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1.  [PSI+] turns 50 
Prion  2015;9(5):318-332.
The year 2015 sees the fiftieth anniversary of the publication of a research paper that underpins much of our understanding of fungal prion biology, namely “ψ, a cytoplasmic suppressor of super-suppressor in yeast” by Brian Cox. Here we show how our understanding of the molecular nature of the [PSI+] determinant evolved from an ‘occult’ determinant to a transmissible amyloid form of a translation termination factor. We also consider the impact studies on [PSI] have had – and continue to have - on prion research. To demonstrate this, leading investigators in the yeast prion field who have made extensive use of the [PSI+] trait in their research, provide their own commentaries on the discovery and significance of [PSI].
PMCID: PMC4964859  PMID: 26645632
yeast; prion; Brian Cox; non-Mendelian inheritance; [PSI+] prion; nonsense suppression
2.  An acid tale of prion formation 
eLife  null;5:e22256.
Some bacteria use lactic acid to communicate with yeast cells.
PMCID: PMC5127635  PMID: 27897124
Dekkera bruxellensis; Staphylococcus gallinarum; lactic acid; prion; catabolite repression; molecular communication; S. cerevisiae
3.  New links between SOD1 and metabolic dysfunction from a yeast model of amyotrophic lateral sclerosis 
Journal of Cell Science  2016;129(21):4118-4129.
A number of genes have been linked to familial forms of the fatal motor neuron disease amyotrophic lateral sclerosis (ALS). Over 150 mutations within the gene encoding superoxide dismutase 1 (SOD1) have been implicated in ALS, but why such mutations lead to ALS-associated cellular dysfunction is unclear. In this study, we identify how ALS-linked SOD1 mutations lead to changes in the cellular health of the yeast Saccharomyces cerevisiae. We find that it is not the accumulation of aggregates but the loss of Sod1 protein stability that drives cellular dysfunction. The toxic effect of Sod1 instability does not correlate with a loss of mitochondrial function or increased production of reactive oxygen species, but instead prevents acidification of the vacuole, perturbs metabolic regulation and promotes senescence. Central to the toxic gain-of-function seen with the SOD1 mutants examined was an inability to regulate amino acid biosynthesis. We also report that leucine supplementation results in an improvement in motor function in a Caenorhabditis elegans model of ALS. Our data suggest that metabolic dysfunction plays an important role in Sod1-mediated toxicity in both the yeast and worm models of ALS.
Summary: In a new yeast model of ALS we have discovered for the first time that mutations in Sod1 can lead to the formation of toxic, soluble proteins that disrupt metabolic regulation.
PMCID: PMC5117206  PMID: 27656112
ALS; SOD1; Metabolism; Vacuole; Yeast
4.  Probing the role of structural features of mouse PrP in yeast by expression as Sup35-PrP fusions 
Prion  2012;6(3):201-210.
The yeast Saccharomyces cerevisiae is a tractable model organism in which both to explore the molecular mechanisms underlying the generation of disease-associated protein misfolding and to map the cellular responses to potentially toxic misfolded proteins. Specific targets have included proteins which in certain disease states form amyloids and lead to neurodegeneration. Such studies are greatly facilitated by the extensive ‘toolbox’ available to the yeast researcher that provides a range of cell engineering options. Consequently, a number of assays at the cell and molecular level have been set up to report on specific protein misfolding events associated with endogenous or heterologous proteins. One major target is the mammalian prion protein PrP because we know little about what specific sequence and/or structural feature(s) of PrP are important for its conversion to the infectious prion form, PrPSc. Here, using a study of the expression in yeast of fusion proteins comprising the yeast prion protein Sup35 fused to various regions of mouse PrP protein, we show how PrP sequences can direct the formation of non-transmissible amyloids and focus in particular on the role of the mouse octarepeat region. Through this study we illustrate the benefits and limitations of yeast-based models for protein misfolding disorders.
PMCID: PMC3399540  PMID: 22449853
PrP; Sup35 fusions; nonsense suppression; prion; yeast (Saccharomyces cerevisiae)
5.  Prion Stability 
Prion  2007;1(3):170-178.
The rate of spontaneous change from ψ to the ψ+ condition, determined in yeast by states of the Sup35p protein, is briefly discussed together with the conditions necessary for such change to occur. Conditions that promote and which affect the rate of induction of ψ+ in Sup35p and of other prion-forming proteins to their respective prion forms are also discussed. These include the influence of the amount of non-prion protein, the presence of other prions, the activity of chaperones, and brief descriptions of the role of native sequences in the proteins and how alteration of sequences in prion-forming proteins influences the rate of induction of [prion+] and amyloid forms.
