interactions between co-existing heterologous amyloid aggregates. Two levels of structural organization, like those previously described for [PSI+], were demonstrated for [PIN+]. In cells with both [PSI+] and [PIN+] the two prions formed separate structures at both levels. Also, the destabilization of [PSI+] by certain [PIN+] variants was shown not to involve alterations in the [PSI+] prion size. Finally, when two variants of the same prion that have aggregates with distinct biochemical characteristics were combined in a single cell, only one aggregate type was propagated. These studies demonstrate the intracellular organization of yeast prions and provide insight into the principles of in vivo amyloid assembly.     

Small heat shock proteins potentiate amyloid dissolution by protein disaggregases from yeast and humans

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.     

Hsp110 chaperones regulate prion formation and propagation in S. cerevisiae by two discrete activities

The cytosolic chaperone network of Saccharomyces cerevisiae is intimately associated with the emergence and maintenance of prion traits. Recently, the Hsp110 protein, Sse1, has been identified as a nucleotide exchange factor (NEF) for both cytosolic Hsp70 chaperone family members, Ssa1 and Ssb1. We have investigated the role of Sse1 in the de novo formation and propagation of [PSI(+)], the prion form of the translation termination factor, Sup35. As observed by others, we find that Sse1 is essential for efficient prion propagation. Our results suggest that the NEF activity is required for maintaining sufficient levels of substrate-free Ssa1. However, Sse1 exhibits an additional NEF-independent activity; it stimulates in vitro nucleation of Sup35NM, the prion domain of Sup35. We also observe that high levels of Sse1, but not of an unrelated NEF, very potently inhibit Hsp104-mediated curing of [PSI(+)]. Taken together, these results suggest a chaperone-like activity of Sse1 that assists in stabilization of early folding intermediates of the Sup35 prion conformation. This activity is not essential for prion formation under conditions of Sup35 overproduction, however, it may be relevant for spontaneous [PSI(+)] formation as well as for protection of the prion trait upon physiological Hsp104 induction.     

New aspects of research upon the yeast Saccharomyces cerevisiae [PSI+] prion

One of the key feature of prions is the ability to be stable in two alternative conformations. Besides the intensively studied mammalian prions, there are also prion proteins present in the yeast Saccharomyces cerevisiae. Research in this field has lead to opposite hypotheses that explain the sense of presence of [PSI+] prion in yeast cells. Some authors postulate e of role of the prions in the evolution of S. cerevisiae, whereas other investigators point out the negative influence of these particles upon the yeast physiology. In recent years, yeast prions are used for anti-prion drug screening, because of common features with mammalian prions. This work presents the most intensively studied fields of the research carried out on [PSI+] prion in yeast.     

Nucleotide exchange factors for Hsp70s are required for [URE3] prion propagation in Saccharomyces cerevisiae

The [URE3] and [PSI(+)] prions are infectious amyloid forms of Ure2p and Sup35p. Several chaperones influence prion propagation: Hsp104p overproduction destabilizes [PSI(+)], whereas [URE3] is sensitive to excess of Ssa1p or Ydj1p. Here, we show that overproduction of the chaperone, Sse1p, can efficiently cure [URE3]. Sse1p and Fes1p are nucleotide exchange factors for Ssa1p. Interestingly, deletion of either SSE1 or FES1 completely blocked [URE3] propagation. In addition, deletion of SSE1 also interfered with [PSI(+)] propagation.     

Prions affect the appearance of other prions: the story of [PIN(+)

Prions are self-propagating protein conformations. Recent research brought insight into prion propagation, but how they first appear is unknown. We previously established that the yeast non-Mendelian trait [PIN(+)] is required for the de novo appearance of the [PSI(+)] prion. Here, we show that the presence of prions formed by Rnq1 or Ure2 is sufficient to make cells [PIN(+)]. Thus, [PIN(+)] can be caused by more than one prion. Furthermore, an unbiased functional screen for [PIN(+)] prions uncovered the known prion gene, URE2, the proposed prion gene, NEW1, and nine novel candidate prion genes all carrying prion domains. Importantly, the de novo appearance of Rnq1::GFP prion aggregates also requires the presence of other prions, suggesting the existence of a general mechanism by which the appearance of prions is enhanced by heterologous prion aggregates.     

