Extraction is near complete for the majority of proteins.

<p>(A) Yeast cells were extracted in a first round with the procedure described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001078#pone-0001078-g001" target="_blank">figure 1</a> and recovered. They were then subjected to a second round of extraction either by boiling in sample buffer also containing 8 M Urea (sample “Urea”), by glass bead lysis followed by boiling in normal sample buffer (sample “Glass Beads”), or by glass bead lysis followed by boiling in sample buffer also containing 8 M Urea (sample “Glass Beads, Urea”). Note that the second extracts are twice concentrated with respect to the first extract. (B) Western blotting analysis of samples from panel A. The western blot using anti-Sup35 antibodies confirms that this particular protein requires Urea for efficient extraction. In contrast, other individually tested proteins were not recovered by a second Urea-based extraction (Pgk1p is shown here as a representative example). (C) The total protein content of mock-extracted and extracted yeast cells was visualized by staining with the fluorescent protein stain Sypro Red. The Sypro Red Signal drops by >95% following application of the extraction procedure.</p

Deletion of <i>hcr1</i> results in accumulation of eRF3 in heavy polysomes.

<p>(<b>A–C</b>) The <i>hcr1Δ</i> strain (H3675) was transformed with either hc <i>HCR1</i> (<b>A</b>), empty vector (<b>B</b>), or hc <i>RLI1</i> (<b>C</b>), and the resulting transformants were grown in SD medium at 30°C to an OD₆₀₀ of ∼1 and cross-linked with 0.5% HCHO prior to harvesting. WCEs were prepared, separated on a 5%–45% sucrose gradient by centrifugation at 39,000 rpm for 2.5 h and subjected to Western blot analysis. Several fractions corresponding to the Top, 40S, 60S, and 80S plus polysomal species were pooled, as indicated. Asterisk indicates a non-specific band. (<b>D</b>) Statistical significance of the eRF3 accumulation in heavy polysomes in the <i>hcr1</i> strain and its partial recovery by hc <i>RLI1</i>. Amounts of each individual factor in all fractions were quantified by fluorescence imaging. Thus obtained values for the fractions containing heavy polysomes (14–18) as well as all remaining fractions (1–13) were added up for each of these two groups. Values (mean±SE; n = 4) given in the table then represent relative amounts of factors in heavy polysomes divided by the compound value of the rest of the gradient. Changes in the redistribution of factors between the heavy polysomes and lighter fractions in all three strain were analyzed by the student's <i>t</i>-test and shown to be statistically significant only for eRF3 as shown in the table. (<b>E</b>) Statistical significance of the eIF3 shift from 40S-containing fractions to the top, which is independent of the effect of hc <i>RLI1</i> on eRF3. Essential the same as in panel D, except that the values for the Top fractions (1–4) as well as the 40S fractions (5–6) were added up for each of these two groups. Values (mean±SE; n = 4) given in the table then represent relative amounts of factors in the Top divided by the 40S group. Changes in the redistribution of factors between the 40S and Top fractions in <i>hcr1Δ</i>+EV or +hc <i>RLI1</i> strains vs. wt were analyzed by the student's <i>t</i>-test and shown to be statistically significant only for eIF3 as shown in the table.</p

Complexes containing eIF3, HCR1, ABCE1/RLI1 and both eRFs, free of ribosomes and RNA, occur <i>in vivo</i>; and the NTD of g/TIF35 and i/TIF34 directly interact with the N and M domains of eRF1.

<p>(<b>A</b>) WCEs were prepared from YDH353 bearing chromosomal Myc-tagged <i>RLI1</i> and immunoprecipitated with or without anti-Myc antibodies. The immune complexes were subjected to Western analysis. In, 5% of input; E, 100% of the elution fraction; W, 5% of the supernatant fraction. Also note that anti-RLI1 and -eRF1 antibodies were raised for the purpose of this study. (<b>B</b>) WCEs were prepared from HCHO-treated (1%) cells bearing wt (H2879) or TAP-tagged (H553) chromosomal alleles of <i>HCR1</i> and incubated with IgG Sepharose 6 Fast Flow beads. The immune complexes were eluted by boiling in the SDS buffer and subjected to Western analysis. In, 1.5% of input; E, 50% of the elution fraction; W, 1.5% of the supernatant fraction. eRF1 is indicated by an asterisk below the immunoglobulins. (<b>C</b>) WCEs from HCHO-treated cells (1%) cells bearing wt (H2879) or TAP-tagged (H555) chromosomal alleles of <i>TIF32</i> were processed as in panel B except that the immune complexes were eluted by TEV protease cleavage. In, 1.5% of input; E, 100% of the elution fraction; W, 1.5% of the supernatant fraction. (<b>D</b>) WCEs from HCHO-treated cells (1%) cells bearing wt (74D-694) or TAP-tagged (H517) chromosomal alleles of <i>SUP35</i> were processed as in panel C. (<b>E</b>) Full-length i/TIF34 (lane 3), g/TIF35 (lane 4), and HCR1 (lane 5) fused to GST, and GST alone (lane 2), were tested for binding to ³⁵S-labeled individual domains of eRF1; 10% of input amounts added to each reaction is shown in lane 1 (In). (<b>F</b>) The RRM (lane 3) and N-terminal (lane 4) domains of g/TIF35 fused to GST, and GST alone (lane 2), were tested for binding to ³⁵S-labeled NM domains of eRF1; 10% of input amounts added to each reaction is shown in lane 1.</p

Increased gene dosage of ABCE/RLI1 suppresses the slow growth and read-through defects of <i>hcr1Δ</i>.

<p>(<b>A</b>) The <i>hcr1Δ</i> strain was transformed with either empty vector (EV), hc <i>HCR1</i> or hc <i>RLI1</i>. The resulting transformants were subjected to a growth spot assay at 30°C for 2 days. (<b>B</b>) The <i>hcr1Δ</i> strain was transformed with hc vectors carrying either wt or mutant <i>HCR1</i> and <i>RLI1</i> alleles, and <i>SUI1</i> (eIF1) and <i>TIF11</i> (eIF1A). The resulting transformants were grown in SD and analyzed for stop codon read-through as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003962#pgen-1003962-g001" target="_blank">Figure 1</a>. Thus obtained values were normalized to the value obtained with the <i>hcr1Δ</i> strain transformed with wt <i>HCR1</i>, which was set to 100%.</p

Model of eIF3 and HCR1 involvement in yeast translation termination.

<p>Upon stop codon entry into the ribosomal A-site the pre-TC forms, composed of the canonical release factors eRF1 and eRF3·GTP, and eIF3 and HCR1. eRFs and eIF3 may associate with the pre-TC as a pre-formed unit or alone. In the pre-TC, eIF3 interacts with the N domain of eRF1, <i>via</i> its two small g/TIF35 and i/TI34 subunits, and modulates, perhaps inhibits its stop codon recognition activity during the proofreading step. Upon stop codon recognition the GTP molecule on eRF3 is hydrolyzed. Subsequently, HCR1 promotes eRF3·GDP ejection to allow the ABCE1/RLI1·ATP recruitment to begin the accommodation phase of termination – the eRF1 GGQ motif is pushed to the peptidyl-transferase center (PTC) – during which HCR1 interacts with ABCE1/RLI1. Subsequently, both factors together with eIF3 participate in ribosomal recycling to enable and promote initiation of the next translational cycle (the elongation step is shown only for illustration purposes).</p