PSI+] maintenance is dependent on the composition, not primary sequence, of the oligopeptide repeat domain

[PSI(+)], the prion form of the yeast Sup35 protein, results from the structural conversion of Sup35 from a soluble form into an infectious amyloid form. The infectivity of prions is thought to result from chaperone-dependent fiber cleavage that breaks large prion fibers into smaller, inheritable propagons. Like the mammalian prion protein PrP, Sup35 contains an oligopeptide repeat domain. Deletion analysis indicates that the oligopeptide repeat domain is critical for [PSI(+)] propagation, while a distinct region of the prion domain is responsible for prion nucleation. The PrP oligopeptide repeat domain can substitute for the Sup35 oligopeptide repeat domain in supporting [PSI(+)] propagation, suggesting a common role for repeats in supporting prion maintenance. However, randomizing the order of the amino acids in the Sup35 prion domain does not block prion formation or propagation, suggesting that amino acid composition is the primary determinant of Sup35's prion propensity. Thus, it is unclear what role the oligopeptide repeats play in [PSI(+)] propagation: the repeats could simply act as a non-specific spacer separating the prion nucleation domain from the rest of the protein; the repeats could contain specific compositional elements that promote prion propagation; or the repeats, while not essential for prion propagation, might explain some unique features of [PSI(+)]. Here, we test these three hypotheses and show that the ability of the Sup35 and PrP repeats to support [PSI(+)] propagation stems from their amino acid composition, not their primary sequences. Furthermore, we demonstrate that compositional requirements for the repeat domain are distinct from those of the nucleation domain, indicating that prion nucleation and propagation are driven by distinct compositional features.     

Yeast [PSI+] "prions" that are crosstransmissible and susceptible beyond a species barrier through a quasi-prion state.

The yeast [PSI(+)] element represents an aggregated form of release factor Sup35p and is inherited by a prion mechanism. A "species barrier" prevents crosstransmission of the [PSI(+)] state between heterotypic Sup35p "prions." Kluyveromyces lactis and Yarrowia lipolytica Sup35 proteins, however, show interspecies [PSI(+)] transmissibility and susceptibility and a high spontaneous propagation rate. Cross-seeding was visualized by coaggregation of differential fluorescence probes fused to heterotypic Sup35 proteins. This coaggregation state, referred to as a "quasi-prion" state, can be stably maintained as a heritable [PSI(+)] element composed of heterologous Sup35 proteins. K. lactis Sup35p was capable of forming [PSI(+)] elements not only in S. cerevisiae but in K. lactis. These two Sup35 proteins contain unique multiple imperfect oligopeptide repeats responsible for crosstransmission and high spontaneous propagation of novel [PSI(+)] elements.     

Insights into intragenic and extragenic effectors of prion propagation using chimeric prion proteins

The study of fungal prion proteins affords remarkable opportunities to elucidate both intragenic and extragenic effectors of prion propagation. The yeast prion protein Sup35 and the self-perpetuating [PSI+] prion state is one of the best characterized fungal prions. While there is little sequence homology among known prion proteins, one region of striking similarity exists between Sup35p and the mammalian prion protein PrP. This region is comprised of roughly five octapeptide repeats of similar composition. The expansion of the repeat region in PrP is associated with inherited prion diseases. In order to learn more about the effects of PrP repeat expansions on the structural properties of a protein that undergoes a similar transition to a self-perpetuating aggregate, we generated chimeric Sup35-PrP proteins. Using both in vivo and in vitro systems we described the effect of repeat length on protein misfolding, aggregation, amyloid formation, and amyloid stability. We found that repeat expansions in the chimeric prion proteins increase the propensity to initiate prion propagation and enhance the formation of amyloid fibers without significantly altering fiber stability.     

Genetic and Environmental Factors Affecting the De Novo Appearance of the [Psi(+)] Prion in Saccharomyces Cerevisiae

