Hsf1 activity in Hsp70 mutants and chaperone AA strains. (A) HSE-YFP reporter assay for Hsf1 activity in Hsp70 deletion strains. Cells were left untreated or were heat shocked at 39°C for 4 h, and YFP levels were measured by flow cytometry and normalized to untreated WT. Three biological replicates are shown, along with the mean (boxes) and SD (error bars). (B) HSE-YFP reporter assay of AA strains in the presence and absence of rapamycin (rapa) normalized to the untagged AA parent strain. Cells were treated with 1 µM rapa for 8 h before the reporter was measured. These data suggest that C-terminal tagging of Ssa1 and Ssa2 compromises their function nearly as severely as knocking them out. Consistent with this interpretation, individual AA tagging of Ssa1 and Ssa2 resulted in mild and moderate increases HSE-YFP levels, respectively, akin to their respective deletions. N-terminal AA tagging of Ssa2 likewise resulted in a constitutive increase in HSE-YFP signal. Error bars represent the SD of the replicates (n = 3 for each strain and condition). HS, heat shocked; NHS, non–heat shocked.