Yeast Prions

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 [b] and [C] prions are enzymes whose precursor activation requires their own active form. [URE3] and [PSI+] are clearly diseases, while [Het-s] and [b] 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 b-sheet structure.     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.Key words: prion, Sup35p, Ure2p, Rnq1p, [PSI⁺], [URE3], [PIN⁺], 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     

Yeast Prions Compared to Functional Prions and Amyloids

Saccharomyces cerevisiae is an occasional host to an array of prions, most based on self-propagating, self-templating amyloid filaments of a normally soluble protein. [URE3] is a prion of Ure2p, a regulator of nitrogen catabolism, while [PSI +] is a prion of Sup35p, a subunit of the translation termination factor Sup35p. In contrast to the functional prions, [Het-s] of Podospora anserina and [BETA] of yeast, the amyloid-based yeast prions are rare in wild strains, arise sporadically, have an array of prion variants for a single prion protein sequence, have a folded in-register parallel β-sheet amyloid architecture, are detrimental to their hosts, arouse a stress response in the host, and are subject to curing by various host anti-prion systems. These characteristics allow a logical basis for distinction between functional amyloids/prions and prion diseases. These infectious yeast amyloidoses are outstanding models for the many common human amyloid-based diseases that are increasingly found to have some infectious characteristics.     

A bipolar personality of yeast prion proteins

Prions are infectious, self-propagating protein conformations. [PSI⁺], [RNQ⁺] and [URE3] are well characterized prions in Saccharomyces cerevisiae and represent the aggregated states of the translation termination factor Sup35, a functionally unknown protein Rnq1, and a regulator of nitrogen metabolism Ure2, respectively. Overproduction of Sup35 induces the de novo appearance of the [PSI⁺] prion in [RNQ⁺] or [URE3] strain, but not in non-prion strain. However, [RNQ⁺] and [URE3] prions themselves, as well as overexpression of a mutant Rnq1 protein, Rnq1Δ100, and Lsm4, hamper the maintenance of [PSI⁺]. These findings point to a bipolar activity of [RNQ⁺], [URE3], Rnq1Δ100, and Lsm4, and probably other yeast prion proteins as well, for the fate of [PSI⁺] prion. Possible mechanisms underlying the apparent bipolar activity of yeast prions will be discussed.     

The yeast prions [PSI+] and [URE3] are molecular degenerative diseases

The yeast prions [URE3] and [PSI] are not found in wild strains, suggesting they are not an advantage. Prion-forming ability is not conserved, even within Saccharomyces, suggesting it is a disease. Prion domains have non-prion functions, explaining some conservation of sequence. However, in spite of the sequence being constrained in evolution by these non-prion functions, the prion domains vary more rapidly than the remainder of the molecule, and these changes produce a transmission barrier, suggesting that these changes were selected to block prion infection. Yeast prions [PSI] and [URE3] induce a cellular stress response (Hsp104 and Hsp70 induction), suggesting the cells are not happy about being infected. Recently, we showed that the array of [PSI] and [URE3] prions includes a majority of lethal or very toxic variants, a result not expected if either prion were an adaptive cellular response to stress.Key words: [URE3], [PSI⁺], prion, Sup35p, Ure2pfMammalian prions are uniformly fatal, but a lethal yeast prion would not be detected by the usual procedure, which requires growth of a colony under some selective condition. As a result, the prion variants commonly studied are quite mild in their effects. This circumstance has led to the suggestion that yeast prions actually benefit their host. Sup35p, the translation termination subunit whose amyloid becomes the [PSI⁺] prion, is essential for growth and Ure2p, the nitrogen regulation protein whose amyloid constitutes the [URE3] prion, is important for growth, with ure2 mutants showing noticeably slowed growth.When yeast prions were discovered,¹ we assumed they were diseases, by analogy with the mammalian diseases and the many non-prion amyloid diseases. Inactivating the essential Sup35p or the desireable Ure2p did not seem like a useful strategy. While control of either protein''s activity might be advantageous, and Ure2p activity control is the key to regulation of nitrogen catabolism, prion formation is a stochastic process, so it makes control of activity of these proteins random instead of appropriate to the circumstances. The [Het-s] prion changed that picture.² Here was a prion necessary for a normal function, heterokaryon incompatibility, and we suggested that it was the first beneficial prion.³     

Yeast prions assembly and propagation: Contributions of the prion and non-prion moieties and the nature of assemblies