It has previously been shown that yeast prion [PSI(+)] is cured by GuHCl, although reports on reversibility of curing were contradictory. Here we show that GuHCl treatment of both [PSI(+)] and [psi(-)] yeast strains results in two classes of [psi(-)] derivatives: Pin(+), in which [PSI(+)] can be reinduced by Sup35p overproduction, and Pin(-), in which overexpression of the complete SUP35 gene does not lead to the [PSI(+)] appearance. However, in both Pin(+) and Pin(-) derivatives [PSI(+)] is reinduced by overproduction of a short Sup35p N-terminal fragment, thus, in principle, [PSI(+)] curing remains reversible in both cases. Neither suppression nor growth inhibition caused by SUP35 overexpression in Pin(+) [psi(-)] derivatives are observed in Pin(-) [psi(-)] derivatives. Genetic analyses show that the Pin(+) phenotype is determined by a non-Mendelian factor, which, unlike the [PSI(+)] prion, is independent of the Sup35p N-terminal domain. A Pin(-) [psi(-)] derivative was also generated by transient inactivation of the heat shock protein, Hsp104, while [PSI(+)] curing by Hsp104 overproduction resulted exclusively in Pin(+) [psi(-)] derivatives. We hypothesize that in addition to the [PSI(+)] prion-determining domain in the Sup35p N-terminus, there is another self-propagating conformational determinant in the C-proximal part of Sup35p and that this second prion is responsible for the Pin(+) phenotype.     

Biochemical and functional analysis of the assembly of full-length Sup35p and its prion-forming domain

The protein Sup35 has prion properties. Its aggregation is at the origin of the [PSI(+)] trait in Saccharomyces cerevisiae. In vitro, the N-terminal domain of Sup35p alone or with the middle domain assembles into fibrils that exhibit the characteristics of amyloids. The vast majority of in vitro studies on the assembly of Sup35p have been performed using Sup35pNM, as fibrils made of Sup35pNM assembled in vitro propagate [PSI(+)] when reintroduced into yeast cells. Little is known about the assembly of full-length Sup35p and the role of the functional C-terminal domain of the protein. Here we report a systematic comparison of the biochemical and assembly properties of full-length Sup35p and Sup35pNM. We show that the native structure of the C-terminal domain is retained within the fibrils. We determined the size of Sup35p nuclei and the critical concentration for assembly that both differ from that of Sup35pNM. We demonstrate that Sup35pNM co-assembles with the full-length protein and that fibrils made of Sup35p or Sup35pNM seed the assembly of soluble Sup35pNM and Sup35p with different efficiencies. Finally, we show that fibrils made of full-length Sup35p induce with higher efficiency [PSI(+)] appearance as compared with those made of Sup35pNM. Our findings reveal differences and similarities in the assembly of Sup35p and its NM fragment and validate the use of Sup35pNM in studying some aspects of Sup35p aggregation but also underline the importance of using full-length Sup35p in studying prion propagation both in vivo and in vitro.     

Probing the role of PrP repeats in conformational conversion and amyloid assembly of chimeric yeast prions

Oligopeptide repeats appear in many proteins that undergo conformational conversions to form amyloid, including the mammalian prion protein PrP and the yeast prion protein Sup35. Whereas the repeats in PrP have been studied more exhaustively, interpretation of these studies is confounded by the fact that many details of the PrP prion conformational conversion are not well understood. On the other hand, there is now a relatively good understanding of the factors that guide the conformational conversion of the Sup35 prion protein. To provide a general model for studying the role of oligopeptide repeats in prion conformational conversion and amyloid formation, we have substituted various numbers of the PrP octarepeats for the endogenous Sup35 repeats. The resulting chimeric proteins can adopt the [PSI+] prion state in yeast, and the stability of the prion state depends on the number of repeats. In vitro, these chimeric proteins form amyloid fibers, with more repeats leading to shorter lag phases and faster assembly rates. Both pH and the presence of metal ions modulate assembly kinetics of the chimeric proteins, and the extent of modulation is highly sensitive to the number of PrP repeats. This work offers new insight into the properties of the PrP octarepeats in amyloid assembly and prion formation. It also reveals new features of the yeast prion protein, and provides a level of control over yeast prion assembly that will be useful for future structural studies and for creating amyloid-based biomaterials.     

Sequestration of Sup35 by aggregates of huntingtin fragments causes toxicity of [PSI+] yeast

Expression of huntingtin fragments with 103 glutamines (HttQ103) is toxic in yeast containing either the [PIN(+)] prion, which is the amyloid form of Rnq1, or [PSI(+)] prion, which is the amyloid form of Sup35. We find that HttQP103, which has a polyproline region at the C-terminal end of the polyQ repeat region, is significantly more toxic in [PSI(+)] yeast than in [PIN(+)], even though HttQP103 formed multiple aggregates in both [PSI(+)] and [PIN(+)] yeast. This toxicity was only observed in the strong [PSI(+)] variant, not the weak [PSI(+)] variant, which has more soluble Sup35 present than the strong variant. Furthermore, expression of the MC domains of Sup35, which retains the C-terminal domain of Sup35, but lacks the N-terminal prion domain, almost completely rescued HttQP103 toxicity, but was less effective in rescuing HttQ103 toxicity. Therefore, the toxicity of HttQP103 in yeast containing the [PSI(+)] prion is primarily due to sequestration of the essential protein, Sup35.     