The second part of this article discusses the conditions which affect the reversion of ψ+ to ψ-, including factors which affect the copy-number of prion “seeds” or propagons and their partition. The principal factor discussed is the activity of the chaperone Hsp104, but the existence of other factors, such protein sequence and of other, less well-studied agents is touched upon and comparisons are made, as appropriate, with studies with other yeast prions.
We conclude with a discussion of models of maintenance, in particular that of Tanaka et al. published in Nature (2006),6 which provides much insight into the phenotypic and genetic parameters of the numerous “variants” of prions increasingly being described in the literature.
PMCID: PMC2634590  PMID: 19164897
Saccharomyces cerevisiae; [PSI]; [URE3]; [PIN]; prion induction; prion curing; prion inheritance; Hsp104
6.  Protein Misfolding and Aggregation in Ageing and Disease 
Prion  2007;1(2):116-120.
Although intensively researched, the fundamental mechanism of protein misfolding that leads to protein aggregation and associated diseases remains somewhat enigmatic. The failure of a protein to correctly fold de novo or to remain correctly folded can have profound consequences on a living system especially when the cellular quality control processes fail to eliminate the rogue proteins. Over 20 different human diseases have now been designated as ‘conformational diseases’ and include neurodegenerative diseases such as Alzheimer's disease (AD), Huntington's disease (HD) and Creutzfeldt Jakob disease (CJD) that are becoming increasingly prevalent in an ageing human population. Such diseases are usually characterised by the deposition of specific misfolded proteins as amyloid fibrils and hence are often referred to as the amyloidoses.
PMCID: PMC2634451  PMID: 19164925
conformational disease; folding intermediates; conformational changes; prion; amyloid; fibrils; toxicity; aggregation propensity; molecular chaperones; therapeutics
7.  The Genetic Control of the Formation and Propagation of the [PSI+] Prion of Yeast 
Prion  2007;1(2):101-109.
It is over 40 years since it was first reported that the yeast Saccahromyces cerevisiae contains two unusual cytoplasmic ‘genetic’ elements: [PSI+] and [URE3]. Remarkably the underlying determinants are protein-based rather than nucleic acid-based, i.e., that they are prions, and we have already learnt much about their inheritance and phenotypic effects from the application of ‘classical’ genetic studies alongside the more modern molecular, cellular and biochemical approaches. Of particular value has been the exploitation of chemical mutagens and ‘antagonistic’ mutants which directly affect the replication and/or transmission of yeast prions. In this Chapter we describe what has emerged from the application of classical and molecular genetic studies, to the most intensively studied of the three native yeast prions, the [PSI+] prion.
PMCID: PMC2634449  PMID: 19164924
yeast; [PSI]; prion; SUP35/eRF3; SUP45/eRF1; antisuppressor; [PSI+] maintenance genes; Hsp104; prion antagonists
8.  Oxidative stress conditions increase the frequency of de novo formation of the yeast [PSI+] prion 
Molecular Microbiology  2015;96(1):163-174.
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.
PMCID: PMC4407919  PMID: 25601439
9.  Specialized Yeast Ribosomes: A Customized Tool for Selective mRNA Translation 
PLoS ONE  2013;8(7):e67609.
Evidence is now accumulating that sub-populations of ribosomes - so-called specialized ribosomes - can favour the translation of subsets of mRNAs. Here we use a large collection of diploid yeast strains, each deficient in one or other copy of the set of ribosomal protein (RP) genes, to generate eukaryotic cells carrying distinct populations of altered ‘specialized’ ribosomes. We show by comparative protein synthesis assays that different heterologous mRNA reporters based on luciferase are preferentially translated by distinct populations of specialized ribosomes. These mRNAs include reporters carrying premature termination codons (PTC) thus allowing us to identify specialized ribosomes that alter the efficiency of translation termination leading to enhanced synthesis of the wild-type protein. This finding suggests that these strains can be used to identify novel therapeutic targets in the ribosome. To explore this further we examined the translation of the mRNA encoding the extracellular matrix protein laminin β3 (LAMB3) since a LAMB3-PTC mutant is implicated in the blistering skin disease Epidermolysis bullosa (EB). This screen identified specialized ribosomes with reduced levels of RP L35B as showing enhanced synthesis of full-length LAMB3 in cells expressing the LAMB3-PTC mutant. Importantly, the RP L35B sub-population of specialized ribosomes leave both translation of a reporter luciferase carrying a different PTC and bulk mRNA translation largely unaltered.