Yeast prions are self-perpetuating protein aggregates that are at the origin of heritable and transmissible non-Mendelian phenotypic traits. Among these, [PSI⁺], [URE3] and [PIN⁺] are the most well documented prions and arise from the assembly of Sup35p, Ure2p and Rnq1p, respectively, into insoluble fibrillar assemblies. Fibril assembly depends on the presence of N- or C-terminal prion domains (PrDs) which are not homologous in sequence but share unusual amino-acid compositions, such as enrichment in polar residues (glutamines and asparagines) or the presence of oligopeptide repeats. Purified PrDs form amyloid fibrils that can convert prion-free cells to the prion state upon transformation. Nonetheless, isolated PrDs and full-length prion proteins have different aggregation, structural and infectious properties. In addition, mutations in the “non-prion” domains (non-PrDs) of Sup35p, Ure2p and Rnq1p were shown to affect their prion properties in vitro and in vivo. Despite these evidences, the implication of the functional non-PrDs in fibril assembly and prion propagation has been mostly overlooked. In this review, we discuss the contribution of non-PrDs to prion assemblies, and the structure-function relationship in prion infectivity in the light of recent findings on Sup35p and Ure2p assembly into infectious fibrils from our laboratory and others.     

The 26S Proteasome Degrades the Soluble but Not the Fibrillar Form of the Yeast Prion Ure2p In Vitro

Yeast prions are self-perpetuating protein aggregates that cause heritable and transmissible phenotypic traits. Among these, [PSI ⁺] and [URE3] stand out as the most studied yeast prions, and result from the self-assembly of the translation terminator Sup35p and the nitrogen catabolism regulator Ure2p, respectively, into insoluble fibrillar aggregates. Protein quality control systems are well known to govern the formation, propagation and transmission of these prions. However, little is known about the implication of the cellular proteolytic machineries in their turnover. We previously showed that the 26S proteasome degrades both the soluble and fibrillar forms of Sup35p and affects [PSI ⁺] propagation. Here, we show that soluble native Ure2p is degraded by the proteasome in an ubiquitin-independent manner. Proteasomal degradation of Ure2p yields amyloidogenic N-terminal peptides and a C-terminal resistant fragment. In contrast to Sup35p, fibrillar Ure2p resists proteasomal degradation. Thus, structural variability within prions may dictate their ability to be degraded by the cellular proteolytic systems.     

Strategies for identifying new prions in yeast

The unexpected discovery of two prions, [URE3] and [PSI⁺], in Saccharomyces cerevisiae led to questions about how many other proteins could undergo similar prion-based structural conversions. However, [URE3] and [PSI⁺] were discovered by serendipity in genetic screens. Cataloging the full range of prions in yeast or in other organisms will therefore require more systematic search methods. Taking advantage of some of the unique features of prions, various researchers have developed bioinformatic and experimental methods for identifying novel prion proteins. These methods have generated long lists of prion candidates. The systematic testing of some of these prion candidates has led to notable successes; however, even in yeast, where rapid growth rate and ease of genetic manipulation aid in testing for prion activity, such candidate testing is laborious. Development of better methods to winnow the field of prion candidates will greatly aid in the discovery of new prions, both in yeast and in other organisms, and help us to better understand the role of prions in biology.Key words: yeast, prion, bioinformatics, Sup35, [PSI⁺], Ure2, [URE3]     

Rnq1 protein protects [<Emphasis Type="Italic">PSI</Emphasis><Superscript>+</Superscript>] prion from effect of the <Emphasis Type="Italic">PNM</Emphasis> mutation

The interaction of [PSI ⁺] and [PIN ⁺] factors in yeast Saccharomyces cerevisiae is known as the first evidence of prions networks. In [PIN ⁺] cells, Rnq1p prion aggregates work as a template for Sup35p aggregation, which is essential for [PSI ⁺] induction. No additional factors are required for subsequent Sup35p aggregation. Nevertheless, several recent reports provide data that indicate a more complex interplay between these prions. Our results show that the presence of Rnq1p in the cell significantly decreases the loss of [PSI ⁺] prion, which is caused by a double mutation in SUP35 (Q61K, Q62K substitutions in the Sup35 protein). These observations support the existence of interaction networks that converge on a strong linkage of prionogenic and prion-like proteins, and the participation of Rnq1 protein in the maintenance of prion [PSI ⁺].     

