A new perspective on Hsp104-mediated propagation and curing of the yeast prion [PSI+]

Most prions in yeast form amyloid fibrils that must be severed by the protein disaggregase Hsp104 to be propagated and transmitted efficiently to newly formed buds. Only one yeast prion, [PSI⁺], is cured by Hsp104 overexpression. We investigated the interaction between Hsp104 and Sup35, the priongenic protein in yeast that forms the [PSI⁺] prion.1 Helsen CW, Glover JR. Insight into molecular basis of curing of [PSI+] prion by overexpression of 104-kDa heat shock protein (Hsp104). J Biol Chem 2012; 287:542 - 56; http://dx.doi.org/10.1074/jbc.M111.302869; PMID: 22081611 [Crossref], [PubMed], [Web of Science ®] , [Google Scholar] We found that a 20-amino acid segment within the highly-charged, unstructured middle domain of Sup35 contributes to the physical interaction between the middle domain and Hsp104. When this segment was deleted from Sup35, the efficiency of [PSI⁺] severing was substantially reduced, resulting in larger Sup35 particles and weakening of the [PSI⁺] phenotype. Furthermore, [PSI⁺] in these cells was completely resistant to Hsp104 curing. The affinity of Hsp104 was considerably weaker than that of model Hsp104-binding proteins and peptides, implying that Sup35 prions are not ideal substrates for Hsp104-mediated remodeling. In light of this finding, we present a modified model of Hsp104-mediated [PSI⁺] propagation and curing that requires only partial remodeling of Sup35 assembled into amyloid fibrils.     

定量研究菲啶对酵母朊病毒[PSI+]的治愈作用

为系统研究菲啶对酵母朊病毒的治愈效果,借助表达融合蛋白GFP-Sup35p的酵母朊病毒模型(NGMC),引入半变性琼脂糖凝胶电泳技术和荧光漂白后恢复技术在蛋白和细胞水平定量分析了菲啶对酵母朊病毒的治愈效果。结果表明,蛋白和细胞水平采用的定量分析方法能够精确定量菲啶对酵母朊病毒的治愈作用,菲啶作用酵母朊病毒[PSI+]1~5 d的治愈率分别为0%、0%、51.7%、87.5%和94.4%。另外,菲啶作用酵母朊病毒[PSI+]细胞1~2 d后出现的粉色菌落中朊病毒的聚集状态与[PSI+]相似,而3~5 d后出现的粉色菌落中朊病毒的状态与[psi-]相似。     

Curing of [PSI+] by Hsp104 Overexpression: Clues to solving the puzzle

The yeast [PSI⁺] prion, which is the amyloid form of Sup35, has the unusual property of being cured not only by the inactivation of, but also by the overexpression of Hsp104. Even though this latter observation was made more than two decades ago, the mechanism of curing by Hsp104 overexpression has remained controversial. This question has been investigated in depth by our laboratory by combining live cell imaging of GFP-labeled Sup35 with standard plating assays of yeast overexpressing Hsp104. We will discuss why the curing of [PSI⁺] by Hsp104 overexpression is not compatible with a mechanism of either inhibition of severing of the prion seeds or asymmetric segregation of the seeds. Instead, our recent data (J. Biol. Chem. 292:8630-8641) indicate that curing is due to dissolution of the prion seeds, which in turn is dependent on the trimming activity of Hsp104. This trimming activity decreases the size of the seeds by dissociating monomers from the fibers, but unlike Hsp104 severing activity, it does not increase the number of prion seeds. Finally, we will discuss the other factors that affect the curing of [PSI⁺] by Hsp104 overexpression and how these factors may relate to the trimming activity of Hsp104.     

Effects of the [PSI+] prion on rates of adaptation in yeast

The [PSI(+)] prion in yeast has been shown to improve short-term growth in some environments, but its effects on rates of adaptation have not been assessed before now. We adapted three yeast genotypes to three novel environments in the presence and the absence of the prion. There were significant differences in adaptation rates between lines with different combinations of genotype, environment, and prion status. We saw no consistent effect, however, of the prion on the rate of adaptation to new environments. A major factor affecting the rate of adaptation was initial fitness in the new environment: lines with low initial fitness evolved faster than lines with high initial fitness.     