Guanidine hydrochloride inhibits the generation of prion "seeds" but not prion protein aggregation in yeast

[PSI(+)] strains of the yeast Saccharomyces cerevisiae replicate and transmit the prion form of the Sup35p protein but can be permanently cured of this property when grown in millimolar concentrations of guanidine hydrochloride (GdnHCl). GdnHCl treatment leads to the inhibition of the replication of the [PSI(+)] seeds necessary for continued [PSI(+)] propagation. Here we demonstrate that the rate of incorporation of newly synthesized Sup35p into the high-molecular-weight aggregates, diagnostic of [PSI(+)] strains, is proportional to the number of seeds in the cell, with seed number declining (and the levels of soluble Sup35p increasing) in the presence of GdnHCl. GdnHCl does not cause breakdown of preexisting Sup35p aggregates in [PSI(+)] cells. Transfer of GdnHCl-treated cells to GdnHCl-free medium reverses GdnHCl inhibition of [PSI(+)] seed replication and allows new prion seeds to be generated exponentially in the absence of ongoing protein synthesis. Following such release the [PSI(+)] seed numbers double every 20 to 22 min. Recent evidence (P. C. Ferreira, F. Ness, S. R. Edwards, B. S. Cox, and M. F. Tuite, Mol. Microbiol. 40:1357-1369, 2001; G. Jung and D. C. Masison, Curr. Microbiol. 43:7-10, 2001), together with data presented here, suggests that curing yeast prions by GdnHCl is a consequence of GdnHCl inhibition of the activity of molecular chaperone Hsp104, which in turn is essential for [PSI(+)] propagation. The kinetics of elimination of [PSI(+)] by coexpression of a dominant, ATPase-negative allele of HSP104 were similar to those observed for GdnHCl-induced elimination. Based on these and other data, we propose a two-cycle model for "prionization" of Sup35p in [PSI(+)] cells: cycle A is the GdnHCl-sensitive (Hsp104-dependent) replication of the prion seeds, while cycle B is a GdnHCl-insensitive (Hsp104-independent) process that converts these seeds to pelletable aggregates.     

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.     

Hsp70 chaperones as modulators of prion life cycle: novel effects of Ssa and Ssb on the Saccharomyces cerevisiae prion [PSI+

[PSI(+)] is a prion isoform of the yeast release factor Sup35. In some assays, the cytosolic chaperones Ssa1 and Ssb1/2 of the Hsp70 family were previously shown to exhibit "pro-[PSI(+)]" and "anti-[PSI(+)]" effects, respectively. Here, it is demonstrated for the first time that excess Ssa1 increases de novo formation of [PSI(+)] and that pro-[PSI(+)] effects of Ssa1 are shared by all other Ssa proteins. Experiments with chimeric constructs show that the peptide-binding domain is a major determinant of differences in the effects of Ssa and Ssb proteins on [PSI(+)]. Surprisingly, overproduction of either chaperone increases loss of [PSI(+)] when Sup35 is simultaneously overproduced. Excess Ssa increases both the average size of prion polymers and the proportion of monomeric Sup35 protein. Both in vivo and in vitro experiments uncover direct physical interactions between Sup35 and Hsp70 proteins. The proposed model postulates that Ssa stimulates prion formation and polymer growth by stabilizing misfolded proteins, which serve as substrates for prion conversion. In the case of very large prion aggregates, further increase in size may lead to the loss of prion activity. In contrast, Ssb either stimulates refolding into nonprion conformation or targets misfolded proteins for degradation, in this way counteracting prion formation and propagation.     

Novel non-Mendelian determinant involved in the control of translation accuracy in Saccharomyces cerevisiae.

Two cytoplasmically inherited determinants related by their manifestation to the control of translation accuracy were previously described in yeast. Cells carrying one of them, [PSI(+)], display a nonsense suppressor phenotype and contain a prion form of the Sup35 protein. Another element, [PIN(+)], determines the probability of de novo generation of [PSI(+)] and results from a prion form of several proteins, which can be functionally unrelated to Sup35p. Here we describe a novel nonchromosomal determinant related to the SUP35 gene. This determinant, designated [ISP(+)], was identified as an antisuppressor of certain sup35 mutations. We observed its loss upon growth on guanidine hydrochloride and subsequent spontaneous reappearance with high frequency. The reversible curability of [ISP(+)] resembles the behavior of yeast prions. However, in contrast to known prions, [ISP(+)] does not depend on the chaperone protein Hsp104. Though manifestation of both [ISP(+)] and [PSI(+)] is related to the SUP35 gene, the maintenance of [ISP(+)] does not depend on the prionogenic N-terminal domain of Sup35p and Sup35p is not aggregated in [ISP(+)] cells, thus ruling out the possibility that [ISP(+)] is a specific form of [PSI(+)]. We hypothesize that [ISP(+)] is a novel prion involved in the control of translation accuracy in yeast.     