Prion Propagation: The Role of Protein Dynamics

The transfer of phenotypes from one individual to another is a fundamental aspect of biology. In addition to traditional nucleic acid-based genetic determinants, unique proteins known as prions can also act as elements of inheritance, infectivity, and disease. Nucleic acids and proteins encode genetic information in distinct ways, either in the sequence of bases in DNA or RNA or in the three dimensional structure of the polypeptide chain. Given these differences in the nature of the genetic repository, the mechanisms underlying the transmission of nucleic acid-based and protein-based phenotypes are necessarily distinct. While the appearance, persistence and transfer of nucleic acid determinants require the synthesis of new polymers, recent studies indicate that prions are propagated through dynamic transitions in the structure of existing protein.Key Words: prion, PrP, [PSI⁺], [URE3], [Het-s], Sup35, Ure2, Het-s, Hsp104     

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.     

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.     

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.     

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.     

Infectious Fold and Amyloid Propagation in Podospora anserina

Amyloid protein aggregation is involved in serious neurodegenerative disorders such as Alzheimer''s disease and transmissible encephalopathies. The concept of an infectious protein (prion) being the scrapie agent was successfully validated for several yeast and fungi proteins. Ure2, Sup35 and Rnq1 in Saccharomyces cerevisiae and HET-s in Podospora anserina have been genetically and biochemically identified as prion proteins. Studies on these proteins have revealed critical information on the mechanisms of prions appearance and propagation. The prion phenotype correlates with the aggregation state of these particular proteins. In vitro, the recombinant prion proteins form amyloid fibers characterized by rich β sheet content. In a previous work on the HET-s prion protein Podospora, we demonstrated the infectivity of HET-s recombinant amyloid aggregates. More recently, the structural analysis of the HET-s prion domain associated with in vivo mutagenesis allowed us to propose a model for the infectious fold of the HET-s prion domain. Further investigations to complete this model are discussed in this review, as are relevant questions about the [Het-s] system of Podospora anserina.Key Words: prion, HET-s, Podospora, amyloid, infectious, β sheet, mutagenesis, fold, propagation     

The [URE3] phenotype: evidence for a soluble prion in yeast

The aggregation of the two yeast proteins Sup35p and Ure2p is widely accepted as a model for explaining the prion propagation of the phenotypes [PSI⁺] and [URE3], respectively. Here, we demonstrate that the propagation of [URE3] cannot simply be the consequence of generating large aggregates of Ure2p, because such aggregation can be found in some conditions that are not related to the prion state of Ure2p. A comparison of [PSI⁺] and [URE3] aggregation demonstrates differences between these two prion mechanisms. Our findings lead us to propose a new unifying model for yeast prion propagation.     

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.     

Requirements of Hsp104p activity and Sis1p binding for propagation of the [RNQ+] prion

The formation and maintenance of prions in the yeast Saccharomyces cerevisiae is highly regulated by the cellular chaperone machinery. The most important player in this regulation is Hsp104p, which is required for the maintenance of all known prions. The requirements for other chaperones, such as members of the Hsp40 or Hsp70 families, vary with each individual prion. [RNQ⁺] cells do not have a phenotype that is amenable to genetic screens to identify cellular factors important in prion propagation. Therefore, we used a chimeric construct that reports the [RNQ⁺] status of cells to perform a screen for mutants that are unable to maintain [RNQ⁺]. We found eight separate mutations in Hsp104p that caused [RNQ⁺] cells to become [rnq⁻]. These mutations also caused the loss of the [PSI⁺] prion. The expression of one of these mutants, Hsp104p-E190K, showed differential loss of the [RNQ⁺] and [PSI⁺] prions in the presence of wild type Hsp104p. Hsp104p-E190K inefficiently propagated [RNQ⁺] and was unable to maintain [PSI⁺]. The mutant was unable to act on other in vivo substrates, as strains carrying it were not thermotolerant. Purified recombinant Hsp104p-E190K showed a reduced level of ATP hydrolysis as compared to wild type protein. This is likely the cause of both prion loss and lack of in vivo function. Furthermore, it suggests that [RNQ⁺] requires less Hsp104p activity to maintain transmissible protein aggregates than Sup35p. Additionally, we show that the L94A mutation in Rnq1p, which reduces its interaction with Sis1p, prevents Rnq1p from maintaining a prion and inducing [PSI⁺].Key words: [RNQ⁺], [PSI⁺], Hsp104p, Sis1p, mutagenesis