Global analysis of protein aggregation in yeast during physiological conditions and arsenite stress.

Protein aggregation is a widespread phenomenon in cells and associated with pathological conditions. Yet, little is known about the rules that govern protein aggregation in living cells. In this study, we biochemically isolated aggregation-prone proteins and used computational analyses to identify characteristics that are linked to physiological and arsenite-induced aggregation in living yeast cells. High protein abundance, extensive physical interactions, and certain structural properties are positively correlated with an increased aggregation propensity. The aggregated proteins have high translation rates and are substrates of ribosome-associated Hsp70 chaperones, indicating that they are susceptible for aggregation primarily during translation/folding. The aggregation-prone proteins are enriched for multiple chaperone interactions, thus high protein abundance is probably counterbalanced by molecular chaperones to allow soluble expression in vivo. Our data support the notion that arsenite interferes with chaperone activity and indicate that arsenite-aggregated proteins might engage in extensive aberrant protein-protein interactions. Expression of aggregation-prone proteins is down-regulated during arsenite stress, possibly to prevent their toxic accumulation. Several aggregation-prone yeast proteins have human homologues that are implicated in misfolding diseases, suggesting that similar mechanisms may apply in disease- and non-disease settings.

Arsenite interferes with protein folding and triggers formation of protein aggregates in yeast.

Several metals and metalloids profoundly affect biological systems, but their impact on the proteome and mechanisms of toxicity are not fully understood. Here, we demonstrate that arsenite causes protein aggregation in Saccharomyces cerevisiae. Various molecular chaperones were found to be associated with arsenite-induced aggregates indicating that this metalloid promotes protein misfolding. Using in vivo and in vitro assays, we show that proteins in the process of synthesis/folding are particularly sensitive to arsenite-induced aggregation, that arsenite interferes with protein folding by acting on unfolded polypeptides, and that arsenite directly inhibits chaperone activity. Thus, folding inhibition contributes to arsenite toxicity in two ways: by aggregate formation and by chaperone inhibition. Importantly, arsenite-induced protein aggregates can act as seeds committing other, labile proteins to misfold and aggregate. Our findings describe a novel mechanism of toxicity that may explain the suggested role of this metalloid in the etiology and pathogenesis of protein folding disorders associated with arsenic poisoning.

The essential iron-sulfur protein Rli1 is an important target accounting for inhibition of cell growth by reactive oxygen species.

Oxidative stress mediated by reactive oxygen species (ROS) is linked to degenerative conditions in humans and damage to an array of cellular components. However, it is unclear which molecular target(s) may be the primary "Achilles' heel" of organisms, accounting for the inhibitory action of ROS. Rli1p (ABCE1) is an essential and highly conserved protein of eukaryotes and archaea that requires notoriously ROS-labile cofactors (Fe-S clusters) for its functions in protein synthesis. In this study, we tested the hypothesis that ROS toxicity is caused by Rli1p dysfunction. In addition to being essential, Rli1p activity (in nuclear ribosomal-subunit export) was shown to be impaired by mild oxidative stress in yeast. Furthermore, prooxidant resistance was decreased by RLI1 repression and increased by RLI1 overexpression. This Rlip1 dependency was abolished during anaerobicity and accentuated in cells expressing a FeS cluster-defective Rli1p construct. The protein's FeS clusters appeared ROS labile during in vitro incubations, but less so in vivo. Instead, it was primarily (55)FeS-cluster supply to Rli1p that was defective in prooxidant-exposed cells. The data indicate that, owing to its essential nature but dependency on ROS-labile FeS clusters, Rli1p function is a primary target of ROS action. Such insight could help inform new approaches for combating oxidative stress-related disease.

Methionine oxidation of Sup35 protein induces formation of the [PSI+] prion in a yeast peroxiredoxin mutant.

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.

Ribosome-associated peroxiredoxins suppress oxidative stress-induced de novo formation of the [PSI+] prion in yeast.

