Heat Shock-Induced Accumulation of Translation Elongation and Termination Factors Precedes Assembly of Stress Granules in <em>S. cerevisiae</em>

<div><p>In response to severe environmental stresses eukaryotic cells shut down translation and accumulate components of the translational machinery in stress granules (SGs). Since they contain mainly mRNA, translation initiation factors and 40S ribosomal subunits, they have been referred to as dominant accumulations of stalled translation preinitiation complexes. Here we present evidence that the robust heat shock-induced SGs of <i>S. cerevisiae</i> also contain translation elongation factors eEF3 (Yef3p) and eEF1Bγ2 (Tef4p) as well as translation termination factors eRF1 (Sup45p) and eRF3 (Sup35p). Despite the presence of the yeast prion protein Sup35 in heat shock-induced SGs, we found out that its prion-like domain is not involved in the SGs assembly. Factors eEF3, eEF1Bγ2 and eRF1 were accumulated and co-localized with Dcp2 foci even upon a milder heat shock at 42°C independently of P-bodies scaffolding proteins. We also show that eEF3 accumulations at 42°C determine sites of the genuine SGs assembly at 46°C. We suggest that identification of translation elongation and termination factors in SGs might help to understand the mechanism of the eIF2α factor phosphorylation-independent repression of translation and SGs assembly.</p> </div

RNP accumulations formed at 42°C serve as “seeds” for genuine SGs.

<p>(A) Distribution of Dcp2-GFP was analyzed in wild-type (CRY564 strain) and <i>edc3</i>Δ<i>lsm4</i>ΔC mutant (CRY977 strain) cells after incubation at 30°C, 37°C and 42°C for 10 minutes. These wild-type cells exhibited increasing number of enlarged accumulations of the Dcp2-GFP fusion protein corresponding to raising temperatures of the heat shock. In contrast, <i>edc3</i>Δ<i>lsm4</i>ΔC mutant cells did not exhibit any cytoplasmic accumulations of the Dcp2-GFP fusion protein upon incubation at 30°C or 37°C. However, these mutant cells displayed accumulations of Dcp2-GFP upon heat shock at 42°C for 10 minutes. (B) The distribution of fusion proteins Pub1-GFP, Ngr1-GFP and Yef3-GFP was analyzed in <i>edc3</i>Δ<i>lsm4</i>ΔC mutant cells (CRY1035, CRY1041 and CRY1146 strains) either at 30°C or upon heat shock at 42°C for 10 minutes. All of these proteins accumulated in the mutant cells at 42°C, thus independently of P-body scaffolding proteins Edc3 and Lsm4. (C) Time lapse experiment using the cells co-expressing Yef3-GFP and Rpg1-RFP fusion proteins (CRY649 strain) showing the formation of Yef3-GFP foci after 10 minutes at 42°C, accumulation of Rpg1-RFP protein during the temperature rise from 42°C to 46°C and after 10 minutes at 46°C. Yef3-GFP foci were first formed at 42°C, whereas the localization of the Rpg1-RFP protein was still uniformly cytoplasmic at this temperature. During heating from 42°C to 46°C and upon cultivation at 46°C, the Rpg1-RFP protein only accumulated at the preformed Yef3-GFP foci. Scale bar 4 µm.</p

Heat shock-induced stress granules contain translation elongation factor eEF3 (Yef3p).

<p>(A) The distribution of fusion proteins Yef3-GFP and Rpg1-RFP (CRY649 strain) was uniformly cytosolic at 30°C. Accumulations of Yef3-GFP fusion protein completely co-localized with accumulations of the stress granule marker protein Rpg1-GFP in cells heat-shocked at 46°C for 10 minutes. The Scatter plot produced by colocalization analysis software shows colocalized pixels along the diagonal. Very high values of the Pearsońs correlation coefficient (R_r), over 0.9, confirmed the high degree of this co-localization. (B) Localization of theYef3-mCherry fusion protein with Ngr1-GFP (CRY1287 strain) and Dcp2-GFP (CRY1559 strain) fusion proteins under control conditions (30°C) and in heat-shocked cells at 46°C for 10 minutes. Yef3-mCherry protein co-localized with heat-induced accumulations of stress granules marker proteins Dcp2-GFP and Ngr1-GFP. (C) Yef3-GFP accumulates in cytoplasmic foci in both, wild-type (CRY510 strain) and <i>gcn2</i>Δ (CRY1292 strain), cells heat-shocked at 46°C. Scale bar 4 µm.</p

Full length Sup35 protein is not necessary for SGs assembly and its localization into SGs.

<p>(A) The distribution of Rpg1-GFP fusion protein under control (30°C) and heat shock (46°C) conditions in either the wild-type strain (Sup35p; CRY1011 strain) or the strain expressing only the N-terminal truncated form of Sup35 protein (Sup35ΔN; CRY1007 strain). SGs (Rpg1-GFP) were formed even in the mutant strain suggesting that the N-terminal part of Sup35 protein is dispensable for the SGs formation. Scale bar 4 µm. (B) Pellet and supernatant fractions from the wild-type (CRY998 strain) and the Sup35 N-terminal truncated mutant strains (CRY1001 strain) were analyzed by SDS-PAGE and Western blotting using the anti-Sup35 antibody. Samples were prepared as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057083#pone.0057083-Grousl1" target="_blank">[2]</a>. The N-terminal truncated form of Sup35 protein was enriched in the pellet fraction of heat-shocked cells, as well as the wild-type form of Sup35 protein, suggesting that only Sup35 C-terminal part is sufficient to localize this protein to the heat-induced stress granules.</p