Prion properties of the Sup35 protein of yeast Pichia methanolica

The Sup35 protein (Sup35p) of Saccharomyces cerevisiae is a translation termination factor of the eRF3 family. The proteins of this family possess a conservative C-terminal domain responsible for translation termination and N-terminal extensions of different structure. The N-terminal domain of Sup35p defines its ability to undergo a heritable prion-like conformational switch, which is manifested as the cytoplasmically inherited [PSI(+)] determinant. Here, we replaced the N-terminal domain of S.cerevisiae Sup35p with an analogous domain from Pichia methanolica. Overexpression of hybrid Sup35p induced the de novo appearance of cytoplasmically inherited suppressor determinants manifesting key genetic and biochemical traits of [PSI(+)]. In contrast to the conventional [PSI(+)], 'hybrid' [PSI(+)] showed lower mitotic stability and preserved their suppressor phenotype upon overexpression of the Hsp104 chaperone protein. The lack of Hsp104 eliminated both types of [PSI(+)]. No transfer of prion state between the two Sup35p variants was observed, which reveals a 'species barrier' for the [PSI(+)] prions. The data obtained show that prion properties are conserved within at least a part of this protein family.     

Analysis of the generation and segregation of propagons: entities that propagate the [PSI+] prion in yeast

The propagation of the prion form of the yeast Sup35p protein, the so-called [PSI(+)] determinant, involves the generation and partition of a small number of particulate determinants that we propose calling "propagons." The numbers of propagons in [PSI(+)] cells can be inferred from the kinetics of elimination of [PSI(+)] during growth in the presence of a low concentration of guanidine hydrochloride (GdnHCl). Using this and an alternative method of counting the numbers of propagons, we demonstrate considerable clonal variation in the apparent numbers of propagons between different [PSI(+)] yeast strains, between different cultures of the same [PSI(+)] yeast strain, and between different cells of the same [PSI(+)] culture. We provide further evidence that propagon generation is blocked by growth in GdnHCl and that it is largely confined to the S phase of the cell cycle. In addition, we show that at low propagon number there is a bias toward retention of propagons in mother cells and that production of new propagons is very rapid when cells with depleted numbers of propagons are rescued into normal growth medium. The implications of our findings with respect to yeast prion propagation mechanisms are discussed.     

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.     

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.     

A mutation within the C-terminal domain of Sup35p that affects [PSI+] prion propagation

The epigenetic factor [PSI+] in the yeast Saccharomyces cerevisiae is due to the prion form of Sup35p. The N-terminal domain of Sup35p (N), alone or together with the middle-domain (NM), assembles in vitro into fibrils that induce [PSI+] when introduced into yeast cells. The Sup35p C-terminal domain (C), involved in translation termination, is essential for growth. The involvement of Sup35p C-terminal domain into [PSI+] propagation is subject to debate. We previously showed that mutation of threonine 341 within Sup35p C-domain affects translation termination efficiency. Here, we demonstrate that mutating threonine 341 to aspartate or alanine results in synthetic lethality with [PSI+] and weakening of [PSI+] respectively. The corresponding Sup35D and Sup35A proteins assemble into wild-type like fibrils in vitro, but with a slower elongation rate. Moreover, cross-seeding between Sup35p and Sup35A is inefficient both in vivo and in vitro, suggesting that the point mutation alters the structural properties of Sup35p within the fibrils. Thus, Sup35p C-terminal domain modulates [PSI+] prion propagation, possibly through a functional interaction with the N and/or M domains of the protein. Our results clearly demonstrate that Sup35p C-terminal domain plays a critical role in prion propagation and provide new insights into the mechanism of prion conversion.     