Prion aggregate structure in yeast cells is determined by the Hsp104-Hsp110 disaggregase machinery

Prions consist of misfolded proteins that have adopted an infectious amyloid conformation. In vivo, prion biogenesis is intimately associated with the protein quality control machinery. Using electron tomography, we probed the effects of the heat shock protein Hsp70 chaperone system on the structure of a model yeast [PSI+] prion in situ. Individual Hsp70 deletions shift the balance between fibril assembly and disassembly, resulting in a variable shell of nonfibrillar, but still immobile, aggregates at the surface of the [PSI+] prion deposits. Both Hsp104 (an Hsp100 disaggregase) and Sse1 (the major yeast form of Hsp110) were localized to this surface shell of [PSI+] deposits in the deletion mutants. Elevation of Hsp104 expression promoted the appearance of this novel, nonfibrillar form of the prion aggregate. Moreover, Sse1 was found to regulate prion fibril length. Our studies reveal a key role for Sse1 (Hsp110), in cooperation with Hsp104, in regulating the length and assembly state of [PSI+] prion fibrils in vivo.     

Variant-specific [PSI+] infection is transmitted by Sup35 polymers within [PSI+] aggregates with heterogeneous protein composition

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.     

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant

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.     

Effects of ubiquitin system alterations on the formation and loss of a yeast prion

The yeast prion [PSI+] is a self-propagating amyloidogenic isoform of the translation termination factor Sup35. Overproduction of the chaperone protein Hsp104 results in loss of [PSI+]. Here we demonstrate that this effect is decreased by deletion of either the gene coding for one of the major yeast ubiquitin-conjugating enzymes, Ubc4, or the gene coding for the ubiquitin-recycling enzyme, Ubp6. The effect of ubc4Delta on [PSI+] loss was increased by depletion of the Hsp70 chaperone Ssb but was not influenced by depletion of Ubp6. This indicates that Ubc4 affects [PSI+] loss via a pathway that is the same as the one affected by Ubp6 but not by Ssb. In the presence of Rnq1 protein, ubc4Delta also facilitates spontaneous de novo formation of [PSI+]. This stimulation is independent of [PIN+], the prion isoform of Rnq1. Numerous attempts failed to detect ubiquitinated Sup35 in the yeast extracts. While ubc4Delta and other alterations of ubiquitin system used in this work cause slight induction of some Hsps, these changes are insufficient to explain their effect on [PSI+]. However, ubc4Delta increases the proportion of the Hsp70 chaperone Ssa bound to Sup35, suggesting that misfolded Sup35 is either more abundant or more accessible to the chaperones in the absence of Ubc4. The proportion of [PSI+] cells containing large aggregated Sup35 structures is also increased by ubc4Delta. We propose that UPS alterations induce an adaptive response, resulting in accumulation of the large "aggresome"-like aggregates that promote de novo prion generation and prion recovery from the chaperone treatment.     

Characterization of Hsp70 binding and nucleotide exchange by the yeast Hsp110 chaperone Sse1

SSE1 and SSE2 encode the essential yeast members of the Hsp70-related Hsp110 molecular chaperone family. Both mammalian Hsp110 and the Sse proteins functionally interact with cognate cytosolic Hsp70s as nucleotide exchange factors. We demonstrate here that Sse1 forms high-affinity (Kd approximately 10-8 M) heterodimeric complexes with both yeast Ssa and mammalian Hsp70 chaperones and that binding of ATP to Sse1 is required for binding to Hsp70s. Sse1.Hsp70 heterodimerization confers resistance to exogenously added protease, indicative of conformational changes in Sse1 resulting in a more compact structure. The nucleotide binding domains of both Sse1/2 and the Hsp70s dictate interaction specificity and are sufficient for mediating heterodimerization with no discernible contribution from the peptide binding domains. In support of a strongly conserved functional interaction between Hsp110 and Hsp70, Sse1 is shown to associate with and promote nucleotide exchange on human Hsp70. Nucleotide exchange activity by Sse1 is physiologically significant, as deletion of both SSE1 and the Ssa ATPase stimulatory protein YDJ1 is synthetically lethal. The Hsp110 family must therefore be considered an essential component of Hsp70 chaperone biology in the eukaryotic cell.     

