Figure 2.
Sis1 promotes interaction between Hsf1 and Hsp70.(A) Anti-FLAG IP of Hsf1-3xFLAG-V5 followed by MS at 30°C and at 39°C for 60 min. IPs were performed in biological triplicates. Levels of interacting proteins were plotted relative to the amount of Hsf1 measured in each replicate to generate a stoichiometryapp value. Mean and SD of the replicates are shown. (B) Sis1 functional complementation assay. HSE-YFP levels were measured in Sis1-AA cells expressing additional copies of untagged Sis1 from the SIS1 promoter: WT Sis1, ΔJ, a mutant of Sis1 in which the HPD motif residues are mutated to alanine (HPD>AAA), or no additional Sis1 (−). Cells were treated with 1 µM rapamycin (+ rapa) for 8 h before the reporter was measured by flow cytometry. YFP levels in each cell were normalized by each cell’s side scatter value (SSC; a proxy for size), and the medians of the resulting distributions are plotted. Error bars represent the SD of the replicates (n = 3 for each strain and condition). (C) Anti-FLAG IP of Hsf1-3xFLAG in cells expressing V5-tagged Sis1, either WT or HPD>AAA. Cells were grown at 30°C in log phase before harvesting. IP eluates were blotted and probed with anti-FLAG (Hsf1) and anti-V5 (Sis1) antibodies. Values plotted below are the ratios of Sis1:Hsf1 in each replicate (repl.); gray boxes show the mean of the replicates. (D) Anti-FLAG/V5 serial IP of Hsf1-3xFLAG-V5 followed by anti-FLAG and anti-Ssa1/2 Western blots. Cells were treated in the absence and presence of 60 min of 1 µM rapa treatment to AA Sis1. Plotted values are normalized ratios of Ssa1/2:Hsf1 in each of three biological replicates; gray boxes show the mean values of the replicates. (E) Dilution series spot assay. Ectopic expression of NLS-Sis1 impairs cells growth; deletion of the CE2 region of Hsf1 rescues growth in NLS-Sis1 cells. Cells were grown for 36 h on YPD + 20 nM estradiol to induce NLS-Sis1. (F) Schematic of the mathematical model of the Hsf1 regulation by the Sis1 and Hsp70. During heat shock, Hsp70 clients accumulate and compete for Sis1 and Hsp70 and release Hsf1. Active Hsf1 then induces expression of Sis1 and Hsp70. (G) Simulations of the mathematical model and corresponding experiments of heat shock time courses of HSE-YFP reporter strains with and without ectopic expression of NLS-Sis1. NLS-Sis1 was induced with 20 nM estradiol for 1 h before the time course. Error bars represent the SD of the replicates (n = 3 for each strain and condition).

Sis1 promotes interaction between Hsf1 and Hsp70. (A) Anti-FLAG IP of Hsf1-3xFLAG-V5 followed by MS at 30°C and at 39°C for 60 min. IPs were performed in biological triplicates. Levels of interacting proteins were plotted relative to the amount of Hsf1 measured in each replicate to generate a stoichiometryapp value. Mean and SD of the replicates are shown. (B) Sis1 functional complementation assay. HSE-YFP levels were measured in Sis1-AA cells expressing additional copies of untagged Sis1 from the SIS1 promoter: WT Sis1, ΔJ, a mutant of Sis1 in which the HPD motif residues are mutated to alanine (HPD>AAA), or no additional Sis1 (−). Cells were treated with 1 µM rapamycin (+ rapa) for 8 h before the reporter was measured by flow cytometry. YFP levels in each cell were normalized by each cell’s side scatter value (SSC; a proxy for size), and the medians of the resulting distributions are plotted. Error bars represent the SD of the replicates (n = 3 for each strain and condition). (C) Anti-FLAG IP of Hsf1-3xFLAG in cells expressing V5-tagged Sis1, either WT or HPD>AAA. Cells were grown at 30°C in log phase before harvesting. IP eluates were blotted and probed with anti-FLAG (Hsf1) and anti-V5 (Sis1) antibodies. Values plotted below are the ratios of Sis1:Hsf1 in each replicate (repl.); gray boxes show the mean of the replicates. (D) Anti-FLAG/V5 serial IP of Hsf1-3xFLAG-V5 followed by anti-FLAG and anti-Ssa1/2 Western blots. Cells were treated in the absence and presence of 60 min of 1 µM rapa treatment to AA Sis1. Plotted values are normalized ratios of Ssa1/2:Hsf1 in each of three biological replicates; gray boxes show the mean values of the replicates. (E) Dilution series spot assay. Ectopic expression of NLS-Sis1 impairs cells growth; deletion of the CE2 region of Hsf1 rescues growth in NLS-Sis1 cells. Cells were grown for 36 h on YPD + 20 nM estradiol to induce NLS-Sis1. (F) Schematic of the mathematical model of the Hsf1 regulation by the Sis1 and Hsp70. During heat shock, Hsp70 clients accumulate and compete for Sis1 and Hsp70 and release Hsf1. Active Hsf1 then induces expression of Sis1 and Hsp70. (G) Simulations of the mathematical model and corresponding experiments of heat shock time courses of HSE-YFP reporter strains with and without ectopic expression of NLS-Sis1. NLS-Sis1 was induced with 20 nM estradiol for 1 h before the time course. Error bars represent the SD of the replicates (n = 3 for each strain and condition).

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