Strain construction                        

Yeast strains and plasmids used in this study are listed in Table S1. All strains are derived from the W303 parent strain. CRISPR-mediated promoter swapping was performed to create the 1xSup35pr-Sis1 (1701) and 1xSup35pr-Sis1-P2A-mscarlet (1661) strains. CRISPR-Cas9 mediated precise, scarless replacement of the native Sis1 promoter with the 600-bp Sup35 promoter. To construct the final non-inducible Sis1 line (2xSup35pr-Sis1, 1761), we incorporated a second copy of Sup35pr-Sis1 at the tryp locus.

In all other cloned lines, genes were tagged at the endogenous locus. Cells were transformed with double-stranded DNA fragments containing ~20 bp homologous flanking regions. This method takes advantage of homology-directed repair mechanisms in S.cerevisiae, as described previously (Longtine et al., 1998).

Cell Growth

For heat shock time courses followed by flow cytometry, cells were cultured in 1xSDC media overnight at room temperature (synthetic media with dextrose and complete amino acids). Before RT-PCR or cell growth assays, cells were cultured in yeast extract peptone dextrose (YPD) media shaking at 30ºC overnight. Cells were subjected to heat shock at 39°C unless otherwise specified.

HSE-YFP reporter heat shock assays                                          

Three biological replicates of each strain were serially diluted five times (1:5) in 1xSDC and grown overnight at room temperature. In the morning, cells had reached logarithmic phase, and 750 µL of each replicate was transferred to a PCR tube and shaken for one hour at 30ºC to aerate. Then, cells were exposed to heat shock at 39ºC. At the pre-determined time points, (0,5,10,15,30,60,90,120,180,240 minutes), 50 µl of cell culture was transferred to a well of a 96-well plate, containing 1xSDC and a final concentration of 50 mg/mL cycloheximide to stop translation. After the time course, cells were incubated at 30ºC for one hour to allow fluorescent reporter maturation before measurement. All experiments were performed using C1000 Touch Thermal Cycler (Bio-Rad). Cell fluorescence was measured by flow cytometry and results were analyzed as described below. 

Anchor-Away Assay

Cells were grown overnight as described above. Rapamycin was added to a final concentration of 10 µM, and the time course was started immediately. During the time course, cells were maintained at 30ºC shaking. At the predetermined time points (0, 15, 30, 45, 60, 75, 90 minutes), 50 µL of cells were transferred to a 96-well plate identically to the heat shock assay described above.

Translation Inhibition Assay (Rapamycin Pre-treatment)

Cells were grown overnight, and in the morning, shaken at 30ºC for 1 hour. Rapamycin was added to a final concentration of 10 µM, and cells were left shaking at 30ºC for 5 minutes. Then, the heat shock time course was performed at 39ºC as described above.

Flow cytometry

HSE-YFP and mscarlet reporter levels in heat shock time course and rapamycin treatment assays were measured at the University of Chicago Cytometry and Antibody Technology Facility. These measurements were performed using the 488-525 FITC fluorescence filter (HSE-YFP) and 561-PE Dazzle (mscarlet) on the BD Fortessa High Throughput Flow Cytometer. The raw fluorescence values were normalized by side scatter in FlowJo. Then, the median fluorescence value was calculated. Each data point represents the average of three biological replicates.

Dilution series spot growth assays          

Yeast strains were grown overnight shaking at 30ºC in YPD. In the morning, they were diluted to and final optical density (OD) of 1 and serially diluted 1:10 in water. Each diluted yeast culture was spotted onto 1xYPD plates. Images were taken after two days of growth at 30º or 37ºC.

Heat Shock Quantitative Growth Assay

Yeast strains were grown overnight at 30ºC in 1xYPD. In the morning, they were diluted to OD= 0.1 in 1xYPD. SpectroStar Nano microplate reader was used to measure cell density every 20 minutes. Each data point represents the mean of three biological replicates.

Glucose Starvation Quantitative Growth Assay

Yeast strains were grown for 24 hours in 2% glucose YEP media then diluted to OD=0.1 and grown overnight again. In the morning, cells were diluted to OD=0.1 in 1xYEP containing 2% glycerol, 2% EtOH and growth was monitored every 20 minutes at 30ºC.

RNA quantification and analysis

​​RT-qPCR was performed as previously described in (Unable to find information for 12149321), except for the following: cells were grown at 30°C in YPD (yeast extract-peptone-dextrose) to a mid-log density (OD₆₀₀ = 0.8). Rapamycin was added to a final concentration of 10 ug/mL for 5 min. At this point, a portion of the culture was maintained at 30°C (0 min HS), and the remainder was subjected to heat shock at 39°C for 5, 15, 60, and 120 min. Cells were flash-frozen immediately after heat shock and then harvested. Total RNA was extracted using hot acid phenol extraction method, followed by ethanol precipitation(Solís et al., 2016). To determine fold change mRNA levels, mean mRNA levels of heat-shocked samples were normalized to the NHS sample(Pfaffl, 2001).

mVenus forward primer: caacattgaagatggtggtgttc

mVenus reverse primer: ctttggataaggcagattgatagg

RNA-Seq Analysis

RNA read counts for 39 Hsf1 target genes were collected in a recent paper and reanalyzed here(Triandafillou et al., 2020). GSE accession number 152916 (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE152916)

Experimental methods are described in detail in the original paper. In brief, cells were exposed to heat shock at 42ºC for 20 minutes. Cells exposed to heat shock and cycloheximide were treated with 200 ug/mL cycloheximide (CHX) simultaneously upon HS. Each data point represents the average read count for a single Hsf1 target gene (two biological replicates). Translation dependence was calculated as 1-(CHX+HS/HS).

Halo-Tagging

To image newly synthesized proteins, we incubated cells with blocker (20uM, 7-bromoheptanol) for 5 minutes, washed two times with 2xSDC media, and then incubated with 0.4 uM of Halo JF646dye at 39ºC, to start HS treatment simultaneously. Under this treatment, only protein induced during heat shock was visualizable. Lattice light sheet microscopy was used to visualize 5–20 cells every 2.5 minutes throughout the 30-minute heat shock. 

Lattice Light Sheet Microscopy and Quantification

Lattice light-sheet imaging was performed at the University of Chicago Integrated Light Microscopy Core (Intelligent Imaging Innovations) and run in SlideBook 6.0 software. Captured images were deconvoluted using Graphics processing unit–based Richardson-Lucy deconvolution with measured PSFs via Brian Northan’s “Ops” implementation (https://github.com/imagej/ops-experiments). 3D reconstructions and videos were assembled using ClearVolume(Royer et al., 2015). The mean Halo intensity at the ring around the Nsr1-marked nucleolus was divided by the mean intensity over the total cell.