Methionine Sulfoxide Reductases Suppress the Formation of the [ PSI+] Prion and Protein Aggregation in Yeast

Abstract

Prions are self-propagating, misfolded forms of proteins associated with various neurodegenerative diseases in mammals and heritable traits in yeast. How prions form spontaneously into infectious amyloid-like structures without underlying genetic changes is poorly understood. Previous studies have suggested that methionine oxidation may underlie the switch from a soluble protein to the prion form. In this current study, we have examined the role of methionine sulfoxide reductases (MXRs) in protecting against de novo formation of the yeast [PSI⁺] prion, which is the amyloid form of the Sup35 translation termination factor. We show that [PSI⁺] formation is increased during normal and oxidative stress conditions in mutants lacking either one of the yeast MXRs (Mxr1, Mxr2), which protect against methionine oxidation by reducing the two epimers of methionine-S-sulfoxide. We have identified a methionine residue (Met124) in Sup35 that is important for prion formation, confirming that direct Sup35 oxidation causes [PSI⁺] prion formation. [PSI⁺] formation was less pronounced in mutants simultaneously lacking both MXR isoenzymes, and we show that the morphology and biophysical properties of protein aggregates are altered in this mutant. Taken together, our data indicate that methionine oxidation triggers spontaneous [PSI⁺] prion formation, which can be alleviated by methionine sulfoxide reductases.

Keywords: methionine oxidation; methionine sulfoxide reductase; oxidative stress; prions; protein aggregation; yeast.

Figure 1

Strains lacking MXRs are unaffected by hydrogen peroxide sensitivity. (A) Wild-type (74D-694) and MXR mutant strains were grown to the exponential phase, and the A₆₀₀ adjusted to 1, 0.1, 0.01, or 0.001 before spotting onto plates containing the indicated concentrations of hydrogen peroxide. (B) Wild-type (BY4741, BY4742) and isogenic MXR mutant strains were tested for hydrogen peroxide sensitivity as described in panel (A).

Figure 2

The frequency of [PSI⁺] prion formation is increased in single MXR mutants. (A) [PSI⁺] prion formation was quantified in the wild-type and MXR mutant strains in the presence or absence of hydrogen peroxide. This was repeated in strains containing wild-type Sup35 or the M124A mutant. Data shown are the means of three independent biological repeat experiments expressed as the number of colonies per viable cell. Error bars denote standard deviation. Significance (one-way ANOVA) is shown compared with the wild-type strain in the absence or presence of hydrogen peroxide; ** p < 0.01, *** p < 0.01. (B) Western blot analysis of the same strains probed with αSup35 or α-Pgk1 as a loading control. Band intensities were quantified and are shown comparing Sup35 with Pgk1 normalized to the untreated wild-type strains (lane 1). (C) Schematic showing domain structure of Sup35. The Sup35 protein can be divided into 3 distinct regions: an N-terminal prion-forming domain (PrD), a highly charged middle region (M), and a C-terminal domain that functions in translation termination (C). Sup35 contains a total of 19 Met residues, including its amino-terminal Met residue. One Met residue is located between the N and M regions (M124), and the other internal 17 Met residues are all located in the C-terminal catalytic region.

Figure 3

Strains lacking MXRs have higher levels of protein aggregation. (A) Hsp104-RFP was visualized in wild-type and MXR mutant cells grown to the exponential phase. Examples of cells containing visible puncta are shown. The percentage of cells containing visible Hsp104-RFP puncta was quantified for each strain. Data shown are the means of three independent biological repeat experiments ± SD. Significance is shown compared with the wild-type strain; * p < 0.05, *** p < 0.001. (B) Protein aggregates were isolated from the same strains and analyzed by SDS-PAGE and silver staining. (C) Proteins within insoluble aggregate fractions were identified by mass spectrometry. The Venn diagrams show pairwise comparisons of the overlaps between the proteins aggregating in wild-type and MXR mutant strains (mxr1, mxr2, mxr1,2). (D) Venn diagrams showing the localization of the proteins that aggregate in the wild-type and MXR mutants.

Figure 4

The localization and physicochemical properties of aggregated proteins are similar in wild-type and MXR mutants. Comparison of the aggregated proteins present in MXR mutants and the wild-type strain. Aggregated proteins were compared with a list of unaggregated proteins identified by mass spectrometry, referred to as the MS set. (A) Grand average of hydrophobicity (GRAVY). (B) The abundance of proteins (molecules/cell) in each set during non-stress conditions. (C) Translational efficiency (TE) expressed as the ratio of ribosome footprint density to mRNA density [61]. (D) Protein size (kDa). Mann–Whitney U-tests were used to assess the statistical significance of observed differences: * p < 0.05, ** p < 0.01, *** p < 0.001. (E) Comparison of the aggregated proteins present in the wild-type and MXR mutants with other datasets including proteins that aggregate during heat-shock [62], proteins that aggregate during AZC stress [59], proteins that aggregate during hydrogen peroxide stress [59], proteins that ag-gregate during postmitotic ageing in yeast [63]. Numbers in brackets indicate the size of each dataset. The significance of overlaps was determined by a hypergeometric test. Blue: p < 0.01, red: p < 0.001.

Figure 5

Visualization of overexpression-induced aggregation of Sup35. (A) Fluorescence micrographs are shown for wild-type and MXR mutant strains containing the Sup35NM-GFP plasmid induced with copper for the indicated times. Representative images are shown for puncta or rod- and ribbon-like aggregates at the indicated time points. The percentage of cells containing visible puncta or rod- and ribbon-like aggregates is indicated for at least 300 cells counted for each strain. (B) The GFP-positive areas were quantified for puncta formed in each strain following induction of Sup35NM-GFP for 20 h. At least 38 puncta were measured from each strain, and statistical significance was determined using Mann–Whitney U-tests (*** p < 0.001). (C) SDS-resistant Sup35 aggregates were detected in the same strains using SDD-AGE. [PSI⁺] and [psi⁻] derivatives of 74D-694 are shown for comparison.

Figure 6

Overexpression-induced [PSI⁺] prion formation is increased in single but not double MXR mutant strains. (A) [PSI⁺] prion formation was quantified in the wild-type and MXR mutant strains containing vector alone or expressing Hsp104 under the control of the constitutively active TDH3 promoter. Prion formation was induced by inducing Sup35NM-GFP expression following 20 h of copper induction. Data shown are the means of three independent biological repeat experiments expressed as the number of [PSI⁺] cells per viable cell. Error bars denote standard deviation. Significance was tested using an unpaired t-test, and pairwise comparisons are for the wild-type strain and MXR mutants containing the vector, or each strain comparing vector with HSP104; * p < 0.05, ** p < 0.01. (B) Western blot analysis of the same strains probed with αSup35 (endogenous Sup35 and NM-GFP), αHsp104 (endogenous Hsp104 and Hsp104-mCherry), or α-Pgk1 as a loading control. (C) Fluorescence micrographs showing examples of puncta or rod- and ribbon-like aggregates formed in the wild-type and MXR mutant strains expressing Hsp104 under the control of the constitutively active TDH3 promoter. Prion formation was induced by inducing Sup35NM-GFP expression following 20 h of copper induction. The percentage of cells containing visible puncta or rod- and ribbon-like aggregates is indicated. (D) The GFP-positive areas were quantified for puncta formed in panel (C). At least 16 puncta were measured from each strain.