Curing of Yeast [URE3] Prion by the Hsp40 Cochaperone Ydj1p Is Mediated by Hsp70

[URE3] is a prion of the yeast Ure2 protein. Hsp40 is a cochaperone that regulates Hsp70 chaperone activity. When overexpressed, the Hsp40 Ydj1p cures yeast of [URE3], but the Hsp40 Sis1p does not. On the basis of biochemical data Ydj1p has been proposed to cure [URE3] by binding soluble Ure2p and preventing it from joining prion aggregates. Here, we mutagenized Ydj1p and find that disrupting substrate binding, dimerization, membrane association, or ability to transfer substrate to Hsp70 had little or no effect on curing. J-domain point mutations that disrupt functional interactions of Ydj1p with Hsp70 abolished curing, and the J domain alone cured [URE3]. Consistent with heterologous J domains possessing similar Hsp70 regulatory activity, the Sis1p J domain also cured [URE3]. We further show that Ydj1p is not essential for [URE3] propagation and that depletion of Ure2p is lethal in cells lacking Ydj1p. Our data imply that curing of [URE3] by overproduced Ydj1p does not involve direct interaction of Ydj1p with Ure2p but rather works through regulation of Hsp70 through a specific J-protein/Hsp70 interaction.     

Prion propagation by Hsp40 molecular chaperones

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.Key words: Hsp40, Ydj1, Sis1, amyloid, prion, Rnq1, J-protein, Hsp70     

Exchangeable chaperone modules contribute to specification of type I and type II Hsp40 cellular function

Hsp40 family members regulate Hsp70s ability to bind nonnative polypeptides and thereby play an essential role in cell physiology. Type I and type II Hsp40s, such as yeast Ydj1 and Sis1, form chaperone pairs with cytosolic Hsp70 Ssa1 that fold proteins with different efficiencies and carry out specific cellular functions. The mechanism by which Ydj1 and Sis1 specify Hsp70 functions is not clear. Ydj1 and Sis1 share a high degree of sequence identity in their amino and carboxyl terminal ends, but each contains a structurally unique and centrally located protein module that is implicated in chaperone function. To test whether the chaperone modules of Ydj1 and Sis1 function in the specification of Hsp70 action, we constructed a set of chimeric Hsp40s in which the chaperone domains of Ydj1 and Sis1 were swapped to form YSY and SYS. Purified SYS and YSY exhibited protein-folding activity and substrate specificity that mimicked that of Ydj1 and Sis1, respectively. In in vivo studies, YSY exhibited a gain of function and, unlike Ydj1, could complement the lethal phenotype of sis1 Delta and facilitate maintenance of the prion [RNQ+]. Ydj1 and Sis1 contain exchangeable chaperone modules that assist in specification of Hsp70 function.     

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.     

Hsp40s Specify Functions of Hsp104 and Hsp90 Protein Chaperone Machines

Hsp100 family chaperones of microorganisms and plants cooperate with the Hsp70/Hsp40/NEF system to resolubilize and reactivate stress-denatured proteins. In yeast this machinery also promotes propagation of prions by fragmenting prion polymers. We previously showed the bacterial Hsp100 machinery cooperates with the yeast Hsp40 Ydj1 to support yeast thermotolerance and with the yeast Hsp40 Sis1 to propagate [PSI⁺] prions. Here we find these Hsp40s similarly directed specific activities of the yeast Hsp104-based machinery. By assessing the ability of Ydj1-Sis1 hybrid proteins to complement Ydj1 and Sis1 functions we show their C-terminal substrate-binding domains determined distinctions in these and other cellular functions of Ydj1 and Sis1. We find propagation of [URE3] prions was acutely sensitive to alterations in Sis1 activity, while that of [PIN⁺] prions was less sensitive than [URE3], but more sensitive than [PSI⁺]. These findings support the ideas that overexpressing Ydj1 cures [URE3] by competing with Sis1 for interaction with the Hsp104-based disaggregation machine, and that different prions rely differently on activity of this machinery, which can explain the various ways they respond to alterations in chaperone function.     

The role of Sse1 in the de novo formation and variant determination of the [PSI+] prion

Yeast prions are a group of non-Mendelian genetic elements transmitted as altered and self-propagating conformations. Extensive studies in the last decade have provided valuable information on the mechanisms responsible for yeast prion propagation. How yeast prions are formed de novo and what cellular factors are required for determining prion "strains" or variants--a single polypeptide capable of existing in multiple conformations to result in distinct heritable phenotypes--continue to defy our understanding. We report here that Sse1, the yeast ortholog of the mammalian heat-shock protein 110 (Hsp110) and a nucleotide exchange factor for Hsp70 proteins, plays an important role in regulating [PSI+] de novo formation and variant determination. Overproduction of the Sse1 chaperone dramatically enhanced [PSI+] formation whereas deletion of SSE1 severely inhibited it. Only an unstable weak [PSI+] variant was formed in SSE1 disrupted cells whereas [PSI+] variants ranging from very strong to very weak were formed in isogenic wild-type cells under identical conditions. Thus, Sse1 is essential for the generation of multiple [PSI+] variants. Mutational analysis further demonstrated that the physical association of Sse1 with Hsp70 but not the ATP hydrolysis activity of Sse1 is required for the formation of multiple [PSI+] variants. Our findings establish a novel role for Sse1 in [PSI+] de novo formation and variant determination, implying that the mammalian Hsp110 may likewise be involved in the etiology of protein-folding diseases.     

