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.
PrP; Sup35 fusions; nonsense suppression; prion; yeast (Saccharomyces cerevisiae)
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.
Saccharomyces cerevisiae; [PSI]; [URE3]; [PIN]; prion induction; prion curing; prion inheritance; Hsp104
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.
conformational disease; folding intermediates; conformational changes; prion; amyloid; fibrils; toxicity; aggregation propensity; molecular chaperones; therapeutics
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.
yeast; [PSI]; prion; SUP35/eRF3; SUP45/eRF1; antisuppressor; [PSI+] maintenance genes; Hsp104; prion antagonists
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.
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.
•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
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.
Methionine; Oxidative Stress; Peroxiredoxin; Prions; Protein Aggregation; Translation; Translation Release Factors; Yeast
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.
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.
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.
Amyloid; Prions; Protein Folding; Translation Release Factors; Yeast; Saccharomyces; Sup35; Prion Interference; Prion Species Barrier
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.
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.
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.
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.
[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.
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