FIGURE 4:

Rgf1p accumulation in the nucleus is Rad24p dependent. (A) Interaction between Rgf1p and Rad24p in yeast two-hybrid screening. Growth in –Leu/–Trp/–His media of yeast cotransformed with pBD-GAL4/rgf1⁺ (pRZ97) and the empty vector pADGAL4 (pGADT7) (lane 1), pAD-GAL4/rad24⁺ (pEM9), and the empty vector pBD-GAL4 (pGBKT7) (lane 2), or pAD-GAL4/rad24⁺ (pEM9) and pBD-GAL4/rgf1⁺ (pRZ97; lane 3), in yeast two-hybrid screenings. The results are representative of three separate cotransformation experiments. (B) GST pull-down assay showing the interaction of Rgf1p and Rad24p. Rad24p-GFP and Rad24p-GFP Rgf1p-GST cells grown to mid log phase were incubated in the presence or absence of 12.5 mM HU for 2.5 h and lysed under native conditions. The complexes precipitated with glutathione–sepharose beads were Western blotted and probed with anti-GFP and anti-GST antibodies to analyze Rad24p-GFP and GST-Rgf1p, respectively (IP). Whole-cell extract (Ext) fractions were assayed with anti-GFP and anti-GST antibodies. (C) Rgf1p-GFP localization in untreated and 12.5 mM HU–treated Rgf1p-GFP cells and rad24Δ Rgf1p-GFP cells. (D) The mutation in NES1 causes nuclear accumulation of Rgf1p-GFP in the absence of Rad24p. Photos for Rgf1p-NES1*-GFP and Rgf1p-NES1*-GFP in rad24Δ exponentially growing cultures. Quantification of the number of cells containing nuclear Rgf1p was performed on a mean of three independent experiments. (E) LMB treatment causes nuclear accumulation of Rgf1p-GFP in the absence of replication damage in wild-type and rad24Δ mutants. Wild-type Rgf1p-GFP and rad24Δ Rgf1p-GFP cells growing in YES medium were mock treated or treated with 100 ng/ml LMB for 30 min.