Peroxiredoxins (Prxs) are ubiquitous antioxidants that protect cells against oxidative stress. We show that the yeast Tsa1/Tsa2 Prxs colocalize to ribosomes and function to protect the Sup35 translation termination factor against oxidative stress-induced formation of its heritable [PSI(+)] prion conformation. In a tsa1 tsa2 [psi(-)] [PIN(+)] strain, the frequency of [PSI(+)] de novo formation is significantly elevated. The Tsa1/Tsa2 Prxs, like other 2-Cys Prxs, have dual activities as peroxidases and chaperones, and we show that the peroxidase activity is required to suppress spontaneous de novo [PSI(+)] prion formation. Molecular oxygen is required for [PSI(+)] prion formation as growth under anaerobic conditions prevents prion formation in the tsa1 tsa2 mutant. Conversely, oxidative stress conditions induced by exposure to hydrogen peroxide elevates the rate of de novo [PSI(+)] prion formation leading to increased suppression of all three termination codons in the tsa1 tsa2 mutant. Altered translational fidelity in [PSI(+)] strains may provide a mechanism that promotes genetic variation and phenotypic diversity (True HL, Lindquist SL (2000) Nature 407:477-483). In agreement, we find that prion formation provides yeast cells with an adaptive advantage under oxidative stress conditions, as elimination of the [PSI(+)] prion from tsa1 tsa2 mutants renders the resulting [psi(-)] [pin(-)] cells hypersensitive to hydrogen peroxide. These data support a model in which Prxs function to protect the ribosomal machinery against oxidative damage, but when these systems become overwhelmed, [PSI(+)] prion formation provides a mechanism for uncovering genetic traits that aid survival during oxidative stress conditions.

Methionine sulphoxide reductases protect iron-sulphur clusters from oxidative inactivation in yeast.

Methionine residues and iron-sulphur (FeS) clusters are primary targets of reactive oxygen species in the proteins of micro-organisms. Here, we show that methionine redox modifications help to preserve essential FeS cluster activities in yeast. Mutants defective for the highly conserved methionine sulphoxide reductases (MSRs; which re-reduce oxidized methionines) are sensitive to many pro-oxidants, but here exhibited an unexpected copper resistance. This phenotype was mimicked by methionine sulphoxide supplementation. Microarray analyses highlighted several Cu and Fe homeostasis genes that were upregulated in the mxrDelta double mutant, which lacks both of the yeast MSRs. Of the upregulated genes, the Cu-binding Fe transporter Fet3p proved to be required for the Cu-resistance phenotype. FET3 is known to be regulated by the Aft1 transcription factor, which responds to low mitochondrial FeS-cluster status. Here, constitutive Aft1p expression in the wild-type reproduced the Cu-resistance phenotype, and FeS-cluster functions were found to be defective in the mxrDelta mutant. Genetic perturbation of FeS activity also mimicked FET3-dependent Cu resistance. 55Fe-labelling studies showed that FeS clusters are turned over more rapidly in the mxrDelta mutant than the wild-type, consistent with elevated oxidative targeting of the clusters in MSR-deficient cells. The potential underlying molecular mechanisms of this targeting are discussed. Moreover, the results indicate an important new role for cellular MSR enzymes in helping to protect the essential function of FeS clusters in aerobic settings.

Oxidative protein damage causes chromium toxicity in yeast.

Oxidative damage in microbial cells occurs during exposure to the toxic metal chromium, but it is not certain whether such oxidation accounts for the toxicity of Cr. Here, a Saccharomyces cerevisiae sod1Delta mutant (defective for the Cu,Zn-superoxide dismutase) was found to be hypersensitive to Cr(VI) toxicity under aerobic conditions, but this phenotype was suppressed under anaerobic conditions. Studies with cells expressing a Sod1p variant (Sod1(H46C)) showed that the superoxide dismutase activity rather than the metal-binding function of Sod1p was required for Cr resistance. To help identify the macromolecular target(s) of Cr-dependent oxidative damage, cells deficient for the reduction of phospholipid hydroperoxides (gpx3Delta and gpx1Delta/gpx2Delta/gpx3Delta) and for the repair of DNA oxidation (ogg1Delta and rad30Delta/ogg1Delta) were tested, but were found not to be Cr-sensitive. In contrast, S. cerevisiae msraDelta (mxr1Delta) and msrbDelta (ycl033cDelta) mutants defective for peptide methionine sulfoxide reductase (MSR) activity exhibited a Cr sensitivity phenotype, and cells overexpressing these enzymes were Cr-resistant. Overexpression of MSRs also suppressed the Cr sensitivity of sod1Delta cells. The inference that protein oxidation is a primary mechanism of Cr toxicity was corroborated by an observed approximately 20-fold increase in the cellular levels of protein carbonyls within 30 min of Cr exposure. Carbonylation was not distributed evenly among the expressed proteins of the cells; certain glycolytic enzymes and heat-shock proteins were specifically targeted by Cr-dependent oxidative damage. This study establishes an oxidative mode of Cr toxicity in S. cerevisiae, which primarily involves oxidative damage to cellular proteins.