Tpk2 and Tpk3 affect the translational response to severe heat stress.

<p>(A) mRNA expression level was determined by q-RT-PCR on samples before and after severe heat stress. The value represents mean +/- SEM, n = 2. <i>ACT1</i> mRNA was used as a control. (B) The RNA collected from sucrose gradient fractions was pooled into monosome (M) and polysome (P) fractions. The mRNA distribution was analyzed by qRT-PCR and quantified relative to a luciferase mRNA control. Translational activity change was calculated as described in Materials and Methods. The values represent mean from two independent samples. The value represents mean +/- SEM, n = 2.</p

Tpk2 and Tpk3 show an opposite role in translational arrest in response to severe heat stress.

<p>Polysomal profile analysis and immunoblots of 15–50% sucrose gradient fractions from WT (A), <i>tpk2</i>Δ and <i>tpk3</i>Δ cells grown to exponential phase in YPD (30°C) and subjected to severe heat stress (46°C for 10 minutes). Free, monosome and polysome regions are indicated over the polysome profile. The numbers represent the polysome/monosome area ratio (mean +/- SEM, n = 3). * <i>p</i> < 0.05 WT and <i>tpk2</i>Δ 30°C <i>versus</i> 46°C; # <i>p</i> < 0.05 <i>tpk3</i>Δ <i>versus</i> WT and <i>tpk2</i>Δ 46°C (ANOVA Bonferroni post-test). Quantification of translation factors in monosome fraction (M) and polysome fraction (P) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.s001" target="_blank">S1E Fig</a>.</p

PKA catalytic subunits differentially affect the SGs and PBs aggregation in response to severe heat stress.

<p>(A) Wild type (WT), <i>tpk1</i>Δ, <i>tpk2</i>Δ and <i>tpk3</i>Δ expressing Pbp1-GFP, Edc3-RFP, Dcp2-RFP, Pab1-GFP or Rpg1-RFP were grown to exponential phase in YPD (30°C) and incubated at 46°C during 10 minutes. PBs and SGs aggregation were analyzed by fluorescence microscopy. The arrows show granular localization. The graph shows the amount of granules/100 cells. Bars represent the mean ± SEM, n = 3. * <i>p</i> < 0.05, 30°C <i>versus</i> 46°C; # <i>p</i> < 0.05, <i>tpk2</i>Δ 46°C or <i>tpk3</i>Δ 46°C <i>versus</i> WT 46°C; & <i>p</i> < 0.05, Edc3 <i>tpk2</i>Δ <i>versus</i> Edc3 WT 30°C; ^ <i>p</i> < 0.05, Pbp1, Rpg1, Dcp2 <i>tpk3</i>Δ <i>versus</i> Pbp1, Rpg1, Dcp2 WT 30°C (ANOVA Bonferroni post-test). <i>tpk3</i>Δ Rpg1-RFP panel results from a montage of images. (B) WT and <i>tpk3</i>Δ mutant cells expressing Tpk2-GFP and eIF4E-RFP were incubated at 46°C for 10 minutes. Tpk2-GFP and eIF4E-RFP co-localization was analyzed by confocal microscopy. (C) Tpk2-GFP enrichment in granular fractions was analyzed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.g002" target="_blank">Fig 2</a>. Representative blots are shown. The graph shows the ratio P/S of the abundance of each protein determined by densitometric quantification of the bands. Values are mean ± SEM, n = 2.</p

Characterization of Tpk2 and Tpk3 granules evoked during mild and severe thermal stress.

<p>(A) Cells co-expressing Tpk2-GFP or Tpk3-GFP and Rpg1-RFP or eIF4E-RFP and Tpk3-GFP and Dcp2-RFP were grown to exponential phase (30°C) and then incubated at 46°C for 10 minutes or 37°C for 30 minutes. Co-localization was determined by confocal microscopy. Arrows indicate Tpk-GFP granular localization and merge. Lower graphs show the quantitation of Tpks, Dcp2, Rpg1, eIF4E and merge granules/100 cells under each thermal stress condition. Values are mean ± SEM, n = 3. * <i>p</i> < 0.05 Tpk3-GFP <i>versus</i> Tpk3-GFP/Dcp2-RFP merge; Tpk3-GFP <i>versus</i> Tpk3-GFP/Rpg1-RFP merge; Tpk3-GFP <i>versus</i> Tpk3-GFP/eIF4E-RFP merge at 37°C (ANOVA-Tukey HSD test). (B) Effect of cycloheximide on the Tpk2 and Tpk3 assembly on heat stress evoked SGs. Cells co-expressing Tpk2-GFP or Tpk3-GFP and eIF4E-RFP were pre incubated or not with cycloheximide 100 μg/ml for 10 minutes before heat stress (CHX). Tpk2-GFP and Tpk3-GFP granule formation was analyzed as described in A. (C) Biochemical analysis of Tpk2 and Tpk3 granules evoked by heat stress. Wild type cells expressing Tpk2-GFP or Tpk3-GFP were grown to exponential phase in YPD and subsequently incubated at 30°C, 37°C for 30 minutes or 46°C for 10 minutes. When indicated, cells expressing Tpk3-GFP incubated at 30°C or 37°C 30 minutes were subsequently cross-linked by treatment with 1% (v/v) formaldehyde. Representative blots are shown. The results for the translation markers in Tpk2-GFP cells or Tpk3-GFP cells were similar. Right graph shows the ratio P/S of the abundance of each protein determined by densitometric quantification of the bands. Values are mean ± SEM, n = 2.</p

PKA catalytic subunits show a different subcellular localization upon mild and severe heat stress.

<p>(A) Subcellular localization of Bcy1-GFP, Tpk1-GFP, Tpk2-GFP or Tpk3-GFP in exponentially growing cells (30°C) and after heat stress at 37°C 30 minutes or 46°C 10 minutes visualized by fluorescence microscopy. Cell nuclei were stained with DAPI. The left graph shows the % of nuclear GFP signal. Values are mean +/- SEM, n = 3. * <i>p</i> < 0.05 Tpk1-GFP 30°C <i>versus</i> 46°C; Tpk2-GFP 30°C <i>versus</i> 37°C and 46°C; Tpk3-GFP 30°C <i>versus</i> 37°C and 46°C (ANOVA Bonferroni post-test). (B) The panels show representative images. Numbers inside each photo indicate total granules/100 cells for each of the conditions tested. The arrows show granular localization in the merge channel. Values are mean +/- SEM, n = 3. * <i>p</i> < 0.05 Tpk2-GFP 30°C <i>versus</i> 46°C; Tpk3-GFP 30°C <i>versus</i> 37°C and 46°C (ANOVA-Tukey HSD test).</p

Characterization of kinase dead <i>tpk2</i> and <i>tpk3</i> granule localization evoked by severe heat stress.

<p>Cells co-expressing Tpk2-GFP, <i>tpk2</i>^<i>dead</i>-GFP, Tpk3-GFP or <i>tpk3</i>^<i>dead</i>-GFP and Dcp2-RFP were subjected to severe heat stress as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0185416#pone.0185416.g002" target="_blank">Fig 2A</a>. Co-localization was determined by confocal microscopy. Arrows indicate Tpk-GFP granular localization and merge.</p