Deletion of <i>GET4</i> or <i>SGT2</i> does not modify heat sensitivity or nuclear localization of Mdy2.

<p>(A) Temperature sensitivity of <i>mdy2</i>Δ cells. Equivalent to 0.4 OD₆₀₀ units of exponentially growing yeast cells diluted serially and spotted on YEPD agar plates. The plates were incubated at 28°C and 38°C for 2 days and pictures were taken. A representative experiment is shown. (B) Temperature sensitivity of <i>mdy2</i>Δ cells grown in liquid medium. Experiments were repeated three times with a similar outcome. The error bars represent standard error of the mean. (C) Mdy2 localizes mainly to the nucleus. Mdy2 in <i>mdy2</i>Δ, <i>get4</i>Δ, and <i>sgt2</i>Δ cells was localized by direct fluorescence of exponentially growing yeast cells using green fluorescence protein (GFP) (<i>green channel</i>). The mutant <i>mdy2</i>Δ was transformed with plasmid MDY2p-GFP-MDY2 (pZH152) or control vector. Nuclear DNA was stained by DAPI to indicate positions of nuclei (<i>blue channel</i>). Green fluorescence images of GFP-Mdy2 were recorded by a Zeiss Axioscope fluorescence microscope. Cells are shown by phase-contrast (PC) images and nuclear DNA by DAPA staining. Strains: WT (W303-1A), <i>mdy2</i>Δ (HZH686), <i>get4</i>Δ (HKA200), <i>sgt2</i>Δ (HLS2002), <i>mdy2</i>Δ <i>get4</i>Δ (HKA227), and <i>mdy2</i>Δ <i>sgt2</i>Δ (HLS2024).</p

Localization of Mdy2 mutants with defective nuclear localization, Mdy2-ΔNLS, and nuclear export, Mdy2-ΔNES.

<p>(<b>A</b>) Mdy2 localizes mainly to the nucleus at 28°C. Mdy2 localize to cytoplasmic granules following heat stress. Mdy2 co-localize with Pab1 following mild and robust heat stress. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a <i>mdy2</i>Δ strain transformed with plasmids containing GFP-Mdy2 and Pab1-RFP (upper panel) or GFP-Mdy2 and Dcp2-RFP (lower panel) after a temperature shift to 38°C (middle panel) and 46°C (right panel). (B) Localization of Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins at permissive temperature. Exponentially growing <i>mdy2</i>Δ cells carrying wild type Mdy2 (<i>MDY2</i>), empty vector (<i>mdy</i>), NLS and NES deletion constructs (<i>mdy2-</i>Δ<i>NLS</i> and <i>mdy2-</i>Δ<i>NES</i>, respectively) fused to the C-terminus of GFP protein in CEN plasmids and expressed under the control of <i>MDY2</i>-own promoter. GFP-fusion proteins were localized by direct fluorescence and nuclear DNA was stained by DAPI. (C) Localization of Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins during heat shock-induced stress. Localization pattern for Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins as indicated in (A) during heat shock-induced stress. Localization of GFP-Mdy2 and GFP-Mdy2-NES to the cytoplasmic granules following heat stress is shown in upper and lower panel.</p

Binding assays of Get4 and Sgt2 with Mdy2-ΔNLS and Mdy2-ΔNES mutant proteins.

<p>(A) HZH686 (W303-1A <i>mdy2</i>Δ<i>)</i> cells were transformed with CEN expression vectors encoding Myc, Myc-tagged Mdy2 (Myc-Mdy2), Myc-Mdy2-NLS, Myc-Mdy2-NES, GST, and GST-tagged Get4 (GST-Get4) under the control of <i>GAL1</i> promoter. Cells were grown in SRG to log phase (see Material and Methods), whole cell extracts were prepared and GST-Get4 was precipitated using Glutathione -Sepharose 4B. The pulldown was then tested for the presence of Mdy2 association by probing a Western blot with anti-Myc Ab (top panel). To monitor pulldown recovery, the level of GST-Mdy2 in the binding assay was measured by probing the same membrane with anti-GST antibody (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for binding assay were measured on a Western blot (bottom panel). (B) HZH686 (W303-1A <i>mdy2</i>Δ<i>)</i> cells were transformed with expression vectors encoding Myc-tagged Mdy2 variants as in (A) and GST-tagged Sgt2 (GST-Sgt2) under the control of <i>GAL1</i> promoter. Cells were grown in SRG to log phase, whole cell extracts were prepared, and GST-Sgt2 was precipitated using Glutathione -Sepharose 4B. The presence of Mdy2 in the pulldown was confirmed by probing a Western blot with anti-Myc antibody (top panel). To monitor binding recovery the level of GST-Sgt2 in the pulldown was measured by probing the same membrane with anti-GST Ab (middle panel). Expression levels of Myc-Mdy2 in the whole cell extracts used for pulldown were measured on Western blots (bottom panel).</p

Identification of a nuclear localization signal (NLS) in the N-terminal domain and a nuclear export signal (NES) in the UBL domain of Mdy2.

<p>(A) Green fluorescence images and nuclear DNA of exponentially growing <i>mdy2</i>Δ cells carrying wild type Mdy2 (<i>MDY2</i>) and different putative NLS deletion constructs of Mdy2 (<i>mdy2</i>-Δ26–36, <i>mdy2</i>-Δ49–61, and <i>mdy2</i>-Δ79–80, respectively) were recorded as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g001" target="_blank">Figure 1C</a>. The NLS sequence of Mdy2 is localized between the amino acids 49 and 61 (panel 3). A putative NES deletion construct of Mdy2 (see schematic) was fused to the C-terminus of GFP protein, expressed under the control of <i>GAL1</i> promoter in <i>mdy2</i>Δ (HZH686) cells, and analyzed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g001" target="_blank">Figure 1B</a>. The NES sequence of Mdy2 is localized between amino acids 118 and 122 (panel 5). (B) Quantitative and statistical analysis of the subcellular localization of Mdy2 mutants with defective nuclear localization, Mdy2-ΔNLS, and nuclear export, Mdy2-ΔNES as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g002" target="_blank">Figure 2D</a>.</p

Mdy2 co-localize and interact with Pab1.

