Nuclear depletion of Sis1 rapidly and specifically activates Hsf1. (A) Cartoon of the chaperone AA approach. Upon addition of rapamycin (+ rapa), chaperones of interest are tethered to a ribosomal protein and depleted from the nucleus. (B) Spinning disc confocal images of cells expressing Hsf1-mKate and GFP-tagged versions of Hsp90 (Hsc/p82) and Sis1. Cells were treated with 1 µM rapa for 30 min before imaging. Scale bar is 2 µm. Lower panel: Indicated strains were serially diluted, spotted, and grown at 30°C for 36 h on YPD with or without 1 µM rapa. (C) HSE-YFP reporter assay of AA strains in the presence and absence of rapa normalized to the untagged AA parent strain. Error bars represent the SD of the replicates (n = 3 for each strain and condition), and statistical significance was determined using a two-tailed t test without assuming equal variance (*, P < 0.05; **, P < 0.01). Cells were treated with 1 µM rapa for 8 h before measuring the reporter. (D) Time course of HSE-YFP levels in the Sis1 and Hsp90 AA strains compared with the untagged parent following addition of 1 µM rapa. Error bars represent the SD of the replicates (n = 3 for each strain and time point). (E) RNA-seq of heat-shocked (HS) cells (15 min at 39°C) versus non–heat-shocked (NHS) cells. Hsf1 target genes are shown in red, Msn2 targets in green, and RPGs in blue. (F) RNA-seq analysis of the Sis1 AA strain in the absence and presence of 1 µM rapa for 30 min. Hsf1 target genes are shown in red, Msn2 targets in green, and RPGs in blue. norm., normalized.