Chimeric yeast prions with unstable inheritance

The Saccharomyces cerevisiae [PSI] factor, a cytoplasmic omnipotent nonsense suppressor, is a conformationally changed (prion) form of translation termination factor eRF3 (Sup35p). Induction and maintenance of the [PSI] factor depend on the prionizing peptide located in the N domain of Sup35p. The N domain of Sup35p was fused with phosphoribosylaminoimidazole carboxylase (Ade2p), a purine biosynthesis enzyme, and the hybrid protein (NM-Sup35p::Ade2p) was tested for induction of the [PSI] factor. Transformation with a centromeric plasmid carrying the gene for NM-Sup35p::Ade2p induced a [PSI]-like factor in yeast cells, which was evident from efficient nonsense suppression. The suppressory effect depended on the presence of the prionizing peptide both in the hybrid protein and in Sup35p synthesized from the chromosomal gene, as well as on the presence of the prion-like [PIN] factor in the cell.     

A role for the proteasome in the turnover of Sup35p and in [PSI+] prion propagation

Yeast prions are superb models for understanding the mechanisms of self‐perpetuating protein aggregates formation. [PSI⁺] stands among the most documented yeast prions and results from self‐assembly of the translation termination factor Sup35p into protein fibrils. A plethora of cellular factors were shown to affect [PSI⁺] formation and propagation. Clearance of Sup35p prion particles is however poorly understood and documented. Here, we investigated the role of the proteasome in the degradation of Sup35p and in [PSI⁺] prion propagation. We found that cells lacking the RPN4 gene, which have reduced intracellular proteasome pools, accumulated Sup35p and have defects in [PSI⁺] formation and propagation. Sup35p is degraded in vitro by the 26S and 20S proteasomes in a ubiquitin‐independent manner, generating an array of amyloidogenic peptides derived from its prion‐domain. We also demonstrate the formation of a proteasome‐resistant fragment spanning residues 83–685 which is devoid of the prion‐domain that is essential for [PSI⁺] propagation. Most important was the finding that the 26S and 20S proteasomes degrade Sup35p fibrils in vitro and abolish their infectivity. Our results point to an overlooked role of the proteasome in clearing toxic protein aggregates, and have important implications for a better understanding of the life cycle of infectious protein assemblies.     

Growth phase‐dependent changes in the size and infectivity of SDS‐resistant Sup35p assemblies associated with the [PSI+] prion in yeast

The translation termination factor Sup35p can form self‐replicating fibrillar aggregates responsible for the [PSI⁺] prion state. Sup35p aggregation yields detergent‐resistant assemblies detectable on agarose gels under semi‐denaturant conditions and fluorescent puncta within the yeast cytosol when the protein is fused to GFP. It is still unclear whether any of these manifestations of [PSI⁺] truly correspond to the Sup35p assemblies that faithfully transmit the [PSI⁺] prion from mother to daughter cells. The infectious titer of prions in cells can be indirectly assessed by the ability of [PSI⁺] cells lysates to induce the prion state when introduced into naïve cells. Here, we report that the dramatic changes in the size and amounts of SDS‐resistant Sup35p that occur during growth do not correlate with the infectious titer. Our results suggest that fluorescent Sup35‐GFP puncta and detergent‐resistant Sup35p assemblies are good indicators of Sup35p conversion to the prion state but not of infectious particles number.     