<p>(A) Mdy2 co-localize with Pab1 following heat stress and treatment with sodium azide. GFP-Mdy2 and Pab1-RFP was visualized by fluorescence microscopy in a <i>mdy2</i>Δ strain transformed with plasmids containing GFP-Mdy2 (upper row), GFP-Mdy2-ΔNLS (middle row) or GFP-Mdy2-ΔNES (lower panel), and Pab1-RFP, after a temperature shift to 46°C (left panel) and after treatment with sodium azide (NaN₃) (right panel). In the overlay pictures (merge), overlap of the colors appears yellow. GFP-Mdy2 and GFP-Mdy2-ΔNES but not GFP-Mdy2-ΔNLS are predominantly nuclear in control (Con) conditions at 28°C (right panel). (B) Mdy2 interacts with Pab1. Cell lysates from the GST-tagged Mdy2 strains were precipitated (P) with Glutathione Sepharose 4B. Following washing, the resin was eluted with glutathione. Eluted proteins were resolved by SDS-PAGE and visualized by immunobloting (control, IB) and Coomassie blue staining (Coomassie). Protein identities were established by mass spectrometry analysis. (C) Extracts from yeast strains HZH686 (W303-1A <i>mdy2</i>Δ<i>)</i> coexpressing GST alone (GST) or GST-tagged Pab1 (GST-Pab1) with Myc alone (Myc), Myc-tagged Mdy2 (Myc-Mdy2), Myc-tagged Mdy2-ΔNLS (Myc-Mdy2-ΔNLS) or Myc-tagged Mdy2-ΔNES (Myc-Mdy2-ΔNES) were subjected to pulldown using Glutathione Sepharose 4B as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g004" target="_blank">Figure 4</a>. The coprecipitation of indicated Myc-tagged Mdy2 proteins in the pulldown was confirmed by probing a Western blot with anti-Myc Ab (top panel, Co-P: Myc). To monitor pulldown recovery, the level of GST-Pab1 in the pulldown was measured by probing the same membrane with anti-GST Ab (second panel from the top, P: GST). Expression levels of indicated Myc-tagged Mdy2 proteins and GST-Pab1 in whole cell extracts (Extract) used for pulldown were measured on Western blots (third and fourth panels from top, IB:Myc and IB:GST, respectively).</p

Deletion of the N-terminal region Mdy2 affects GFP-Mdy2 nuclear localization and heat sensitivity.

<p>(A) Mdy2 and different C-, N-, and NC-terminal deletion fragments of Mdy2 open reading frame (see schematics) fused to the C-terminus of GFP protein expressed under the control of <i>GAL1</i> promoter in <i>mdy2</i>Δ (HZH686) cells. Temperature sensitivity recorded as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g001" target="_blank">Figure 1A</a>. Representative experiments are shown. (B) Protein expression level of GFP-Mdy2 variants shows no difference in mutant cells. The left panel shows Western blot of total protein extracts from GFP-Mdy2, GFP-Mdy2- 1–149, GFP-Mdy2- 74–212, and GFP-Mdy2- 74–149 expressing yeast cells. GFP-Mdy2 was detected using anti-GFP antibody. Protein expression of actin as internal standard was performed using anti-actin antibody, clone C4/MAB1501 (left panel). Quantitative densitometry of protein expression showed no changes in the protein levels of GFP-Mdy2 variants. GFP-Mdy2 was set to 1 (right panel). (C) Visualization of exponentially growing indicated yeast cells was performed using fluorescence microscopy as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g001" target="_blank">Figure 1B</a>. (D) Quantitative and statistical analysis of the subcellular localization of GFP-Mdy2 variants. About 100 cells from three independent experiments were counted. The graphs show the percentage of cells demonstrating nuclear or cytosolic GFP-Mdy2 variant protein distribution.</p

Heat sensitivity of Mdy2 mutants with defective nuclear localization, Mdy2-ΔNLS, and nuclear export, Mdy2-ΔNES.

<p>(A) Protein expression level of GFP-Mdy2 and different putative NLS and NES constructs of Mdy2 is equal. Western blotting analysis of NLS and NES deletion constructs (<i>mdy2-</i>Δ<i>NLS</i> and <i>mdy2-</i>Δ<i>NES</i>, respectively) in CEN plasmids expressed under the control of <i>MDY2</i> promoter in <i>mdy2</i>Δ (HZH686) cells (left panel). Quantitative densitometry of protein expression showed no changes in GFP-Mdy2 variants. GFP-Mdy2 was set to 1 (right panel). (B) Temperature sensitivity of <i>mdy2</i>Δ cells carrying wild type Mdy2 (<i>MDY2</i>), empty vector (<i>mdy2</i>Δ), NLS and NES deletion constructs (<i>mdy2-</i>Δ<i>NLS</i> and <i>mdy2-</i>Δ<i>NES</i>, respectively) in CEN plasmids expressed under the control of <i>MDY2</i> promoter recorded as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052956#pone-0052956-g001" target="_blank">Figure 1A</a>. Mdy2 mutants with a defect in nuclear localization <i>(mdy2-</i>Δ<i>NLS)</i> revealed an enhanced growth defect at elevated temperature (third row). Representative experiments are shown. (C) Temperature sensitivity of <i>mdy2</i> mutant cells grown in liquid medium.</p