PMCID: PMC3704640  PMID: 23861776
10.  Structural Definition Is Important for the Propagation of the Yeast [PSI+] Prion 
Molecular Cell  2013;50(5):675-685.
Prions are propagated in Saccharomyces cerevisiae with remarkable efficiency, yet we know little about the structural basis of sequence variations in the prion protein that support or prohibit propagation of the prion conformation. We show that certain single-amino-acid substitutions in the prion protein Sup35 impact negatively on the maintenance of the associated prion-based [PSI+] trait by combining in vivo phenotypic analysis with solution NMR structural studies. A clear correlation is observed between mutationally induced conformational differences in one of the oligopeptide repeats (R2) in the N terminus of Sup35 and the relative ability to propagate [PSI+]. Strikingly, substitution of one of a Gly-Gly pair with highly charged residues that significantly increase structural definition of R2 lead to a severe [PSI+] propagation defect. These findings offer a molecular explanation for the dominant-negative effects of such psi-no-more (PNM) mutations and demonstrate directly the importance of localized structural definition in prion propagation.
Graphical Abstract
•Mutations in a Gly58-Gly59 pair in Sup35 inhibit propagation of the [PSI+] prion•Biochemistry of the amino acid introduced dictates the resulting phenotype•Prion propagation defects correlate with local changes in Sup35 structure•Increased local structural definition is associated with impaired prion propagation
PMCID: PMC3679450  PMID: 23746351
11.  Methionine Oxidation of Sup35 Protein Induces Formation of the [PSI+] Prion in a Yeast Peroxiredoxin Mutant 
The Journal of Biological Chemistry  2011;286(45):38924-38931.
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.
PMCID: PMC3234717  PMID: 21832086
Methionine; Oxidative Stress; Peroxiredoxin; Prions; Protein Aggregation; Translation; Translation Release Factors; Yeast
12.  The prion hypothesis: from biological anomaly to basic regulatory mechanism 
Prions are unusual proteinaceous infectious agents that are typically associated with a class of fatal degenerative diseases of the mammalian brain. However, the discovery of fungal prions, which are not associated with disease, suggests that we must now consider the impact of these factors on basic cellular physiology in a different light. Fungal prions are epigenetic determinants that can alter a range of cellular processes, including metabolism and gene expression pathways, and these changes can lead to a range of prion-associated phenotypes. The mechanistic similarities between prion propagation in mammals and fungi suggest that prions are not a biological anomaly but instead are a new appreciated and perhaps ubiquitous regulatory mechanism.
PMCID: PMC3003427  PMID: 21081963
13.  Molecular Basis for Transmission Barrier and Interference between Closely Related Prion Proteins in Yeast* 
The Journal of Biological Chemistry  2011;286(18):15773-15780.
Replicating amyloids, called prions, are responsible for transmissible neurodegenerative diseases in mammals and some heritable phenotypes in fungi. The transmission of prions between species is usually inhibited, being highly sensitive to small differences in amino acid sequence of the prion-forming proteins. To understand the molecular basis of this prion interspecies barrier, we studied the transmission of the [PSI+] prion state from Sup35 of Saccharomyces cerevisiae to hybrid Sup35 proteins with prion-forming domains from four other closely related Saccharomyces species. Whereas all the hybrid Sup35 proteins could adopt a prion form in S. cerevisiae, they could not readily acquire the prion form from the [PSI+] prion of S. cerevisiae. Expression of the hybrid Sup35 proteins in S. cerevisiae [PSI+] cells often resulted in frequent loss of the native [PSI+] prion. Furthermore, all hybrid Sup35 proteins showed different patterns of interaction with the native [PSI+] prion in terms of co-polymerization, acquisition of the prion state, and induced prion loss, all of which were also dependent on the [PSI+] variant. The observed loss of S. cerevisiae [PSI+] can be related to inhibition of prion polymerization of S. cerevisiae Sup35 and formation of a non-heritable form of amyloid. We have therefore identified two distinct molecular origins of prion transmission barriers between closely sequence-related prion proteins: first, the inability of heterologous proteins to co-aggregate with host prion polymers, and second, acquisition by these proteins of a non-heritable amyloid fold.
PMCID: PMC3091186  PMID: 21454674
Amyloid; Prions; Protein Folding; Translation Release Factors; Yeast; Saccharomyces; Sup35; Prion Interference; Prion Species Barrier
14.  Decoding accuracy in eRF1 mutants and its correlation with pleiotropic quantitative traits in yeast 
Nucleic Acids Research  2010;38(16):5479-5492.
