FIGURE 6.

Tor1 and Gad8 are required for induction of MBF-targeted genes independent of Yox1 phosphorylation. A, TORC2-Gad8 is required for induction of MBF-targeted genes in response to HU. Expression levels of cdc22⁺, cdt2⁺, cdc18⁺, cig2⁺, and mik1⁺ in WT, Δgad8, or Δtor1 cells were determined by qRT-PCR. Total RNA was prepared from untreated cells (−) or cells treated with 12 mm HU for 3 h (+). The level of act1⁺ mRNA was used as a reference. Each value is the mean of at least three independent assays, and the error bars indicate S.D. B, the expression level of MBF-targeted genes in Δcds1Δtor1 double mutant cells is similar to that in Δcds1 or Δtor1 single mutant cells. C, Yox1 is normally phosphorylated in TORC2 or Gad8 mutant cells. Total protein extracts were prepared by trichloroacetic acid extractions from WT, Δgad8, or Δtor1 strains expressing genomic Yox1-MYC. Cells were grown in rich medium (−) or treated with 12 mm HU for 1 h (+). Antibody against Act1 was used as a loading control.