Induction of [<i>PSI</i>⁺] prion formation by overexpression of Sup35.

<p><b>A.</b> Fluorescence micrographs are shown for [<i>PIN</i>⁺][<i>psi</i>^-] versions of wild-type, <i>abp1</i>, <i>crn1</i> and <i>pan1</i> mutant strains containing the Sup35NM-GFP plasmid induced with copper for 24 hours. Representative Images are shown for rod/ribbon-like and foci aggregate formation. The numbers indicate the percentage of cells containing each type of aggregate from an average of 300 cells counted. The scale bar is 3μm. <b>B.</b> Western blot analysis of the wild-type and <i>abp1</i> mutant strain following induction of Sup35NM-GFP. Blots were probed with αSup35 or α-Pgk1 as a loading control <b>(C)</b> [<i>PSI</i>⁺] prion formation was quantified in the wild-type and <i>abp1</i> mutant strain containing the <i>Sup35NM-GFP</i> plasmid following 24 h of copper induction. Data shown are the means of three independent biological repeat experiments expressed as the number of colonies per 10⁴ viable cells. Error bars denote standard deviation; * marks statistical significance at p<0.01.</p

Analysis of protein oxidation and aggregation in an <i>abp1</i> mutant.

<p><b>A.</b> Mutants disrupting the cortical actin cytoskeleton dot not affect Sup35 protein oxidative damage. The wild-type, <i>abp1</i>, <i>crn1</i> and <i>pan1</i> mutant strains were treated with 100μM hydrogen peroxide for 20 hours and protein carbonylation used as a measure of protein oxidative damage. Protein extracts were treated with the carbonyl-specific probe, DNPH, and analyzed by Western blot analysis using an antibody against DNPH. <b>B.</b> Subcellular distribution of Sup35 in wild-type and <i>abp1</i> mutant cells grown in the presence or absence of 100μM hydrogen peroxide for 20 hours. Total denotes total crude extract; Soluble, soluble fraction; Pellet, SDS-resistant high molecular weight fraction. <b>C.</b> Representative fluorescent micrographs are shown for wild-type and <i>abp1</i> mutant cells expressing GFP–Ubc9^ts. Strains were grown in SRaf media before switching to SGal media to induce GFP–Ubc9^ts expression for 3 hours. This was followed by a 37°C heat shock for 30 minutes to trigger the misfolding and aggregation of Ubc9. Quantification of the frequency of GFP–Ubc9 foci is expressed as the percentage of cells containing fluorescence foci out of approximately 300 cells counted. Error bars denote standard deviation.</p

Reduced frequency of [<i>PSI</i>⁺] formation in cortical actin cytoskeleton mutants.

<p><b>A.</b> The frequency of <i>de novo</i> [<i>PSI</i>⁺] prion formation was quantified in wild-type <i>abp1</i>, <i>crn1</i>, and <i>pan1</i> mutant strains grown in the presence or absence of 100μM hydrogen peroxide for 20 hours. [<i>PSI</i>⁺] formation was quantified using the <i>ade1-14</i> mutant allele by growth on media lacking adenine and differentiated from nuclear gene mutations by their irreversible elimination in GdnHCL. Data shown are the means of at least three independent biological repeat experiments expressed as the number of colonies per 10⁵ viable cells. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>B.</b> [<i>PSI</i>⁺] propagation was scored by culturing [<i>PSI</i>⁺]-versions of wild-type and <i>abp1</i> mutant strains for four days. [<i>PSI</i>⁺] maintenance was quantified using the <i>ade1-14</i> mutant allele as above and is expressed as the percentage of [<i>PSI</i>⁺] cells from three independent biological repeat experiments. <b>C.</b> The frequency of <i>de novo</i> [<i>PSI</i>⁺] prion formation was quantified in wild-type, <i>tsa1 tsa2</i> and <i>tsa1 tsa2 abp1</i> mutants. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>D.</b> <i>De novo</i> [<i>PIN</i>⁺] prion formation was quantified in the wild-type and <i>abp1</i> mutant strain grown in the presence or absence of 100μM hydrogen peroxide for 20 hours. Data shown are the means of three independent biological repeats expressed as the percentage of viable cells forming [<i>PIN</i>⁺] colonies. Error bars denote standard deviation (*p<0.01; # p<0.05).</p

Identification of Sup35-interacting proteins in wild-type and <i>tsa1 tsa2</i> mutant strains.

<p><b>A.</b> Functional categorisation of Sup35-associated proteins. Results are ordered on MIPS category classification numbers and overarching categories are in capitals. Where an overarching category was enriched, sub-categories within the overarching category were omitted from the graph. Confidence of each classification category is shown as Bonferroni corrected <i>p</i>-values. <b>B.</b> Sup35-TAP was immunoprecipitated from the wild-type and <i>tsa1 tsa2</i> mutant and possible interactions examined using immuno-blot analysis with the indicated antibodies. Sup35-TAP co-immunoprecipitates Abp1, Arp3 and Sap190 in a <i>tsa1 tsa2</i> mutant. Sup35-TAP co-immunoprecipitates Act1 in both wild-type and <i>tsa1 tsa2</i> mutant strains. Total denotes whole cell extracts and IP denotes immunoprecipitates.</p

Latrunculin A treatment reduces oxidant-induced [<i>PSI</i>⁺] formation.