Chaperones that cure yeast artificial [PSI+] and their prion-specific effects

The [PSI(+)] nonsense-suppressor determinant of Saccharomyces cerevisiae results from the ability of Sup35 (eRF3) translation termination factor to undergo prion-like aggregation [1]. Although this process is autocatalytic, in vivo it depends on the chaperone Hsp104, whose lack or overexpression can cure [PSI(+)] [2]. Overproduction of the chaperone protein Ssb1 increased the [PSI(+)] curing by excess Hsp104, although it had no effect on its own, and excess chaperone protein Ssa1 protected [PSI(+)] against Hsp104 [3,4]. We used an artificial [PSI(+)(PS)] based on the Sup35 prion-forming domain from yeast Pichia methanolica [5] to find other prion-curing factors. Both [PSI(+)(PS)] and [PSI(+)] have prion 'strains', differing in their suppressor efficiency and mitotic stability. We show that [PSI(+)(PS)] and a 'weak' strain of [PSI(+)] can be cured by overexpression of chaperones Ssa1, Ssb1 and Ydj1. The ability of different chaperones to cure [PSI(+)(PS)] showed significant prion strain specificity, which could be related to variation in Sup35 prion structure. Our results imply that homologs of these chaperones may be active against mammalian prion and amyloid diseases.     

The [PSI+] prion of yeast: a problem of inheritance

The [PSI(+)] prion of the yeast Saccharomyces cerevisiae was first identified by Brian Cox some 40 years ago as a non-Mendelian genetic element that modulated the efficiency of nonsense suppression. Following the suggestion by Reed Wickner in 1994 that such elements might be accounted for by invoking a prion-based model, it was subsequently established that the [PSI(+)] determinant was the prion form of the Sup35p protein. In this article, we review how a combination of classical genetic approaches and modern molecular and biochemical methods has provided conclusive evidence of the prion basis of the [PSI(+)] determinant. In so doing we have tried to provide a historical context, but also describe the results of more recent experiments aimed at elucidating the mechanism by which the [PSI(+)] (and other yeast prions) are efficiently propagated in dividing cells. While understanding of the [PSI(+)] prion and its mode of propagation has, and will continue to have, an impact on mammalian prion biology nevertheless the very existence of a protein-based mechanism that can have a beneficial impact on a cell's fitness provides equally sound justification to fully explore yeast prions.     

The role of the N-terminal oligopeptide repeats of the yeast Sup35 prion protein in propagation and transmission of prion variants

The cytoplasmic [PSI+] determinant of Saccharomyces cerevisiae is the prion form of the Sup35 protein. Oligopeptide repeats within the Sup35 N-terminal domain (PrD) presumably are required for the stable [PSI+] inheritance that in turn involves fragmentation of Sup35 polymers by the chaperone Hsp104. The nonsense suppressor [PSI+] phenotype can vary in efficiency probably due to different inheritable Sup35 polymer structures. Here we study the ability of Sup35 mutants with various deletions of the oligopeptide repeats to support [PSI+] propagation. We define the minimal region of the Sup35-PrD necessary to support [PSI+] as amino acids 1-64, which include the first two repeats, although a longer fragment, 1-83, is required to maintain weak [PSI+] variants. Replacement of wild-type Sup35 with deletion mutants decreases the strength of the [PSI+] phenotype. However, with one exception, reintroducing the wild-type Sup35 restores the original phenotype. Thus, the specific prion fold defining the [PSI+] variant can be preserved by the mutant Sup35 protein despite the change of phenotype. Coexpression of wild-type and mutant Sup35 containing three, two, one, or no oligopeptide repeats causes variant-specific [PSI+] elimination. These data suggest that [PSI+] variability is primarily defined by differential folding of the Sup35-PrD oligopeptide-repeat region.     

Dependence and independence of [PSI(+)] and [PIN(+)]: a two-prion system in yeast?

The [PSI(+)] prion can be induced by overproduction of the complete Sup35 protein, but only in strains carrying the non-Mendelian [PIN(+)] determinant. Here we demonstrate that just as [psi (-)] strains can exist as [PIN(+)] and [pin(-)] variants, [PSI(+)] can also exist in the presence or absence of [PIN(+)]. [PSI(+)] and [PIN(+)] tend to be cured together, but can be lost separately. [PSI(+)]-related phenotypes are not affected by [PIN(+)]. Thus, [PIN(+)] is required for the de novo formation of [PSI(+)], not for [PSI(+)] propagation. Although [PSI(+)] induction is shown to require [PIN(+)] even when the only overexpressed region of Sup35p is the prion domain, two altered prion domain fragments circumventing the [PIN(+)] requirement are characterized. Finally, in strains cured of [PIN(+)], prolonged incubation facilitates the reappearance of [PIN(+)]. Newly appearing [PIN(+)] elements are often unstable but become stable in some mitotic progeny. Such reversibility of curing, together with our previous demonstration that the inheritance of [PIN(+)] is non-Mendelian, supports the hypothesis that [PIN(+)] is a prion. Models for [PIN(+)] action, which explain these findings, are discussed.