Figure 4.

Disruption of Ssa1-binding sites in full-length Hsf1 results in constitutive activation and severe slow growth phenotypes. A, immunoblotting of cell lysates from the indicated strains expressing plasmid-based HSF1 alleles with a C-terminal FLAG tag, lacking one (hsf1-mN, hsf1-mC) or both (hsf1-mNmC) of the Ssa1-binding sites. Steady-state levels of Hsp70 (Ssa1) and Hsp90 relative to WT cells and normalized to the load control, PGK, were determined by averaging densitometric quantitation of three independent experiments and are shown below the respective panels. B, the same strains were plated onto YPD solid medium as indicated in the key and grown at 30 or 37 °C for 2 days. C, quantitative RT-PCR of the Hsf1 target genes BTN2, HSP82, SSA3, and SSA4 is shown as -fold increase in gene expression relative to the WT strain. Results from three independent experiments are shown. *, p < 0.05; **, p < 0.005; ***, p < 0.0005.