SWI], the prion formed by the chromatin remodeling factor Swi1, is highly sensitive to alterations in Hsp70 chaperone system activity

The yeast prion [SWI+], formed of heritable amyloid aggregates of the Swi1 protein, results in a partial loss of function of the SWI/SNF chromatin-remodeling complex, required for the regulation of a diverse set of genes. Our genetic analysis revealed that [SWI+] propagation is highly dependent upon the action of members of the Hsp70 molecular chaperone system, specifically the Hsp70 Ssa, two of its J-protein co-chaperones, Sis1 and Ydj1, and the nucleotide exchange factors of the Hsp110 family (Sse1/2). Notably, while all yeast prions tested thus far require Sis1, [SWI+] is the only one known to require the activity of Ydj1, the most abundant J-protein in yeast. The C-terminal region of Ydj1, which contains the client protein interaction domain, is required for [SWI+] propagation. However, Ydj1 is not unique in this regard, as another, closely related J-protein, Apj1, can substitute for it when expressed at a level approaching that of Ydj1. While dependent upon Ydj1 and Sis1 for propagation, [SWI+] is also highly sensitive to overexpression of both J-proteins. However, this increased prion-loss requires only the highly conserved 70 amino acid J-domain, which serves to stimulate the ATPase activity of Hsp70 and thus to stabilize its interaction with client protein. Overexpression of the J-domain from Sis1, Ydj1, or Apj1 is sufficient to destabilize [SWI+]. In addition, [SWI+] is lost upon overexpression of Sse nucleotide exchange factors, which act to destabilize Hsp70's interaction with client proteins. Given the plethora of genes affected by the activity of the SWI/SNF chromatin-remodeling complex, it is possible that this sensitivity of [SWI+] to the activity of Hsp70 chaperone machinery may serve a regulatory role, keeping this prion in an easily-lost, meta-stable state. Such sensitivity may provide a means to reach an optimal balance of phenotypic diversity within a cell population to better adapt to stressful environments.     

Prion diseases of yeast: amyloid structure and biology

Prion "variants" or "strains" are prions with the identical protein sequence, but different characteristics of the prion infection: e.g. different incubation periods for scrapie strains or different phenotype intensities for yeast prion variants. We have shown that infectious amyloids of the yeast prions [PSI+], [URE3] and [PIN+] each have an in-register parallel β-sheet architecture. Moreover, we have pointed out that this amyloid architecture can explain how one protein can faithfully transmit any of several conformations to new protein monomers. This explains how proteins can be genes.     

Antagonistic interactions between yeast [PSI(+)] and [URE3] prions and curing of [URE3] by Hsp70 protein chaperone Ssa1p but not by Ssa2p

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.     

Molecular chaperone Hsp104 can promote yeast prion generation

[URE3] is an amyloid-based prion of Ure2p, a regulator of nitrogen catabolism in Saccharomyces cerevisiae. The Ure2p of the human pathogen Candida albicans can also be a prion in S. cerevisiae. We find that overproduction of the disaggregating chaperone, Hsp104, increases the frequency of de novo [URE3] prion formation by the Ure2p of S. cerevisiae and that of C. albicans. This stimulation is strongly dependent on the presence of the [PIN(+)] prion, known from previous work to enhance [URE3] prion generation. Our data suggest that transient Hsp104 overproduction enhances prion generation through persistent effects on Rnq1 amyloid, as well as during overproduction by disassembly of amorphous Ure2 aggregates (generated during Ure2p overproduction), driving the aggregation toward the amyloid pathway. Overproduction of other major cytosolic chaperones of the Hsp70 and Hsp40 families (Ssa1p, Sse1p, and Ydj1p) inhibit prion formation, whereas another yeast Hsp40, Sis1p, modulates the effects of Hsp104p on both prion induction and prion curing in a prion-specific manner. The same factor may both enhance de novo prion generation and destabilize existing prion variants, suggesting that prion variants may be selected by changes in the chaperone network.     