Prion-Dependent Lethality of sup45 Mutants in Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae translation termination factors eRF1 (Sup45) and eRF3 (Sup35) are encoded by the essential genes SUP45 and SUP35 respectively. Heritable aggregation of Sup35 results in formation of the yeast prion [PSI⁺]. It is known that combination of [PSI⁺] with some mutant alleles of the SUP35 or SUP45 genes in one and the same haploid yeast cell causes synthetic lethality. In this study, we perform detailed analysis of synthetic lethality between various sup45 nonsense and missense mutations on one hand, and different variants of [PSI⁺] on the other hand. Synthetic lethality with sup45 mutations was detected for [PSI⁺] variants of different stringencies. Moreover, we demonstrate for the first time that in some combinations, synthetic lethality is dominant and occurs at the postzygotic stage after only a few cell divisions. The tRNA suppressor SUQ5 counteracts the prion-dependent lethality of the nonsense alleles but not of the missense alleles of SUP45, indicating that the lethal effect is due to the depletion of Sup45. Synthetic lethality is also suppressed in the presence of the C-proximal fragment of Sup35 (Sup35C) that lacks the prion domain and cannot be included into the prion aggregates. Remarkably, the production of Sup35C in a sup45 mutant strain is also accompanied by an increase in the Sup45 levels, suggesting that translationally active Sup35 up-regulates Sup45 or protects it from degradation.Key Words: Sup45, Sup35, eRF1, eRF3, amyloid, [PSI⁺], translation termination, Saccharomyces cerevisiae     

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.     

Yeast J-protein Sis1 prevents prion toxicity by moderating depletion of prion protein

[PSI⁺] is a prion of Saccharomyces cerevisiae Sup35, an essential ribosome release factor. In [PSI⁺] cells, most Sup35 is sequestered into insoluble amyloid aggregates. Despite this depletion, [PSI⁺] prions typically affect viability only modestly, so [PSI⁺] must balance sequestering Sup35 into prions with keeping enough Sup35 functional for normal growth. Sis1 is an essential J-protein regulator of Hsp70 required for the propagation of amyloid-based yeast prions. C-terminally truncated Sis1 (Sis1JGF) supports cell growth in place of wild-type Sis1. Sis1JGF also supports [PSI⁺] propagation, yet [PSI⁺] is highly toxic to cells expressing only Sis1JGF. We searched extensively for factors that mitigate the toxicity and identified only Sis1, suggesting Sis1 is uniquely needed to protect from [PSI⁺] toxicity. We find the C-terminal substrate-binding domain of Sis1 has a critical and transferable activity needed for the protection. In [PSI⁺] cells that express Sis1JGF in place of Sis1, Sup35 was less soluble and formed visibly larger prion aggregates. Exogenous expression of a truncated Sup35 that cannot incorporate into prions relieved [PSI⁺] toxicity. Together our data suggest that Sis1 has separable roles in propagating Sup35 prions and in moderating Sup35 aggregation that are crucial to the balance needed for the propagation of what otherwise would be lethal [PSI⁺] prions.     

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.Key Words: Hsp104, reverse genetics, hexamer, nonsense suppression, yeast prion [PSI⁺], thermotolerance     

Oxidative stress conditions increase the frequency of de novo formation of the yeast [PSI+] prion

Prions are self‐perpetuating amyloid protein aggregates which underlie various neurodegenerative diseases in mammals and heritable traits in yeast. The molecular basis of how yeast and mammalian prions form spontaneously into infectious amyloid‐like structures is poorly understood. We have explored the hypothesis that oxidative stress is a general trigger for prion formation using the yeast [PSI⁺] prion, which is the altered conformation of the Sup35 translation termination factor. We show that the frequency of [PSI⁺] prion formation is elevated under conditions of oxidative stress and in mutants lacking key antioxidants. We detect increased oxidation of Sup35 methionine residues in antioxidant mutants and show that overexpression of methionine sulphoxide reductase abrogates both the oxidation of Sup35 and its conversion to the [PSI⁺] prion. [PSI⁺] prion formation is particularly elevated in a mutant lacking the Sod1 Cu,Zn‐superoxide dismutase. We have used fluorescence microscopy to show that the de novo appearance of [PSI⁺] is both rapid and increased in frequency in this mutant. Finally, electron microscopy analysis of native Sup35 reveals that similar fibrillar structures are formed in both the wild‐type and antioxidant mutants. Together, our data indicate that oxidative stress is a general trigger of [PSI⁺] formation, which can be alleviated by antioxidant defenses.     