Differences in the curing of [PSI+] prion by various methods of Hsp104 inactivation

[PSI(+)] yeast, containing the misfolded amyloid conformation of Sup35 prion, is cured by inactivation of Hsp104. There has been controversy as to whether inactivation of Hsp104 by guanidine treatment or by overexpression of the dominant negative Hsp104 mutant, Hsp104-2KT, cures [PSI(+)] by the same mechanism- inhibition of the severing of the prion seeds. Using live cell imaging of Sup35-GFP, overexpression of Hsp104-2KT caused the foci to increase in size, then decrease in number, and finally disappear when the cells were cured, similar to that observed in cells cured by depletion of Hsp104. In contrast, guanidine initially caused an increase in foci size but then the foci disappeared before the cells were cured. By starving the yeast to make the foci visible in cells grown with guanidine, the number of cells with foci was found to correlate exactly with the number of [PSI(+)] cells, regardless of the curing method. Therefore, the fluorescent foci are the prion seeds required for maintenance of [PSI(+)] and inactivation of Hsp104 cures [PSI(+)] by preventing severing of the prion seeds. During curing with guanidine, the reduction in seed size is an Hsp104-dependent effect that cannot be explained by limited severing of the seeds. Instead, in the presence of guanidine, Hsp104 retains an activity that trims or reduces the size of the prion seeds by releasing Sup35 molecules that are unable to form new prion seeds. This Hsp104 activity may also occur in propagating yeast.     

Structure-based view on [PSI+] prion properties

ABSTRACT

Yeast [PSI⁺] prion is one of the most suitable and well characterized system for the investigation of the prion phenomenon. However, until recently, the lack of data on the 3D arrangement of Sup35p prion fibrils hindered progress in this area. The recent arrival in this field of new experimental techniques led to the parallel and in-register superpleated β-structure as a consensus model for Sup35p fibrils. Here, we analyzed the effect of amino acid substitutions of the Sup35 protein through the prism of this structural model. Application of a newly developed computational approach, called ArchCandy, gives us a better understanding of the effect caused by mutations on the fibril forming potential of Sup35 protein. This bioinformatics tool can be used for the design of new mutations with desired modification of prion properties. Thus, we provide examples of how today, having progress toward elucidation of the structural arrangement of Sup35p fibrils, researchers can advance more efficiently to a better understanding of prion [PSI⁺] stability and propagation.     

Nucleocytoplasmic trafficking of the molecular chaperone Hsp104 in unstressed and heat-shocked cells

Hsp104 is a molecular chaperone in yeast that restores solubility and activity to inactivated proteins after severe heat shock. We investigated the mechanisms that influence Hsp104 subcellular distribution in both unstressed and heat-shocked cells. In unstressed cells, Hsp104 and a green fluorescent protein-Hsp104 fusion protein were detected in both the nucleus and the cytoplasm. We demonstrate that a 17-amino-acid sequence of Hsp104 nuclear localization sequence 17 (NLS17) is sufficient to target a reporter molecule to the nucleus and is also necessary for normal Hsp104 subcellular distribution. The nuclear targeting function of NLS17 is genetically dependent on KAP95 and KAP121. In addition, wild-type Hsp104, but not an NLS17-mutated Hsp104 variant, accumulated in the nucleus of cells depleted for the general export factor Xpo1. Interestingly, severe, nonlethal heat shock enhances the nuclear levels of Hsp104 in an NLS17-independent manner. Under these conditions, we demonstrate that karyopherin-mediated nuclear transport is impaired, while the integrity of the nuclear-cytoplasmic barrier remains intact. Based on these observations, we propose that Hsp104 continues to access the nucleus during severe heat shock using a karyopherin-independent mechanism.     

