Whi3 localizes to stress granules during heat shock.

<p>(A) Cells co-expressing Pub1-mCh and Whi3-GFP were heat shocked at 46°C for 15 minutes. Heat shock caused Whi3 and Pub1 to colocalize in distinct cytoplasmic foci. When cells were returned to 30°C, these foci dissolved. Scale bar represents 5 µm. (B) Pelleting assay results support the observation that Whi3 is enriched in insoluble stress granules during heat shock. Both Whi3FL-3xGFP and Pub1-mCh are enriched in soluble (S) fractions at 30°C, but become enriched in the insoluble pellet (P) fraction during heat shock at 46°C. Housekeeping gene Pgk1 does not change solubility in response to stress. (C) Whi3ΔQrich-3xGFP colocalizes with stress granule marker Pub1-mCherry during heat shock. (D) Whi3ΔQrich exhibits a similar solubility profile to Whi3FL-3xGFP. (E) Whi3ΔRRM-3xGFP colocalizes with stress granule marker Pub1-mCherry during heat shock. (F) Whi3ΔRRM exhibits a similar solubility profile to Whi3FL-3xGFP.</p

Whi3 nucleates stress granules during mild heat shock.

<p>(A) Cells coexpressing Whi3FL-3xGFP and P-body marker Edc3-mCh were incubated at various temperatures for 15 minutes. Whi3 does not colocalize with P-bodies during non-stress conditions (30°C). As temperatures increase, both Whi3 and Edc3 form foci. Though some Whi3 foci are distinct from Edc3 foci (arrowheads), colocalization between Whi3 and Edc3 increases as temperature increases. Scale bar represents 5 µm. (B) The experiment described in (A) was repeated in cells coexpressing Whi3FL-3xGFP and stress granule marker Pub1-mCh. Though Whi3 begins localizing to foci at 37°C, Pub1 foci do not form until 42°C. Pub1 and Whi3 foci colocalize at temperatures at or above 42°C.</p

<i>whi3</i>Δ cells have a novel zinc sensitivity phenotype.

<p>(A) BY4741 cells were spotted in serial dilutions on control plates or plates containing 10 mM or 15 mM ZnCl₂. <i>whi3</i>Δcells exhibited a novel zinc sensitivity phenotype. <i>zap1</i>Δ cells grow slowly on control media because they are zinc starved, but grow normally on plates supplemented with zinc. (B) The zinc sensitivity phenotype of <i>whi3</i>Δ cells is not exacerbated by overexpression of Zap1 (OE). <i>whi3</i>Δ cells expressing vector (V) are equally zinc-sensitive as those overexpressing Zap1.</p

Whi3 localizes to stress granules during glucose deprivation.

<p>(A) Cells coexpressing Whi3-GFP and stress granule marker Pub1-mCherry were deprived of glucose for 10 minutes. Glucose deprivation caused Whi3 and Pub1 to colocalize in distinct cytoplasmic foci. Readdition of glucose caused foci to dissolve. Scale bar represents 5 µm. (B) Treatment with cycloheximide (CHX) inhibits stress granule formation by preventing polysome disassembly. Unlike cells treated with drug vehicle alone, cells treated with CHX form neither Pub1-mCh nor Whi3-GFP foci. (C) Cells lacking the scaffolding protein eIF4GII do not form stress granules. Neither Whi3-mCh nor Pab1-GFP forms foci in <i>eif4gii</i>Δ cells. (D) Stress granule marker Pub1-mCh is still able to localize to foci in the absence of Whi3.</p

Comparison of multiple data sets reveals a high-confidence Whi3 RNA target set.

<p>Heat map showing the overlap among Whi3 targets identified in the Whi3 IP presented in this study or from the Colomina data set <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084060#pone.0084060-Colomina1" target="_blank">[11]</a>. Each row is a Whi3 target mRNA identified in at least one of the two data sets with a false discovery rate (FDR) of <10%. The first two columns show the SAM reported false discovery rates for each mRNA in either our Whi3 IP data set or the Colomina data set, with FDRs ranging from >10% in black through red, orange, yellow and 0% in light yellow. The third column indicates in blue which mRNAs contain the (U)GCAU Whi3 interaction motif. The fourth column indicates in red which mRNAs we defined as the top 100 high-confidence Whi3-interacting RNAs.</p

Whi3 changes steady-state levels of its target mRNAs without affecting translation rates.

<p>(A) Boxplot of log base 2 of the fold change in the average number of ribosomes bound to each mRNA in a Whi3 deletion versus a wild-type strain. The top 100 high-confidence Whi3 targets are plotted on the left, compared to all other mRNAs on the right, showing that there is no significant difference in the change in ribosome number in response to Whi3 deletion between these two sets of mRNAs. (B) Wild-type or <i>whi3</i>Δ cells were grown to mid-log phase in YPD, then incubated for 15 minutes at 30°C or 46°C. RT-PCR analysis examined levels of several RNA targets identified in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084060#pone-0084060-g005" target="_blank">Figure 5</a>. Data are averages of three biological replicates; error bars represent SEM. ****, p<0.0001, Sidak’s post test correction for multiple comparison. (C) Pooled data from B. Normalized mRNA abundance is plotted for Whi3 targets or <i>APE3</i>, grouped by strain background, wild-type or <i>whi3</i>Δ. ****, p<0.0001, 2-way ANOVA.</p

Quantification of temperature course data.

