Slow-growth phenotype of the Cet1p-Ceg1p mislocalization strains.

<p>The yeast strain HC101, harboring <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutants (without <i>CEN TRP1 hCAP1</i>) were grown in YPD medium and put on agar plates. These plates were incubated at 25, 30, or 37°C for 2 days.</p

Importance of the Cet1p-Ceg1p interaction for nuclear localization of the Cet1p-Ceg1p complex.

<p>(A) GST pull-down assay. A GST pull-down assay was performed using GST (or GST-Ceg1p OB domain) and Cet1p (or Cet1(4A)p) translated in rabbit reticulocyte lysate with [³⁵S]-methionine. GST pull-downs were separated by SDS-PAGE and detected with an imaging plate (left) and CBB staining (right). (B) Triphosphatase activity of Cet1(4A)p. The reaction was performed as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0078000#s2" target="_blank">Materials and Methods</a> using [γ-³²P]-terminated poly(A), and either 2.5, 5, 10, 20, 40, 80 nM Cet1(4A)p or Cet1p. The reaction products were analyzed by PEI-cellulose TLC. The autoradiogram of the thin-layer plate is shown. (C) Localization of Ceg1p-GFP in cells expressing Cet1(4A)p. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both <i>2 μ URA3 CET1</i> or <i>CET1(4A)</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The cell nucleus was stained with DAPI. (D) Localization of Cet1(4A)p-GFP. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both <i>CEN HIS3 CET1(4A)-GFP</i> and <i>2 μ URA3 CEG1</i> plasmids. The cell nucleus was stained with DAPI. (E) Lethal phenotype of the yeast strain expressing Cet1(4A)p and Ceg1p-GFP. The yeast strain used in (C) and control strains were streaked on agar plates with or without 0.075% 5-FAA. These plates were incubated at 30°C for 2 days. (F) Localization of Ceg1p-GFP in cells expressing Cet1p triphosphatase active site mutant. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with <i>CEN HIS3 CET1(E305,307A)-GFP</i> or both <i>2 μ URA3 CET1(E305,307A)</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The cell nucleus was stained with DAPI.</p

Nuclear accumulation of Cet1p-GFP in the <i>cet1</i>

<p>Δ<b><i>ceg1</i></b>Δ <b>strain.</b> (A) Complementation of <i>cet1</i>Δ and <i>ceg1</i>Δ by Cet1p-GFP and Ceg1p-GFP, respectively. Yeast strains HC101 and HC201 were transformed with a <i>CEN HIS3 CET1-GFP</i> and <i>CEN HIS3 CEG1-GFP</i> plasmid, respectively. His⁺ isolates and control strains were streaked on agar plates with or without 0.075% 5-FAA. These plates were incubated at 30°C for 2 days. (B) Localization of Cet1p-GFP and Ceg1p-GFP in the <i>cet1</i>Δ<i>ceg1</i>Δ strain. Yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with <i>CEN HIS3 CET1-GFP</i>, <i>CEN HIS3 CEG1-GFP</i>, or both <i>2 μ URA3 CET1</i> and <i>CEN HIS3 CEG1-GFP</i> plasmids. The yeast strains were grown in YPD medium and fixed by ethanol. The cell nucleus was stained with DAPI after fixation.</p

Characterization of the amino acid sequence required for nuclear localization of the Cet1p-Ceg1p complex.

<p>(A) Cet1p N-terminal deletion mutants. Localization results shown in (B) are summarized. N: localized in the nucleus. N+C: localized in both the nucleus and the cytoplasm. (B) Localization of Cet1p-GFP mutants in the <i>cet1</i>Δ<i>ceg1</i>Δ strain. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with the <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutant plasmid indicated in (A). The cell nucleus was stained with DAPI. (C) Amino acid sequence between residues 217 and 253 of Cet1p and Cet1p N-terminal deletion mutants. The underline indicates the WAQKW motif for Ceg1p binding. (D) Localization of the Cet1p-GFP N-terminal truncated mutants. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with the <i>CEN HIS3 CET1-GFP</i> N-terminal deletion mutant plasmid indicated in (C). The cell nucleus was stained with DAPI. (E) Localization of Ceg1p-GFP in cells expressing Cet1p N-terminal deletion mutants. The yeast strain <i>cet1</i>Δ<i>ceg1</i>Δ was transformed with both a <i>2 μ URA3 CET1</i> N-terminal deletion mutant plasmid and the <i>CEN HIS3 CEG1-GFP</i> plasmid. The cell nucleus was stained with DAPI. (F) Interaction between His-Cet1(228–549)p and GST-Ceg1. GST pull-down assay was performed using GST or GST-Ceg1p OB domain and Cet1p, Cet1(4A)p, or Cet1(228–549)p translated in rabbit reticulocyte lysate with [³⁵S]-methionine. GST pull-downs were separated by SDS-PAGE and detected with an imaging plate (left) and CBB staining (right).</p