Substoichiometric Hsp104 regulates the genesis and persistence of self-replicable amyloid seeds of Sup35 prion domain

Prion-like self-perpetuating conformational conversion of proteins is involved in both transmissible neurodegenerative diseases in mammals and non-Mendelian inheritance in yeast. The transmissibility of amyloid-like aggregates is dependent on the stoichiometry of chaperones such as heat shock proteins (Hsps), including disaggregases. To provide the mechanistic underpinnings of the formation and persistence of prefibrillar amyloid seeds, we investigated the role of substoichiometric Hsp104 on the in vitro amyloid aggregation of the prion domain (NM-domain) of Saccharomyces cerevisiae Sup35. At low substoichiometric concentrations, we show Hsp104 exhibits a dual role: it considerably accelerates the formation of prefibrillar species by shortening the lag phase but also prolongs their persistence by introducing unusual kinetic halts and delaying their conversion into mature amyloid fibers. Additionally, Hsp104-modulated amyloid species displayed a better seeding capability compared to NM-only amyloids. Using biochemical and biophysical tools coupled with site-specific dynamic readouts, we characterized the distinct structural and dynamical signatures of these amyloids. We reveal that Hsp104-remodeled amyloidogenic species are compositionally diverse in prefibrillar aggregates and are packed in a more ordered fashion compared to NM-only amyloids. Finally, we show these Hsp104-remodeled, conformationally distinct NM aggregates display an enhanced autocatalytic self-templating ability that might be crucial for phenotypic outcomes. Taken together, our results demonstrate that substoichiometric Hsp104 promotes compositional diversity and conformational modulations during amyloid formation, yielding effective prefibrillar seeds that are capable of driving prion-like Sup35 propagation. Our findings underscore the key functional and pathological roles of substoichiometric chaperones in prion-like propagation.     

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI⁺] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates.     

Patterns of [PSI+] aggregation allow insights into cellular organization of yeast prion aggregates

The yeast prion phenomenon is very widespread and mounting evidence suggests that it has an impact on cellular regulatory mechanisms related to phenotypic responses to changing environments. Studying the aggregation patterns of prion amyloids during different stages of the prion life cycle is a first key step to understand major principles of how and where cells generate, organize and turn-over prion aggregates. The induction of the [PSI⁺] state involves the actin cytoskeleton and quality control compartments such as the Insoluble Protein Deposit (IPOD). An initially unstable transitional induction state can be visualized by overexpression of the prion determinant and displays characteristic large ring- and ribbon-shaped aggregates consisting of poorly fragmented bundles of very long prion fibrils. In the mature prion state, the aggregation pattern is characterized by highly fragmented, shorter prion fibrils that form aggregates, which can be visualized through tagging with fluorescent proteins. The number of aggregates formed varies, ranging from a single large aggregate at the IPOD to multiple smaller ones, depending on several parameters discussed. Aggregate units below the resolution of light microscopy that are detectable by fluorescence correlation spectroscopy are in equilibrium with larger aggregates in this stage and can mediate faithful inheritance of the prion state. Loss of the prion state is often characterized by reduced fragmentation of prion fibrils and fewer, larger aggregates.     

Effects of mutations in yeast prion [PSI+] on amyloid toxicity manifested in Escherichia coli strain BL21

We previously showed that over production of a fusion protein in which the prion domain of Saccharomyces cerevisiae [PSI+] is connected to glutathione S-transferase (GST-Sup35NM) causes a marked decrease in the colony forming ability of Escherichia coli strain BL21 after reaching stationary phase. Evidence indicated that the observed toxicity was attributable to intracellular formation of fibrous aggregates of GST-Sup35NM. In this report, we describe the isolation of plasmids that encode mutant forms of GST-Sup35NM which do not confer the toxicity to E. coli strain BL21. Each of the four spontaneous mutant-forms of GST-Sup35NM obtained revealed amino acid substitutions. One substitution was located in the N domain, and the others in the M domain. Congo red binding assay indicated that none of these mutant protein underwent conformational alteration in vitro. From these results, we conclude that the M domain, in collaboration with the N domain, plays an essential role in aggregation of Sup35NM. In addition, our data demonstrate the usefulness of the E. coli expression system in studying aggregate-forming proteins.     

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.     