Influence of prion variant and yeast strain variation on prion-molecular chaperone requirements

Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ⁺], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI⁺] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.Key words: Hsp40, Ssa, heat-shock, protein misfolding, cell stress, Hsp104, PIN, saccharomyces, neurodegenerative disease     

Influence of prion variant and yeast strain variation on prion-molecular chaperone requirements

Prions of budding yeast serve as a tractable model of amyloid behavior. Here we address the issue of the effect of yeast strain variation on prion stability, focusing also on the effect of amyloid conformation and the involvement of the co-chaperone Sis1, an essential J-protein partner of Hsp70. We found, despite an initial report to the contrary, that yeast strain background has little effect on the requirement for particular Sis1 domains for stable propagation of the prion [RNQ⁺], if the level of Sis1 expression is controlled. On the other hand, some variation in prion behavior was observed between yeast strains, in particular, the stability of certain [PSI⁺] variants. Future examination of such yeast strain-specific phenomena may provide useful insights into the basis of prion/chaperone dynamics.     

Variant‐specific and reciprocal Hsp40 functions in Hsp104‐mediated prion elimination

The amyloid‐based prions of Saccharomyces cerevisiae are heritable aggregates of misfolded proteins, passed to daughter cells following fragmentation by molecular chaperones including the J‐protein Sis1, Hsp70 and Hsp104. Overexpression of Hsp104 efficiently cures cell populations of the prion [PSI⁺] by an alternative Sis1‐dependent mechanism that is currently the subject of significant debate. Here, we broadly investigate the role of J‐proteins in this process by determining the impact of amyloid polymorphisms (prion variants) on the ability of well‐studied Sis1 constructs to compensate for Sis1 and ask whether any other S. cerevisiae cytosolic J‐proteins are also required for this process. Our comprehensive screen, examining all 13 members of the yeast cytosolic/nuclear J‐protein complement, uncovered significant variant‐dependent genetic evidence for a role of Apj1 (antiprion DnaJ) in this process. For strong, but not weak [PSI⁺] variants, depletion of Apj1 inhibits Hsp104‐mediated curing. Overexpression of either Apj1 or Sis1 enhances curing, while overexpression of Ydj1 completely blocks it. We also demonstrated that Sis1 was the only J‐protein necessary for the propagation of at least two weak [PSI⁺] variants and no J‐protein alteration, or even combination of alterations, affected the curing of weak [PSI⁺] variants, suggesting the possibility of biochemically distinct, variant‐specific Hsp104‐mediated curing mechanisms.     

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.     

In vivo bipartite interaction between the Hsp40 Sis1 and Hsp70 in Saccharomyces cerevisiae

The essential Hsp40, Sis1, is a J-protein cochaperone for the Ssa class of Hsp70's of Saccharomyces cerevisiae. Sis1 is required for the maintenance of the prion [RNQ(+)], as Sis1 lacking its 55-amino-acid glycine-rich region (G/F) does not maintain [RNQ(+)]. We report that overexpression of Sis1DeltaG/F in an otherwise wild-type strain had a negative effect on both cell growth and [RNQ(+)] maintenance, while overexpression of wild-type Sis1 did not. Overexpression of the related Hsp40 Ydj1 lacking its G/F region did not cause inhibition of growth, indicating that this dominant effect of Sis1DeltaG/F is not a characteristic shared by all Hsp40's. Analysis of small deletions within the SIS1 G/F region indicated that the observed dominant effects were caused by the absence of sequences known to be important for Sis1's unique cellular functions. These inhibitory effects of Sis1DeltaG/F were obviated by alterations in the N-terminal J-domain of Sis1 that affect interaction with Ssa's ATPase domain. In addition, a genetic screen designed to isolate additional mutations that relieved these inhibitory effects identified two residues in Sis1's carboxy-terminal domain. These alterations disrupted the interaction of Sis1 with the 10-kD carboxy-terminal regulatory domain of Ssa1, indicating that Sis1 has a bipartite interaction with Ssa in vivo.     

