Figure 9.

Tti2 is required for stress response. (A) Yeast strains CY7245 (DED1-TTI2 YCplac33), CY7236 (DED1-TTI2 GAL1-htt25Q), CY7237 (DED1-tti2_L187P GAL1-htt25Q), CY7238 (DED1-TTI2 GAL1-htt103Q), CY7239 (DED1-tti2_L187P GAL1-htt103Q), and CY7241 (DED1-tti2_L187P YCplac33) were grown to stationary phase in minimal medium containing 2% galactose and lacking uracil. Cell densities were normalized then cells were spotted in 10-fold serial dilutions onto a YP plate containing galactose, and grown at 32° for 2 d. (B) CY6070, C6973, and a Δhsp42 strain were grown to stationary phase in YP medium containing raffinose, diluted 1:20 in YPD, grown for 8 hr at 30° then harvested. Protein was extracted by bead lysis, and 10 µg of protein loaded for each sample. Western blotting was performed with anti-Hsp42 antibody. Lane 7 was left blank to avoid overflow into the Δhsp42 lane, and the bottom of the gel was stained with CBB as a loading control. (C) Yeast strain CY7086 (WT-TTI2) and BY4742 (no tag control) were grown to stationary phase in YPD, diluted 1:20, grown for 8 hr then heat shocked at 42° for 30 min. Protein extraction and Western blotting were done as described in (B), except with anti-Myc antibodies. The bottom of the gel was stained with CBB, and shown as a loading control. (D) The same CY7086 cell lysate from (C) was separated a second time by SDS-PAGE. Protein lysates were then Western blotted with anti-Hsp42 antibody. Protein extract from a Δhsp42 strain (Cashikar et al. 2005) was used as a negative control.