<p><b>A.</b> Representative fluorescence micrographs are shown for a [<i>PIN</i>⁺][<i>psi</i>^-] strain treated with 10 μM LTA for 20 hours. Cells were fixed, and stained with rhodamine phalloidin to visualize the cortical actin cytoskeleton. <b>B.</b> Fluorescence micrographs are shown for a [<i>PIN</i>⁺][<i>psi</i>^-] strain treated with 100 μM hydrogen peroxide for 20 hours in the presence or absence of 10 μM LTA. The aggregate frequency is shown as the percentage of cells containing visible Sup35 foci from approximately 1000 cells counted. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>C.</b> [<i>PSI</i>⁺] prion formation was quantified in a [<i>PIN</i>⁺][<i>psi</i>^-] strain treated with 100μM hydrogen peroxide for 20 hours in the presence or absence of 10 μM LTA. Data shown are the means of three independent biological repeats expressed as the number of [<i>PSI</i>⁺] colonies per 10⁵ viable cells. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>(D)</b> [<i>PSI</i>⁺] prion formation was quantified in a [<i>PIN</i>⁺][<i>psi</i>^-] strain containing the <i>Sup35NM-GFP</i> plasmid following 1 and 24 h of copper induction in the presence or absence of 10 μM LTA. Data shown are the means of three independent biological repeats expressed as the number of [<i>PSI</i>⁺] colonies per 10⁴ viable cells. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>E.</b> Western blot analysis of cells grown under the same conditions as for panel D. Blots were probed with α-Sup35 and α-Pgk1 as a loading control.</p

Loss of <i>ABP1</i> reduces the co-localization of Sup35 with Rnq1 aggregates.

<p><b>A.</b> Fluorescence micrographs are shown for the wild-type and <i>abp1</i> mutant strain treated with 100 μM hydrogen peroxide for 20 hours. Sup35-NMGFP was induced with copper for 2 hours to visualize aggregate formation and the frequency is shown as the percentage of cells containing visible Sup35 foci from approximately 1000 cells counted. Error bars denote standard deviation; * marks statistical significance at p<0.01. <b>B.</b> The wild-type and <i>abp1</i> mutant strains containing Sup35NM-RFP and Rnq1-GFP were grown for 20 hours in SRaff media in the presence or absence of 100μM hydrogen peroxide before switching to SGal media for three hours to induce the expression of Sup35NM-RFP and Rnq1-GFP. White arrows show examples where Sup35NM-RFP and Rnq1-GFP co-localize, whereas, grey arrows show examples where Sup35NM-RFP does not localize with Rnq1-GFP. The frequency of co-localization of Sup35NM-RFP and Rnq1-GFP is shown as a percentage of co-localization for at least 17 Sup35NM-RFP foci scored in triplicate experiments. * marks statistical significance at p<0.01</p

Yeast variant ribosome screen.

<p>(<b>A</b>) Five individual luciferase reporters were generated, all sharing identical promoter (ADH1P) and terminator (ADH1T) sequences. PTC codons (X) are depicted by a red arrow and the length of the coding sequence is indicated in base pairs. Co-transformed reporter pairs are indicated by the black arrows. NB: Not to scale. (<b>B</b>) Luciferase activity (relative light units) reports expression level of REN (yellow) and FF (green) reporters for the REN/FF, REN/FFPTC, REN/LA3FF and REN/LA3PTCFF reporter pairs. A possible change in reporter expression level between wild type strain and one of the 124 RP deletion strains (<i>RPX/Δrpx</i>) is reported by an altered luciferase readout (red arrow). This allows the identification of specialized ribosomes for selected modulation of a target mRNA; in this example, the LA3PTCFF mRNA.</p

The location of specialized RPs on the yeast ribosome on the small (A) and large (B) ribosomal subunits.

<p>(<b>A</b>) On the small ribosomal subunit (SSU) RPs that increase translation of REN and FF reporter, but not PTC reporter mRNAs are highlighted in green. (<b>B</b>) The location of a RP on the large ribosomal subunit (LSU) that gave increased LAMB3-PTC reporter mRNA translation is highlighted in green. NB: Ribosome structure information <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0067609#pone.0067609-Armache1" target="_blank">[48]</a>: USCF Chimera; PDB models: 3IZS, 3IZF, 3IZB and 3IZE.</p

Luciferase expression profiles in 124 yeast strains each with a different individual specialized ribosome variant.

<p>The luciferase readouts of each of the four individual FF-based reporters, co-expressed with the REN reporter, are shown as follows: (<b>A</b>) FF/REN and FFPTC/REN; (<b>B</b>) LA3FF/REN and LA3PTCFF/REN. The luciferase readouts were monitored in all 124 yeast strains each with a different individual specialized ribosome variant, and sorted according to expression strength: lowest levels to the left, highest on the right. The REN profiles are shown in yellow and the individual FF-based reporters are shown in green. For each reporter spectrum the luciferase activities for the wild type parent strain are indicated as a red dot, the grand mean of the given spectrum of assays as a black dot and standard deviation of the grand mean as black triangles.</p

Supplemental Figure 1 from The control of translational accuracy is a determinant of healthy ageing in yeast

Translational accuracy in an rcm1 deletion strain