The ribosome-associated complex antagonizes prion formation in yeast

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

The ribosome-associated complex antagonizes prion formation in yeast

Abstract

The number of known fungal proteins capable of switching between alternative stable conformations is steadily increasing, suggesting that a prion-like mechanism may be broadly utilized as a means to propagate altered cellular states. To gain insight into the mechanisms by which cells regulate prion formation and toxicity we examined the role of the yeast ribosome-associated complex (RAC) in modulating both the formation of the [PSI⁺] prion – an alternative conformer of Sup35 protein – and the toxicity of aggregation-prone polypeptides. The Hsp40 RAC chaperone Zuo1 anchors the RAC to ribosomes and stimulates the ATPase activity of the Hsp70 chaperone Ssb. We found that cells lacking Zuo1 are sensitive to over-expression of some aggregation-prone proteins, including the Sup35 prion domain, suggesting that co-translational protein misfolding increases in Δzuo1 strains. Consistent with this finding, Δzuo1 cells exhibit higher frequencies of spontaneous and induced prion formation. Cells expressing mutant forms of Zuo1 lacking either a C-terminal charged region required for ribosome association, or the J-domain responsible for Ssb ATPase stimulation, exhibit similarly high frequencies of prion formation. Our findings are consistent with a role for the RAC in chaperoning nascent Sup35 to regulate folding of the N-terminal prion domain as it emerges from the ribosome.     

Biotin-assisted folding of streptavidin on the yeast surface

Yeast surface display allows heterologously expressed proteins to be targeted to the exterior of the cell wall and thus has a potential as a biotechnology platform. In this study, we report the successful display of functional streptavidin on the yeast surface. Streptavidin binds the small molecule biotin with high affinity (K(d) ≈ 10(-14)M) and is used widely in applications that require stable noncovalent interaction, including immobilization of biotinylated compounds on a solid surface. As such, engineering functional streptavidin on the yeast surface may find novel uses in future biotechnology applications. Although the molecule does not require any post-translational modification, streptavidin is difficult to fold in bacteria. We show that Saccharomyces cerevisiae can fold the protein correctly if induced at 20°C. Contrary to a previous report, coexpression of anchored and soluble streptavidin subunits is not necessary, as expressing the anchored subunit alone is sufficient to form a functional complex. For unstable monomer mutants, however, addition of free biotin during protein induction is necessary to display a functional molecule, suggesting that biotin helps the monomer fold. To show that surface displayed streptavidin can be used to immobilize other biomolecules, we used it to capture biotinylated antibody, which is then used to immunoprecipitate a protein target.     

A Systematic Evaluation of the Function of the Protein-Remodeling Factor Hsp104 in [PSI+] Prion Propagation in S. cerevisiae by Comprehensive Chromosomal Mutations

The yeast prion [PSI⁺] represents an aggregated state of the translational release factor Sup35 (eRF3) and deprives termination complexes of functional Sup35, resulting in nonsense codon suppression. Protein-remodeling factor Hsp104 is involved in thermotolerance and [PSI⁺] propagation, however the structure-and-function relationship of Hsp104 for [PSI⁺] remains unclear. In this study, we engineered 58 chromosomal hsp104 mutants that affect residues considered structurally or functionally relevant to Hsp104 remodeling activity, yet most remain to be examined for their significance to [PSI⁺] in the same genetic background. Many of these hsp104 mutants were affected both in thermotolerance and [PSI⁺] propagation. However, nine mutants were impaired exclusively for [PSI⁺], while two mutants were impaired exclusively for thermotolerance. Mutations exclusively affecting [PSI⁺] are clustered around the lateral channel of the Hsp104 hexamer. These findings suggest that Hsp104 possesses shared as well as distinct remodeling activities for stress-induced protein aggregates and [PSI⁺] prion aggregates and that the lateral channel plays a role specific to [PSI⁺] prion propagation.     

