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

AbstractThe [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

Itt1p, a novel protein inhibiting translation termination in Saccharomyces cerevisiae

BACKGROUND: Termination of translation in eukaryotes is controlled by two interacting polypeptide chain release factors, eRFl and eRF3. eRFl recognizes nonsense codons UAA, UAG and UGA, while eRF3 stimulates polypeptide release from the ribosome in a GTP- and eRFl – dependent manner. Recent studies has shown that proteins interacting with these release factors can modulate the efficiency of nonsense codon readthrough. RESULTS: We have isolated a nonessential yeast gene, which causes suppression of nonsense mutations, being in a multicopy state. This gene encodes a protein designated Itt1p, possessing a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes. Overexpression of Itt1p decreases the efficiency of translation termination, resulting in the readthrough of all three types of nonsense codons. Itt1p interacts in vitro with both eRFl and eRF3. Overexpression of eRFl, but not of eRF3, abolishes the nonsense suppressor effect of overexpressed Itt1p. CONCLUSIONS: The data obtained demonstrate that Itt1p can modulate the efficiency of translation termination in yeast. This protein possesses a zinc finger domain characteristic of the TRIAD proteins of higher eukaryotes, and this is a first observation of such protein being involved in translation

Fungal Prions: Structure, Function and Propagation

Prions are not uniquely associated with rare fatal neurodegenerative diseases in the animal kingdom; prions are also found in fungi and in particular the yeast Saccharomyces cerevisiae. As with animal prions, fungal prions are proteins able to exist in one or more self-propagating alternative conformations, but show little primary sequence relationship with the mammalian prion protein PrP. Rather, fungal prions represent a relatively diverse collection of proteins that participate in key cellular processes such as transcription and translation. Upon switching to their prion form, these proteins can generate stable, sometimes beneficial, changes in the host cell phenotype. Much has already been learnt about prion structure, and propagation and de novo generation of the prion state through studies in yeast and these findings are reviewed here

Amyloid Fragmentation and Disaggregation in Yeast and Animals

Amyloids are filamentous protein aggregates that are associated with a number of incurable diseases, termed amyloidoses. Amyloids can also manifest as infectious or heritable particles, known as prions. While just one prion is known in humans and animals, more than ten prion amyloids have been discovered in fungi. The propagation of fungal prion amyloids requires the chaperone Hsp104, though in excess it can eliminate some prions. Even though Hsp104 acts to disassemble prion fibrils, at normal levels it fragments them into multiple smaller pieces, which ensures prion propagation and accelerates prion conversion. Animals lack Hsp104, but disaggregation is performed by the same complement of chaperones that assist Hsp104 in yeast—Hsp40, Hsp70, and Hsp110. Exogenous Hsp104 can efficiently cooperate with these chaperones in animals and promotes disaggregation, especially of large amyloid aggregates, which indicates its potential as a treatment for amyloid diseases. However, despite the significant effects, Hsp104 and its potentiated variants may be insufficient to fully dissolve amyloid. In this review, we consider chaperone mechanisms acting to disassemble heritable protein aggregates in yeast and animals, and their potential use in the therapy of human amyloid diseases

Mapping of Prion Structures in the Yeast Rnq1

The Rnq1 protein is one of the best-studied yeast prions. It has a large potentially prionogenic C-terminal region of about 250 residues. However, a previous study indicated that only 40 C-terminal residues form a prion structure. Here, we mapped the actual and potential prion structures formed by Rnq1 and its variants truncated from the C-terminus in two [RNQ+] strains using partial proteinase K digestion. The location of these structures differed in most cases from previous predictions by several computer algorithms. Some aggregation patterns observed microscopically for the Rnq1 hybrid proteins differed significantly from those previously observed for Sup35 prion aggregates. The transfer of a prion from the full-sized Rnq1 to its truncated versions caused substantial alteration of prion structures. In contrast to the Sup35 and Swi1, the terminal prionogenic region of 72 residues was not able to efficiently co-aggregate with the full-sized Rnq1 prion. GFP fusion to the Rnq1 C-terminus blocked formation of the prion structure at the Rnq1 C-terminus. Thus, the Rnq1-GFP fusion mostly used in previous studies cannot be considered a faithful tool for studying Rnq1 prion properties

A conditional-lethal translation termination defect in a sup45 mutant of the yeast Saccharomyces cerevisiae

Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational-termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRF1), which has a stop-codon-dependent peptidyl-release activity. Using a conditional lethal mutant allele of SUP45 (sup45-2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor-tRNA-mediated readthrough of stop codons is used to show that a translating lysate from a sup45-2 mutant strain exhibits a termination defect when heated for short periods to greater than the non-permissive temperature (37 degrees C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli, The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non-permissive temperature. Cell-free translation lysates from the sup45-2 strain do not show a defect in sense-codon translation at the non-permissive temperature. These data demonstrate that yeast eRF1 plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues

Structural Bases of Prion Variation in Yeast

Amyloids are protein aggregates with a specific filamentous structure that are related to a number of human diseases, and also to some important physiological processes in animals and other kingdoms of life. Amyloids in yeast can stably propagate as heritable units, prions. Yeast prions are of interest both on their own and as a model for amyloids and prions in general. In this review, we consider the structure of yeast prions and its variation, how such structures determine the balance of aggregated and soluble prion protein through interaction with chaperones and how the aggregated state affects the non-prion functions of these proteins

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