Brian S. Cox

The elimination of the yeast [PSI+] prion by guanidine hydrochloride is the result of Hsp104 inactivation

In the yeast Saccharomyces cerevisiae, Sup35p (eRF3), a subunit of the translation termination complex, can take up a prion‐like, self‐propagating conformation giving rise to the non‐Mendelian [PSI+] determinant. The replication of [PSI+] prion seeds can be readily blocked by growth in the presence of low concentrations of guanidine hydrochloride (GdnHCl), leading to the generation of prion‐free [psi–] cells. Here, we provide evidence that GdnHCl blocks seed replication in vivo by inactivation of the molecular chaperone Hsp104. Although growth in the presence of GdnHCl causes a modest increase in HSP104 expression (20–90%), this is not sufficient to explain prion curing. Rather, we show that GdnHCl inhibits two different Hsp104‐dependent cellular processes, namely the acquisition of thermotolerance and the refolding of thermally denatured luciferase. The inhibitory effects of GdnHCl protein refolding are partially suppressed by elevating the endogenous cellular levels of Hsp104 using a constitutive promoter. The kinetics of GdnHCl‐induced [PSI+] curing could be mimicked by co‐expression of an ATPase‐negative dominant HSP104 mutant in an otherwise wild‐type [PSI+] strain. We suggest that GdnHCl inactivates the ATPase activity of Hsp104, leading to a block in the replication of [PSI+] seeds.

Translation termination efficiency can be regulated in Saccharomyces cerevisiae by environmental stress through a prion-mediated mechanism

[PSI+] is a protein‐based heritable phenotype of the yeast Saccharomyces cerevisiae which reflects the prion‐like behaviour of the endogenous Sup35p protein release factor. [PSI+] strains exhibit a marked decrease in translation termination efficiency, which permits decoding of translation termination signals and, presumably, the production of abnormally extended polypeptides. We have examined whether the [PSI+]‐induced expression of such an altered proteome might confer some selective growth advantage over [psi−] strains. Although otherwise isogenic [PSI+] and [psi−] strains show no difference in growth rates under normal laboratory conditions, we demonstrate that [PSI+] strains do exhibit enhanced tolerance to heat and chemical stress, compared with [psi−] strains. Moreover, we also show that the prion‐like determinant [PSI+] is able to regulate translation termination efficiency in response to environmental stress, since growth in the presence of ethanol results in a transient increase in the efficiency of translation termination and a loss of the [PSI+] phenotype. We present a model to describe the prion‐mediated regulation of translation termination efficiency and discuss its implications in relation to the potential physiological role of prions in S.cerevisiae and other fungi.

Dissection and Design of Yeast Prions

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.

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

ABSTRACT [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.

Repair systems in Saccharomyces

Summary Radiation-sensitive mutations of yeast in multiple-mutant combinations interact with one another to affect survival. From these interactions, and from the effects of these mutations on other genetic events such as recombination or mutation, it is possible to deduce a scheme representing the pathways by which repair is effected. The biochemical roles of these pathways can be determined by simple assay systems. For example, one pathway controlled by 4 loci is concerned with the early stages of excision of pyrimidine dialers. Some of the loci used in this investigation affect the rate and duality of UV-induced mutation and so indicate what part repair might play in this process.

Propagation of yeast prions

Prion proteins have been implicated in various human neurodegenerative disorders and form amyloid deposits in the diseased brain. Uniquely, prion proteins seem to be able to propagate this altered conformational state, generating more of the prion form of the protein and acting as infectious agents. The discovery in yeast of prion proteins that can be inherited stably through generations of cell division provides us with an experimental model that is allowing the mysteries of how prions are propagated to be unravelled.

Yeast as an honorary mammal.

Editorial to Special Issue on Yeast DNA Repair and Human Implications: Past, Present and Future Perspectives on DNA Repair in Yeast -

Mechanism of inhibition of Psi+ prion determinant propagation by a mutation of the N-terminus of the yeast Sup35 protein.

The SUP35 gene of Saccharomyces cerevisiae encodes the polypeptide chain release factor eRF3. This protein (also called Sup35p) is thought to be able to undergo a heritable conformational switch, similarly to mammalian prions, giving rise to the cytoplasmically inherited Ψ+ determinant. A dominant mutation (PNM2 allele) in the SUP35 gene causing a Gly58→Asp change in the Sup35p N‐terminal domain eliminates Ψ+. Here we observed that the mutant Sup35p can be converted to the prion‐like form in vitro, but such conversion proceeds slower than that of wild‐type Sup35p. The overexpression of mutant Sup35p induced the de novo appearance of Ψ+ cells containing the prion‐like form of mutant Sup35p, which was able to transmit its properties to wild‐type Sup35p both in vitro and in vivo. Our data indicate that this Ψ+‐eliminating mutation does not alter the initial binding of Sup35p molecules to the Sup35p Ψ+‐specific aggregates, but rather inhibits its subsequent prion‐like rearrangement and/or binding of the next Sup35p molecule to the growing prion‐like Sup35p aggregate.

The genetic control of dark recombination in yeast

Abstract The post-UV effects of dark holding and photoreactivating light on survival and intragenic recombination in UV-sensitive strains of yeast have been studied. The results indicate that two liquid holding dark repair pathways exist in this organism: the excision pathway which does not lead to intragenic recombination (“silent” repair) and a dark recombination pathway which does lead to intragenic recombination. A third pathway also exists which is plating-dependent and leads to intragenic recombination via a postreplication pathway. Some of the UV-sensitive mutants isolated by various workers have been characterised with respect to their responses to UV and post-UV treatments and allocated to positions on a generalised scheme for repair and intragenic recombination.

The number and transmission of [PSI+] prion seeds (Propagons) in the yeast Saccharomyces cerevisiae

Background 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. Conclusions 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.