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

(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 cet1Δceg1Δ strain. The yeast strain cet1Δceg1Δ was transformed with the CEN HIS3 CET1-GFP 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 cet1Δceg1Δ was transformed with the CEN HIS3 CET1-GFP 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 cet1Δceg1Δ was transformed with both a 2 μ URA3 CET1 N-terminal deletion mutant plasmid and the CEN HIS3 CEG1-GFP 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).