The effects of glutamine/asparagine content on aggregation and heterologous prion induction by yeast prion-like domains

Prion-like domains are low complexity, intrinsically disordered domains that compositionally resemble yeast prion domains. Many prion-like domains are involved in the formation of either functional or pathogenic protein aggregates. These aggregates range from highly dynamic liquid droplets to highly ordered detergent-insoluble amyloid-like aggregates. To better understand the amino acid sequence features that promote conversion to stable, detergent-insoluble aggregates, we used the prediction algorithm PAPA to identify predicted aggregation-prone prion-like domains with a range of compositions. While almost all of the predicted aggregation-prone domains formed foci when expressed in cells, the ability to form the detergent-insoluble aggregates was highly correlated with glutamine/asparagine (Q/N) content, suggesting that high Q/N content may specifically promote conversion to the amyloid state in vivo. We then used this data set to examine cross-seeding between prion-like proteins. The prion protein Sup35 requires the presence of a second prion, [PIN⁺], to efficiently form prions, but this requirement can be circumvented by the expression of various Q/N-rich protein fragments. Interestingly, almost all of the Q/N-rich domains that formed SDS-insoluble aggregates were able to promote prion formation by Sup35, highlighting the highly promiscuous nature of these interactions.     

N-terminal domain of yeast Hsp104 chaperone is dispensable for thermotolerance and prion propagation but necessary for curing prions by Hsp104 overexpression

Hsp104 is a hexameric protein chaperone that resolubilizes stress-damaged proteins from aggregates. Hsp104 promotes [PSI(+)] prion propagation by breaking prion aggregates, which propagate as amyloid fibers, into more numerous prion "seeds." Inactivating Hsp104 cures cells of [PSI(+)] and other amyloid-like yeast prions. Overexpressing Hsp104 also eliminates [PSI(+)], presumably by completely resolubilizing prion aggregates. Inexplicably, however, excess Hsp104 does not cure the other prions. Here we identify missense mutations in Hsp104's amino-terminal domain (NTD), which is conserved among Hsp100 proteins but whose function is unknown, that improve [PSI(+)] propagation. Hsp104Delta147, engineered to lack the NTD, supported [PSI(+)] and functioned normally in thermotolerance and protein disaggregation. Hsp104Delta147 failed to cure [PSI(+)] when overexpressed, however, implying that excess Hsp104 does not eliminate [PSI(+)] by direct dissolution of prion aggregates. Curing of [PSI(+)] by overexpressing catalytically inactive Hsp104 (Hsp104KT), which interferes with endogenous Hsp104, did not require the NTD. We further found that Hsp104 mutants defective in threading peptides through the hexamer pore had reduced ability to support [PSI(+)] in proportion to protein resolubilization defects, suggesting that [PSI(+)] propagation depends on this threading and that Hsp104 "breaks" prion aggregates by extracting protein monomers from the amyloid fibers.     

The [PSI+] prion of Saccharomyces cerevisiae can be propagated by an Hsp104 orthologue from Candida albicans

The molecular chaperone Hsp104 is not only a key component of the cellular machinery induced to disassemble aggregated proteins in stressed cells of Saccharomyces cerevisiae but also plays an essential role in the propagation of the [PSI⁺], [URE3], and [RNQ/PIN⁺] prions in this organism. Here we demonstrate that the fungal pathogen Candida albicans carries an 899-residue stress-inducible orthologue of Hsp104 (CaHsp104) that shows a high degree of amino acid identity to S. cerevisiae Hsp104 (ScHsp104). This identity is significantly lower in the N- and C-terminal regions implicated in substrate recognition and cofactor binding, respectively. CaHsp104 is able to provide all known functions of ScHsp104 in an S. cerevisiae hsp104 null mutant, i.e., tolerance to high-temperature stress, reactivation of heat-denatured proteins, and propagation of the [PSI⁺] prion. As also observed for ScHsp104, overexpression of CaHsp104 leads to a loss of the [PSI⁺] prion. However, unlike that of ScHsp104, CaHsp104 function is resistant to guanidine hydrochloride (GdnHCl), an inhibitor of the ATPase activity of this chaperone. These findings have implications both in terms of the mechanism of inhibition of Hsp104 by GdnHCl and in the evolution of the ability of fungal species to propagate prions.