<p>(A) Double-blinded quantification of data from experiments presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084060#pone-0084060-g003" target="_blank">Figure 3</a>. Whi3-3xGFP, Pub1-mCh, and Edc3-mCh foci were manually counted using a binning method, as automated analysis was not possible. Edc3-mCh forms foci at all temperatures, though foci become enriched as temperatures increase. Pub1-mCh remains diffusely cytosolic until 42°C, at which point it localizes to foci. Whi3FL-3xGFP forms increasing numbers of foci starting at 37°C. An average of 300 cells were analyzed for each protein at each temperature. (B) Double-blinded colocalization analysis of experiments presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084060#pone-0084060-g003" target="_blank">Figure 3</a>. Using ImageJ software <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0084060#pone.0084060-Schneider1" target="_blank">[49]</a>, a line was drawn through a cell, avoiding the vacuole, and a plot of signal intensity was generated. The analysis was repeated in the corresponding image and a Pearson’s correlation coefficient between the two lines was calculated. Data presented are the average of 20 cells; error bars represent SEM. Pub1-mCh and Whi3FL-3xGFP exhibit high correlation at 30°C when both are diffusely cytosolic, but correlation decreases at 37°C and 39°C when Whi3 begins localizing to foci. Pub1 and Whi3 correlate more robustly once Pub1 foci begin to form at 42°C. Edc3-mCh and Whi3FL-3xGFP correlate poorly at 30°C when Edc3 is in foci, but become more highly correlated as temperatures increase.</p

The Molecular Mechanism of Substrate Engagement and Immunosuppressant Inhibition of Calcineurin

<div><p>Ser/thr phosphatases dephosphorylate their targets with high specificity, yet the structural and sequence determinants of phosphosite recognition are poorly understood. Calcineurin (CN) is a conserved Ca²⁺/calmodulin-dependent ser/thr phosphatase and the target of immunosuppressants, FK506 and cyclosporin A (CSA). To investigate CN substrate recognition we used X-ray crystallography, biochemistry, modeling, and in vivo experiments to study A238L, a viral protein inhibitor of CN. We show that A238L competitively inhibits CN by occupying a critical substrate recognition site, while leaving the catalytic center fully accessible. Critically, the 1.7 Å structure of the A238L-CN complex reveals how CN recognizes residues in A238L that are analogous to a substrate motif, “LxVP.” The structure enabled modeling of a peptide substrate bound to CN, which predicts substrate interactions beyond the catalytic center. Finally, this study establishes that “LxVP” sequences and immunosuppressants bind to the identical site on CN. Thus, FK506, CSA, and A238L all prevent “LxVP”-mediated substrate recognition by CN, highlighting the importance of this interaction for substrate dephosphorylation. Collectively, this work presents the first integrated structural model for substrate selection and dephosphorylation by CN and lays the groundwork for structure-based development of new CN inhibitors.</p> </div

Potential interaction modes of CN substrates/regulators with CN.

<p>(A) The CN-RII peptide complex obtained by MD. Colors as in <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio-1001492-g002" target="_blank">Figure 2A, C</a>. CN is shown in surface representation and the RII peptide in dark green with the LxVP motif (LDVP) and phospho-Ser95 as green sticks. LDVP is bound to the LxVP binding pocket (light green), and phospho-Ser95 is bound in the CN active site (cyan). (B) Electrostatic interactions between CN and the RII peptide. The CN electrostatic surface has positively and negatively charged areas colored blue and red, respectively. The LxVP motif and residues in RII that participate in polar interactions with CN are shown as green sticks. (C) Features of selected CN substrates and regulators, including substrates tested in this work (NFAT, Crz1, and the RII peptide). PxIxIT and LxVP motifs are highlighted in yellow and green, respectively, with intervening residues in grey. Regions containing S-T residues that are dephosphorylated by CN are pink. (D) Potential modes of interaction of CN with various binding partners. CN is shown in grey, with the active site in cyan, the PxIxIT docking site in yellow, and the LxVP docking site in green. CN binding partners are shown in blue, with PxIxIT and LxVP motifs in purple and phosphorylated regions shown as red circles. The residues between the two CN docking motifs, or between one docking motif and regions dephosphorylated by CN, are represented as coils, as they are predicted to be unstructured in solution. A238L is the CN-A238L crystal structure.</p

A238L interacts with CN via an LxVP and a PxIxIT motif.

<p>(A) C-terminal residues (200–239) of A238L showing putative docking site FLCVK (aa 228–232). Underlined residues were fused to GST. (B) Recombinant CN was incubated with GST fused to 15 amino acids encoding the LxVP motif of NFATc1 or the FLCVK sequence in A238L. CN co-purifies with both motifs; this interaction is disrupted by incubation with excess peptide LxVPc1 encoding the LxVP motif from NFATc1, but not LxVPmut. CN fails to co-purify with GST fused to mutated FLCVK sequence (FLCVK mutated to AACAA). (C) β-galactosidase activity of extracts from yeast strains that harbor 2xCDRE-lacZ, a CN-dependent reporter gene, and GST or GST-A238L truncations are shown. We added 50 mM CaCl₂ to the cell culture 2.5 h before harvesting to induce CN-dependent activation of the Crz1 transcription factor (<a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492-Stathopoulos1" target="_blank">[22]</a>; see also <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s007" target="_blank">Text S1</a>). Error bars indicate ± s.d. from three independent experiments. (D) Secondary plot of K<i>_i</i>^app as a function of [RII] for A238L_200–239 inhibition of CN. Data show a linear dependence characteristic of competitive inhibition, with K<i>_i</i> = 0.37 nM. K<i>_i</i>^app values were obtained from the nonlinear fit of <a href="http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001492#pbio.1001492.s001" target="_blank">Figure S1B</a>. Points represent averages ± s.e.m. (E) Isothermal titration calorimetry confirming that purified A238L_200–239 binds to CN.</p