Translation termination in eukaryotes typically requires the decoding of one of three stop codons UAA, UAG or UGA by the eukaryotic release factor eRF1. The molecular mechanisms that allow eRF1 to decode either A or G in the second nucleotide, but to exclude UGG as a stop codon, are currently not well understood. Several models of stop codon recognition have been developed on the basis of evidence from mutagenesis studies, as well as studies on the evolutionary sequence conservation of eRF1. We show here that point mutants of Saccharomyces cerevisiae eRF1 display significant variability in their stop codon read-through phenotypes depending on the background genotype of the strain used, and that evolutionary conservation of amino acids in eRF1 is only a poor indicator of the functional importance of individual residues in translation termination. We further show that many phenotypes associated with eRF1 mutants are quantitatively unlinked with translation termination defects, suggesting that the evolutionary history of eRF1 was shaped by a complex set of molecular functions in addition to translation termination. We reassess current models of stop-codon recognition by eRF1 in the light of these new data.
PMCID: PMC2938225  PMID: 20444877
15.  The Number and Transmission of [PSI+] Prion Seeds (Propagons) in the Yeast Saccharomyces cerevisiae 
PLoS ONE  2009;4(3):e4670.
Yeast (Saccharomyces cerevisiae) prions are efficiently propagated and the on-going generation and transmission of prion seeds (propagons) to daughter cells during cell division ensures a high degree of mitotic stability. The reversible inhibition of the molecular chaperone Hsp104p by guanidine hydrochloride (GdnHCl) results in cell division-dependent elimination of yeast prions due to a block in propagon generation and the subsequent dilution out of propagons by cell division.
Principal Findings
Analysing the kinetics of the GdnHCl-induced elimination of the yeast [PSI+] prion has allowed us to develop novel statistical models that aid our understanding of prion propagation in yeast cells. Here we describe the application of a new stochastic model that allows us to estimate more accurately the mean number of propagons in a [PSI+] cell. To achieve this accuracy we also experimentally determine key cell reproduction parameters and show that the presence of the [PSI+] prion has no impact on these key processes. Additionally, we experimentally determine the proportion of propagons transmitted to a daughter cell and show this reflects the relative cell volume of mother and daughter cells at cell division.
While propagon generation is an ATP-driven process, the partition of propagons to daughter cells occurs by passive transfer via the distribution of cytoplasm. Furthermore, our new estimates of n0, the number of propagons per cell (500–1000), are some five times higher than our previous estimates and this has important implications for our understanding of the inheritance of the [PSI+] and the spontaneous formation of prion-free cells.
PMCID: PMC2650407  PMID: 19262693
16.  The [PSI+] Prion of Saccharomyces cerevisiae Can Be Propagated by an Hsp104 Orthologue from Candida albicans 
Eukaryotic Cell  2006;5(2):217-225.
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.
PMCID: PMC1405891  PMID: 16467463
17.  Guanidine Hydrochloride Inhibits the Generation of Prion “Seeds” but Not Prion Protein Aggregation in Yeast 
Molecular and Cellular Biology  2002;22(15):5593-5605.
[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.
PMCID: PMC133959  PMID: 12101251
18.  Dissection and Design of Yeast Prions 
PLoS Biology  2004;2(4):e86.
Many proteins can misfold into β-sheet-rich, self-seeding polymers (amyloids). Prions are exceptional among such aggregates in that they are also infectious. In fungi, prions are not pathogenic but rather act as epigenetic regulators of cell physiology, providing a powerful model for studying the mechanism of prion replication. We used prion-forming domains from two budding yeast proteins (Sup35p and New1p) to examine the requirements for prion formation and inheritance. In both proteins, a glutamine/asparagine-rich (Q/N-rich) tract mediates sequence-specific aggregation, while an adjacent motif, the oligopeptide repeat, is required for the replication and stable inheritance of these aggregates. Our findings help to explain why although Q/N-rich proteins are relatively common, few form heritable aggregates: prion inheritance requires both an aggregation sequence responsible for self-seeded growth and an element that permits chaperone-dependent replication of the aggregate. Using this knowledge, we have designed novel artificial prions by fusing the replication element of Sup35p to aggregation-prone sequences from other proteins, including pathogenically expanded polyglutamine.
Artificial prions - infectious, misfolded proteins - can be created by fusing the replication element of one prion to aggregation sequences from another
PMCID: PMC374241  PMID: 15045026

Results 1-18 (18)