Yeast [PSI+] prion aggregates are formed by small Sup35 polymers fragmented by Hsp104

The yeast [PSI+] determinant is related to formation of large prion-like aggregates of the conformationally altered Sup35 protein. Here, we show that these aggregates are composed of small Sup35 prion polymers and associated proteins. In contrast to other protein complexes of yeast lysates, but similarly to amyloid fibers, these polymers are insoluble in SDS at room temperature. The polymers on average are about 30-fold smaller than the aggregates and comprise from 8 to 50 Sup35 monomers. The size of polymers is characteristic of a given [PSI+] variant and differs between the variants. Blocked expression of Hsp104 chaperone causes gradual increase in the size of prion polymers, while inactivation of Hsp104 by guanidine HCl completely stops their fragmentation, which shows indispensability of Hsp104 for this process.     

Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104)

Yeast prions are a powerful model for understanding the dynamics of protein aggregation associated with a number of human neurodegenerative disorders. The AAA+ protein disaggregase Hsp104 can sever the amyloid fibrils produced by yeast prions. This action results in the propagation of "seeds" that are transmitted to daughter cells during budding. Overexpression of Hsp104 eliminates the [PSI+] prion but not other prions. Using biochemical methods we identified Hsp104 binding sites in the highly charged middle domain of Sup35, the protein determinant of [PSI+]. Deletion of a short segment of the middle domain (amino acids 129-148) diminishes Hsp104 binding and strongly affects the ability of the middle domain to stimulate the ATPase activity of Hsp104. In yeast, [PSI+] maintained by Sup35 lacking this segment, like other prions, is propagated by Hsp104 but cannot be cured by Hsp104 overexpression. These results provide new insight into the enigmatic specificity of Hsp104-mediated curing of yeast prions and sheds light on the limitations of the ability of Hsp104 to eliminate aggregates produced by other aggregation-prone proteins.     

Tah1, A Key Component of R2TP Complex that Regulates Assembly of snoRNP,is Involved in De Novo Generation and Maintenance of Yeast Prion [URE3]

The cellular chaperone machinery plays key role in the de novo formation and propagation of yeast prions (infectious protein). Though the role of Hsp70s in the prion maintenance is well studied, how Hsp90 chaperone machinery affects yeast prions remains unclear. In the current study, we examined the role of Hsp90 and its co-chaperones on yeast prions [PSI⁺] and [URE3]. We show that the overproduction of Hsp90 co-chaperone Tah1, cures [URE3] which is a prion form of native protein Ure2 in yeast. The Hsp90 co-chaperone Tah1 is involved in the assembly of small nucleolar ribonucleoproteins (snoRNP) and chromatin remodelling complexes. We found that Tah1 deletion improves the frequency of de novo appearance of [URE3]. The Tah1 was found to interact with Hsp70. The lack of Tah1 not only represses antagonizing effect of Ssa1 Hsp70 on [URE3] but also improves the prion strength suggesting role of Tah1 in both fibril growth and replication. We show that the N-terminal tetratricopeptide repeat domain of Tah1 is indispensable for [URE3] curing. Tah1 interacts with Ure2, improves its solubility in [URE3] strains, and affects the kinetics of Ure2 fibrillation in vitro. Its inhibitory role on Ure2 fibrillation is proposed to influence [URE3] propagation. The present study shows a novel role of Tah1 in yeast prion propagation, and that Hsp90 not only promotes its role in ribosomal RNA processing but also in the prion maintenance.SummaryPrions are self-perpetuating infectious proteins. What initiates the misfolding of a protein into its prion form is still not clear. The understanding of cellular factors that facilitate or antagonize prions is crucial to gain insight into the mechanism of prion formation and propagation. In the current study, we reveal that Tah1 is a novel modulator of yeast prion [URE3]. The Hsp90 co-chaperone Tah1, is required for the formation of small nucleolar ribonucleoprotein complex. We show that the absence of Tah1 improves the induction of [URE3] prion. The overexpressed Tah1 cures [URE3], and this function is promoted by Hsp90 chaperones. The current study thus provides a novel cellular factor and the underlying mechanism, involved in the prion formation and propagation