Over‐expression of the molecular chaperone Hsp104 in Saccharomyces cerevisiae results in the malpartition of [PSI+] propagons

The ability of a yeast cell to propagate [PSI⁺], the prion form of the Sup35 protein, is dependent on the molecular chaperone Hsp104. Inhibition of Hsp104 function in yeast cells leads to a failure to generate new propagons, the molecular entities necessary for [PSI⁺] propagation in dividing cells and they get diluted out as cells multiply. Over‐expression of Hsp104 also leads to [PSI⁺] prion loss and this has been assumed to arise from the complete disaggregation of the Sup35 prion polymers. However, in conditions of Hsp104 over‐expression in [PSI⁺] cells we find no release of monomers from Sup35 polymers, no monomerization of aggregated Sup35 which is not accounted for by the proportion of prion‐free [psi^‐] cells present, no change in the molecular weight of Sup35‐containing SDS‐resistant polymers and no significant decrease in average propagon numbers in the population as a whole. Furthermore, they show that over‐expression of Hsp104 does not interfere with the incorporation of newly synthesised Sup35 into polymers, nor with the multiplication of propagons following their depletion in numbers while growing in the presence of guanidine hydrochloride. Rather, they present evidence that over‐expression of Hsp104 causes malpartition of [PSI⁺] propagons between mother and daughter cells in a sub‐population of cells during cell division thereby generating prion‐free [psi^?] cells.     

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 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.     

Prion and Nonprion Amyloids: A Comparison Inspired by the Yeast Sup35 Protein

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.Key Words: amyloid, prion, Rnq1, Sup35, Ure2, translation termination, yeast     

The small heat shock protein Hsp31 cooperates with Hsp104 to modulate Sup35 prion aggregation

The yeast homolog of DJ-1, Hsp31, is a multifunctional protein that is involved in several cellular pathways including detoxification of the toxic metabolite methylglyoxal and as a protein deglycase. Prior studies ascribed Hsp31 as a molecular chaperone that can inhibit α-Syn aggregation in vitro and alleviate its toxicity in vivo. It was also shown that Hsp31 inhibits Sup35 aggregate formation in yeast, however, it is unknown if Hsp31 can modulate [PSI⁺] phenotype and Sup35 prionogenesis. Other small heat shock proteins, Hsp26 and Hsp42 are known to be a part of a synergistic proteostasis network that inhibits Sup35 prion formation and promotes its disaggregation. Here, we establish that Hsp31 inhibits Sup35 [PSI⁺] prion formation in collaboration with a well-known disaggregase, Hsp104. Hsp31 transiently prevents prion induction but does not suppress induction upon prolonged expression of Sup35 indicating that Hsp31 can be overcome by larger aggregates. In addition, elevated levels of Hsp31 do not cure [PSI⁺] strains indicating that Hsp31 cannot intervene in a pre-existing prion oligomerization cycle. However, Hsp31 can modulate prion status in cooperation with Hsp104 because it inhibits Sup35 aggregate formation and potentiates [PSI⁺] prion curing upon overexpression of Hsp104. The absence of Hsp31 reduces [PSI⁺] prion curing by Hsp104 without influencing its ability to rescue cellular thermotolerance. Hsp31 did not synergize with Hsp42 to modulate the [PSI⁺] phenotype suggesting that both proteins act on similar stages of the prion cycle. We also showed that Hsp31 physically interacts with Hsp104 and together they prevent Sup35 prion toxicity to greater extent than if they were expressed individually. These results elucidate a mechanism for Hsp31 on prion modulation that suggest it acts at a distinct step early in the Sup35 aggregation process that is different from Hsp104. This is the first demonstration of the modulation of [PSI⁺] status by the chaperone action of Hsp31. The delineation of Hsp31's role in the chaperone cycle has implications for understanding the role of the DJ-1 superfamily in controlling misfolded proteins in neurodegenerative disease and cancer.     