Translation elongation and termination factors accumulate in the same foci at 42

<p>°<b>C.</b> (A) The distribution of Yef3-GFP or Yef3-mCherry fusion protein was analyzed in wild-type cells (CRY510 strain: CRY1339 strain) heat-shocked at 42°C for 10 minutes. Whereas the Yef3-GFP and Yef3-mCherry fusion protein were uniformly cytosolic at 30°C, they formed discrete cytoplasmic foci in the cells heat-shocked at 42°C for 10 minutes. (B) Distribution of the Yef3-GFP protein (CRY510 strain) was analyzed in control, heat-shocked (at 42°C) and recovering cells. Yef3-GFP accumulations were dissolved within 20 minutes of the cell recovery from the stress. (C) The distribution of Sup35-GFP (CRY412 strain), Sup45-GFP (CRY1552 strain) and Tef4-GFP (CRY554 strain) was analyzed under control conditions at 30°C and under heat shock at 42°C. Whereas Sup35-GFP protein remained uniformly cytosolic even upon the heat shock at 42°C, Sup45-GFP and Tef4-GFP proteins accumulated into discrete cytoplasmic foci (D) Yef3-mCherry fusion protein was co-localized either with Tef4-GFP (CRY1315 strain), Dcp2-GFP (CRY1559 strain) or Ngr1-GFP (CRY1287 strain) fusion proteins, as well as Sup45-GFP fusion protein was co-localized with Dcp2-mCherry fusion protein (CRY1636 strain), in cells heat-shocked at 42°C.</p

MS identification of Yef3 (eEF3) protein.

<p>MS identification of Yef3 (eEF3) protein.</p

Heat shock at 42°C alters translation and triggers accumulation of mRNA in Yef3 foci.

<p>(A) Distribution of <i>PGK1</i> mRNA (<i>PGK1</i> mRNA/U1A-GFP reporter system) at 30°C or upon heat shock at 42°C was analyzed in cells expressing Yef3-mCherry fusion protein (CRY1618 strain). Fine granules of <i>PGK1</i> mRNA (U1A-GFP) were present at 30°C in a majority of cells. The heat shock at 42°C induced an accumulation of some of these granules into larger clusters and formations of Yef3 foci. Arrows point to obviously overlapping signals of both fusions. Scale bar 4 µm. (B) Polysome profiles of wild-type strain (BY4741) at permissive temperature and after heat shock at 42°C for 10 minutes. Translation profile was altered in heat-shocked cells. P/M stands for polysome/monosome ratio. (C) Distribution of Yef3-GFP (CRY510 strain) was analyzed in cells heat-shocked at 42°C for 10 minutes with or without preincubation in the presence of cycloheximide (CYH). When this drug was added before the heat shock, no accumulations of Yef3-GFP protein were formed. Scale bar 4 µm.</p

Yeast strains.

<p>Yeast strains.</p

Factors eEF1Bγ2 (Tef4p), eRF1 (Sup45p) and eRF3 (Sup35p) are novel components of stress granules.

<p>(A) The distribution of Tef4-GFP (CRY554 strain), Sup35-GFP (CRY412 strain) and Sup45-GFP (CRY1552 strain) proteins was analyzed in control (30°C) and heat-shocked (46°C) cells. All of these fusion proteins accumulated in discrete cytoplasmic foci in heat-shocked cells. (B) Subcellular localization of Eft1-GFP (eEF2) (CRY552 strain), Tif11-GFP (eIF1A) (CRY521 strain) and Tef1-GFP (eEF1A) (CRY1764 strain) fusion proteins upon control and heat shock conditions. The distribution of Eft1-GFP, Tif11-GFP and Tef1-GFP remained diffusely cytosolic even upon the stress condition. (C) Tef4-GFP fusion protein co-localized with accumulations of the Yef3-mCherry fusion protein (CRY1315 strain). Similarly, Sup35-GFP and Sup45-GFP co-localized with the stress granule marker protein Rpg1-RFP in cells heat-shocked at 46°C for 10 minutes (strains CRY1364; CRY1627 strains). High values of the Pearsońs correlation coefficient (R_r) confirmed the high degree of this co-localization. Scale bar 4 µm. (D) Pellet and supernatant fractions from the appropriate cell lysates (strains CRY554; CRY412; CRY1552 strains) were analyzed by SDS-PAGE and Western blotting. Samples were prepared as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057083#pone.0057083-Grousl1" target="_blank">[2]</a>. All tested fusion proteins were clearly accumulated in the pellet fraction of cells heat-shocked at 46°C for 10 minutes.</p

Translation elongation factor eEF3 (Yef3p) accumulates in foci upon robust heat shock.

<p>(A) Pellet fractions from the <i>edc3</i>Δ<i>lsm4</i>ΔC cell (CRY1043 strain) lysates taken upon control (30°C) and heat shock (42°C and 46°C) conditions were separated by SDS-PAGE and stained with Coomassie Brilliant Blue. Yef3 protein, identified by MS analysis, is enriched in the pellet fraction from the cells heat-shocked at 46°C for 10 minutes. (B) Pellet and supernatant fractions from the lysates of cells expressing Yef3-GFP (CRY510 strain) were analyzed by SDS-PAGE and Western blotting. Samples were prepared as described elsewhere <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0057083#pone.0057083-Grousl1" target="_blank">[2]</a>. The Yef3-GFP fusion protein was accumulated in the pellet fraction of heat-shocked cells. (C) The Yef3 fusions were accumulated at cytoplasmic foci in wild-type cells (CRY510 and CRY1339 strains) as well as in the <i>edc3</i>Δ<i>lsm4</i>ΔC mutant cells (CRY1146 strain) heat-shocked at 46°C for 10 minutes. Scale bar 4 µm.</p