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

(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 Materials and Methods 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 cet1Δceg1Δ was transformed with both 2 μ URA3 CET1 or CET1(4A) and CEN HIS3 CEG1-GFP plasmids. The cell nucleus was stained with DAPI. (D) Localization of Cet1(4A)p-GFP. The yeast strain cet1Δceg1Δ was transformed with both CEN HIS3 CET1(4A)-GFP and 2 μ URA3 CEG1 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 cet1Δceg1Δ was transformed with CEN HIS3 CET1(E305,307A)-GFP or both 2 μ URA3 CET1(E305,307A) and CEN HIS3 CEG1-GFP plasmids. The cell nucleus was stained with DAPI.