Specificity of class II Hsp40 Sis1 in maintenance of yeast prion [RNQ+

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

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

Structural variants of yeast prions show conformer‐specific requirements for chaperone activity

Molecular chaperones monitor protein homeostasis and defend against the misfolding and aggregation of proteins that is associated with protein conformational disorders. In these diseases, a variety of different aggregate structures can form. These are called prion strains, or variants, in prion diseases, and cause variation in disease pathogenesis. Here, we use variants of the yeast prions [RNQ+] and [PSI+] to explore the interactions of chaperones with distinct aggregate structures. We found that prion variants show striking variation in their relationship with Hsp40s. Specifically, the yeast Hsp40 Sis1 and its human orthologue Hdj1 had differential capacities to process prion variants, suggesting that Hsp40 selectivity has likely changed through evolution. We further show that such selectivity involves different domains of Sis1, with some prion conformers having a greater dependence on particular Hsp40 domains. Moreover, [PSI+] variants were more sensitive to certain alterations in Hsp70 activity as compared to [RNQ+] variants. Collectively, our data indicate that distinct chaperone machinery is required, or has differential capacity, to process different aggregate structures. Elucidating the intricacies of chaperone‐client interactions, and how these are altered by particular client structures, will be crucial to understanding how this system can go awry in disease and contribute to pathological variation.     

Hsp40 interacts directly with the native state of the yeast prion protein Ure2 and inhibits formation of amyloid-like fibrils

Ure2 is the protein determinant of the [URE3] prion phenotype in Saccharomyces cerevisiae and consists of a flexible N-terminal prion-determining domain and a globular C-terminal glutathione transferase-like domain. Overexpression of the type I Hsp40 member Ydj1 in yeast cells has been found to result in the loss of [URE3]. However, the mechanism of prion curing by Ydj1 remains unclear. Here we tested the effect of overexpression of Hsp40 members Ydj1, Sis1, and Apj1 and also Hsp70 co-chaperones Cpr7, Cns1, Sti1, and Fes1 in vivo and found that only Ydj1 showed a strong curing effect on [URE3]. We also investigated the interaction of Ydj1 with Ure2 in vitro. We found that Ydj1 was able to suppress formation of amyloid-like fibrils of Ure2 by delaying the process of fibril formation, as monitored by thioflavin T binding and atomic force microscopy imaging. Controls using bovine serum albumin, Sis1, or the human Hsp40 homologues Hdj1 or Hdj2 showed no significant inhibitory effect. Ydj1 was only effective when added during the lag phase of fibril formation, suggesting that it interacts with Ure2 at an early stage in fibril formation and delays the nucleation process. Using surface plasmon resonance and size exclusion chromatography, we demonstrated a direct interaction between Ydj1 and both wild type and N-terminally truncated Ure2. In contrast, Hdj2, which did not suppress fibril formation, did not show this interaction. The results suggest that Ydj1 inhibits Ure2 fibril formation by binding to the native state of Ure2, thus delaying the onset of oligomerization.     

Molecular chaperones and the assembly of the prion Sup35p, an in vitro study

The protein Sup35 from Saccharomyces cerevisiae possesses prion properties. In vivo, a high molecular weight form of Sup35p is associated to the [PSI+] factor. The continued propagation of [PSI+] is highly dependent on the expression levels of molecular chaperones from the Hsp100, 70 and 40 families; however, so far, their role in this process is unclear. We have developed a reproducible in vitro system to study the effects of molecular chaperones on the assembly of full-length Sup35p. We show that Hsp104p greatly stimulates the assembly of Sup35p into fibrils, whereas Ydj1p has inhibitory effect. Hsp82p, Ssa1p and Sis1p, individually, do not affect assembly. In contrast, Ssa1p together with either of its Hsp40 cochaperones blocks Sup35p polymerization. Furthermore, Ssa1p and Ydj1p or Sis1p can counteract the stimulatory activity of Hsp104p, by forming complexes with Sup35p oligomers, in an ATP-dependent manner. Our observations reveal the functional differences between Hsp104p and the Hsp70-40 systems in the assembly of Sup35p into fibrils and bring new insight into the mechanism by which molecular chaperones influence the propagation of [PSI+].     

Molecular dynamics simulations of Hsp40 J-domain mutants identifies disruption of the critical HPD-motif as the key factor for impaired curing in vivo of the yeast prion [URE3]

Genetic screens using Saccharomyces cerevisiae have identified an array of Hsp40 (Ydj1p) J-domain mutants that are impaired in the ability to cure the yeast [URE3] prion through disrupting functional interactions with Hsp70. However, biochemical analysis of some of these Hsp40 J-domain mutants has so far failed to provide major insight into the specific functional changes in Hsp40-Hsp70 interactions. To explore the detailed structural and dynamic properties of the Hsp40 J-domain, 20 ns molecular dynamic simulations of 4 mutants (D9A, D36A, A30T, and F45S) and wild-type J-domain were performed, followed by Hsp70 docking simulations. Results demonstrated that although the Hsp70 interaction mechanism of the mutants may vary, the major structural change was targeted to the critical HPD motif of the J-domain. Our computational analysis fits well with previous yeast genetics studies regarding highlighting the importance of J-domain function in prion propagation. During the molecular dynamics simulations several important residues were identified and predicted to play an essential role in J-domain structure. Among these residues, Y26 and F45 were confirmed, using both in silico and in vivo methods, as being critical for Ydj1p function.     

Functional Diversification of Hsp40: Distinct J-Protein Functional Requirements for Two Prions Allow for Chaperone-Dependent Prion Selection

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.     

Prion propagation by Hsp40 molecular chaperones

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.     

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.