Physical properties of polymorphic yeast prion amyloid fibers

Amyloid fibers play important roles in many human diseases and natural biological processes and have immense potential as novel nanomaterials. We explore the physical properties of polymorphic amyloid fibers formed by yeast prion protein Sup35. Amyloid fibers that conferred distinct prion phenotypes ([PSI⁺]), strong (S) versus weak (W) nonsense suppression, displayed different physical properties. Both S[PSI⁺] and W[PSI⁺] fibers contained structural inhomogeneities, specifically local regions of static curvature in S[PSI⁺] fibers and kinks and self-cross-linking in W[PSI⁺] fibers. Force-extension experiments with optical tweezers revealed persistence lengths of 1.5 μm and 3.3 μm and axial stiffness of 5600 pN and 9100 pN for S[PSI⁺] and W[PSI⁺] fibers, respectively. Thermal fluctuation analysis confirmed the twofold difference in persistence length between S[PSI⁺] and W[PSI⁺] fibers and revealed a torsional stiffness of kinks and cross-links of ∼100–200 pN·nm/rad.     

Qualitative and quantitative multiplexed proteomic analysis of complex yeast protein fractions that modulate the assembly of the yeast prion Sup35p

Background

The aggregation of the baker''s yeast prion Sup35p is at the origin of the transmissible [PSI⁺] trait. We and others have shown that molecular chaperones modulate Sup35p aggregation. However, other protein classes might be involved in [PSI⁺] formation.

Results

We designed a functional proteomic study that combines two techniques to identify modulators of Sup35p aggregation and describe the changes associated to [PSI⁺] formation. The first allows measuring the effect of fractionated Saccharomyces cerevisiae cytosolic extracts from [PSI⁺] and [psi⁻] yeast cells on Sup35p assembly. The second is a multiplex qualitative and quantitative comparison of protein composition of active and inactive fractions using a gel-free and label-free LC-MS approach. We identify changes in proteins involved in translation, folding, degradation, oxido-reduction and metabolic processes.

Conclusion

Our functional proteomic study provides the first inventory list of over 300 proteins that directly or indirectly affect Sup35p aggregation and [PSI⁺] formation. Our results highlight the complexity of the cellular changes accompanying [PSI⁺] formation and pave the way for in vitro studies aimed to document the effect of individual and/or combinations of proteins identified here, susceptible of affecting Sup35p assembly.     

Autophagy protects against de novo formation of the [PSI+] prion in yeast

Prions are self-propagating, infectious proteins that underlie several neurodegenerative diseases. The molecular basis underlying their sporadic formation is poorly understood. We show that autophagy protects against de novo formation of [PSI⁺], which is the prion form of the yeast Sup35 translation termination factor. Autophagy is a cellular degradation system, and preventing autophagy by mutating its core components elevates the frequency of spontaneous [PSI⁺] formation. Conversely, increasing autophagic flux by treating cells with the polyamine spermidine suppresses prion formation in mutants that normally show a high frequency of de novo prion formation. Autophagy also protects against the de novo formation of another prion, namely the Rnq1/[PIN⁺] prion, which is not related in sequence to the Sup35/[PSI⁺] prion. We show that growth under anaerobic conditions in the absence of molecular oxygen abrogates Sup35 protein damage and suppresses the high frequency of [PSI⁺] formation in an autophagy mutant. Autophagy therefore normally functions to remove oxidatively damaged Sup35, which accumulates in cells grown under aerobic conditions, but in the absence of autophagy, damaged/misfolded Sup35 undergoes structural transitions favoring its conversion to the propagatable [PSI⁺] form.     

Yeast Prions: Evolution of the Prion Concept

Prions (infectious proteins) analogous to the scrapie agent have been identified in Saccharomyces cerevisiae and Podospora anserina based on their special genetic characteristics. Each is a protein acting as a gene, much like nucleic acids have been shown to act as enzymes. The [URE3], [PSI⁺], [PIN⁺] and [Het-s] prions are self-propagating amyloids of Ure2p, Sup35p, Rnq1p and the HET-s protein, respectively. The [β] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI⁺] are clearly diseases, while [Het-s] and [β] carry out normal cell functions. Surprisingly, the prion domains of Ure2p and Sup35p can be randomized without loss of ability to become a prion. Thus amino acid content and not sequence determine these prions. Shuffleability also suggests amyloids with a parallel in-register β-sheet structure.Key Words: Ure2, Sup35, Rnq1, HETs, PrP, prion, amyloid     

Increased [PSI+] Appearance by Fusion of Rnq1 with the Prion Domain of Sup35 in Saccharomyces cerevisiae

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 were originally defined as proteinaceous infectious particles responsible for transmissible spongiform encephalopathies in mammals (reviewed in reference 23). Since a non-Mendelian genetic element, [URE3], was identified as a yeast prion (37), however, this concept has been expanded to include protein-based genetic elements. In addition to [URE3], there are at least two more proteinaceous genetic elements in Saccharomyces cerevisiae, namely, [PSI⁺] and [RNQ⁺] (20, 22, 28). [Het-s] was also identified as a prion in the filamentous fungus Podospora anserina (2).Despite the absence of any structural and functional homologies between various prion proteins from different organisms, they share a common feature, i.e., prion proteins can adopt two distinct conformational states. One of these, the aggregated prion state, can stimulate the soluble, nonprion conformation to convert into the prion form. Gaining intermolecular β-sheet structures, purified yeast prion proteins aggregate and form amyloid fibers in vitro (8, 12, 28, 32). Protein extract from yeast cells in the prion state can facilitate the in vitro polymerization of soluble prion protein from nonprion cells (21), and amyloid fibers of purified yeast prion proteins can convert the cells into the prion state when introduced into yeast cells, demonstrating the protein-only hypothesis (15, 31). Thus, intracellular prion aggregates are thought to have the same structural basis as amyloid fibers formed in vitro.Yeast prion biology has provided invaluable insights into the prion concept at the molecular level. Because of its experimental convenience, [PSI⁺] has been investigated most intensively among various yeast prions. [PSI⁺] results from the aggregation of Sup35 protein, which is essential for terminating the translation at stop codons. When Sup35 is in the [PSI⁺] aggregated state, ribosomes often fail to release polypeptides at stop codons, causing a non-Mendelian trait which is easily detected by nonsense suppression. ade1 or ade2 nonsense mutants are used as marker genes to determine the [PSI⁺] state. These mutants cannot grow on adenine-deficient medium and form red colonies on medium supplemented with a limiting amount of adenine, such as yeast extract-peptone-dextrose (YPD). ade mutants in the [PSI⁺] state, however, can grow on adenine-deficient medium and form white colonies, as they produce functional Ade1 or Ade2 by virtue of a nonsense mutation readthrough. To sustain propagation, all yeast prions need the disaggregation activity of Hsp104, which can be inhibited by guanidine hydrochloride (GuHCl) (9). Since yeast prions are cured by growth on guanidine-containing medium, prion phenotypes can easily be distinguished from chromosomal suppressor mutants.Sup35 (eRF3) of S. cerevisiae has a prion-determining N-terminal domain (N), a highly charged middle domain (M) that confers solubility on the molecule, and an essential C-terminal domain that binds guanine nucleotides and stimulates the polypeptide release reaction catalyzed by Sup45 (eRF1) (17, 29, 33). The de novo appearance of [PSI⁺] can be induced by overexpression of SUP35 or its prion domain-containing fragments (NM) (6). [PSI⁺] induction, however, can be achieved only in [RNQ⁺] cells that harbor the prion state of the Rnq1 protein (4, 19). Two hypotheses about how [RNQ⁺] can affect the appearance of [PSI⁺] have been suggested. One is an inhibitor titration model that postulates the molecules preventing the aggregation of Sup35 and the recruitment of these inhibitors to Rnq1 aggregates in [RNQ⁺] cells. The other is a cross-seeding model in which Rnq1 aggregates directly catalyze the polymerization of Sup35. In vitro cross-seeding between different amyloidogenic proteins was reported, and Rnq1 amyloid fiber can also act as a seed for Sup35 polymerization in vitro (7, 13). These in vitro data support the possibility of cross-seeding between Rnq1 and Sup35. However, because the milieu of cytoplasm is very different from that of a test tube, whether this cross-seeding really occurs in vivo is still obscure. For this study, we developed a new, robust [PSI⁺] induction method that confirms the cross-seeding events in the cytoplasmic environment.     

Heterologous Aggregates Promote De Novo Prion Appearance via More than One Mechanism

Prions are self-perpetuating conformational variants of particular proteins. In yeast, prions cause heritable phenotypic traits. Most known yeast prions contain a glutamine (Q)/asparagine (N)-rich region in their prion domains. [PSI⁺], the prion form of Sup35, appears de novo at dramatically enhanced rates following transient overproduction of Sup35 in the presence of [PIN⁺], the prion form of Rnq1. Here, we establish the temporal de novo appearance of Sup35 aggregates during such overexpression in relation to other cellular proteins. Fluorescently-labeled Sup35 initially forms one or a few dots when overexpressed in [PIN⁺] cells. One of the dots is perivacuolar, colocalizes with the aggregated Rnq1 dot and grows into peripheral rings/lines, some of which also colocalize with Rnq1. Sup35 dots that are not near the vacuole do not always colocalize with Rnq1 and disappear by the time rings start to grow. Bimolecular fluorescence complementation failed to detect any interaction between Sup35-VN and Rnq1-VC in [PSI ⁺][PIN ⁺] cells. In contrast, all Sup35 aggregates, whether newly induced or in established [PSI ⁺], completely colocalize with the molecular chaperones Hsp104, Sis1, Ssa1 and eukaryotic release factor Sup45. In the absence of [PIN⁺], overexpressed aggregating proteins such as the Q/N-rich Pin4C or the non-Q/N-rich Mod5 can also promote the de novo appearance of [PSI ⁺]. Similar to Rnq1, overexpressed Pin4C transiently colocalizes with newly appearing Sup35 aggregates. However, no interaction was detected between Mod5 and Sup35 during [PSI⁺] induction in the absence of [PIN ⁺]. While the colocalization of Sup35 and aggregates of Rnq1 or Pin4C are consistent with the model that the heterologous aggregates cross-seed the de novo appearance of [PSI ⁺], the lack of interaction between Mod5 and Sup35 leaves open the possibility of other mechanisms. We also show that Hsp104 is required in the de novo appearance of [PSI⁺] aggregates in a [PIN ⁺]-independent pathway.     

Effect of Charged Residues in the N-domain of Sup35 Protein on Prion [PSI+] Stability and Propagation

Recent studies have shown that Sup35p prion fibrils probably have a parallel in-register β-structure. However, the part(s) of the N-domain critical for fibril formation and maintenance of the [PSI⁺] phenotype remains unclear. Here we designed a set of five SUP35 mutant alleles (sup35^KK) with lysine substitutions in each of five N-domain repeats, and investigated their effect on infectivity and ability of corresponding proteins to aggregate and coaggregate with wild type Sup35p in the [PSI⁺] strain. Alleles sup35-M1 (Y46K/Q47K) and sup35-M2 (Q61K/Q62K) led to prion loss, whereas sup35-M3 (Q70K/Q71K), sup35-M4 (Q80K/Q81K), and sup35-M5 (Q89K/Q90K) were able to maintain the [PSI⁺] prion. This suggests that the critical part of the parallel in-register β-structure for the studied [PSI⁺] prion variant lies in the first 63–69 residues. Our study also reveals an unexpected interplay between the wild type Sup35p and proteins expressed from the sup35^KK alleles during prionization. Both Sup35-M1p and Sup35-M2p coaggregated with Sup35p, but only sup35-M2 led to prion loss in a dominant manner. We suggest that in the fibrils, Sup35p can bind to Sup35-M1p in the same conformation, whereas Sup35-M2p only allowed the Sup35p conformation that leads to the non-heritable fold. Mutations sup35-M4 and sup35-M5 influence the structure of the prion forming region to a lesser extent, and can lead to the formation of new prion variants.