Regulation of the Hsf1-dependent transcriptome via conserved bipartite contacts with Hsp70 promotes survival in yeast

Protein homeostasis and cellular fitness in the presence of proteotoxic stress is promoted by heat shock factor 1 (HSF1), which controls basal and stress-induced expression of molecular chaperones and other targets. The major heat shock proteins Hsp70 and Hsp90 in turn participate in a negative feedback loop that ensures appropriate coordination of the heat shock response with environmental conditions. Features of this regulatory circuit in the budding yeast Saccharomyces cerevisiae have been recently defined, most notably regarding direct interaction between Hsf1 and the constitutively expressed Hsp70 Ssa1. We sought to further explore the complex nature of Ssa1/Hsf1 regulation. Ssa1 is found to interact independently with both the previously defined CE2 site in the Hsf1 carboxyl-terminal transcriptional activation domain as well as a novel site that we identify within the amino-terminal activation domain. Consistent with both sites bearing a recognition signature for Hsp70, we demonstrate that Ssa1 contacts Hsf1 using its substrate binding domain and abolishing either regulatory site results in loss of Ssa1 association. Removing Hsp70 regulation of Hsf1 results in global dysregulation of Hsf1 transcriptional activity, with synergistic effects when both sites are disrupted together on both gene expression and cellular fitness. Finally, we find that Hsp70 interacts with both transcriptional activation domains of Hsf1 in the related yeast Lachancea kluyveri, implying a conserved mechanism of regulation to promote cellular proteostasis. Overall design: Three biological replicates were analyzed for four different yeast strains at 30°C, and one strain (wild type) at 37°C for 15 min

在蛋白毒性应激条件下，热休克因子1（Heat Shock Factor 1, HSF1）可促进蛋白质稳态（Protein homeostasis）与细胞适合度，其能够调控分子伴侣（molecular chaperones）及其他靶基因的基础表达与应激诱导型表达。主要热休克蛋白Hsp70与Hsp90则参与构成负反馈环路（negative feedback loop），确保热休克反应与环境条件的恰当协调。该调控环路在出芽酵母酿酒酵母（Saccharomyces cerevisiae）中的特征已于近期被阐明，其中最为突出的是Hsf1与组成型表达的Hsp70亚型Ssa1之间的直接相互作用。本研究旨在进一步探究Ssa1与Hsf1之间调控关系的复杂机制。研究发现，Ssa1可独立结合Hsf1羧基末端（carboxyl-terminal）转录激活结构域（transcriptional activation domain）中此前已被定义的CE2位点，以及我们在其氨基末端（amino-terminal）激活结构域内鉴定出的全新位点。鉴于两个位点均带有Hsp70的识别特征序列，本研究证实Ssa1通过其底物结合结构域（substrate binding domain）与Hsf1发生结合；且任意一个调控位点的失活都会导致Ssa1与Hsf1的结合丧失。解除Hsp70对Hsf1的调控会导致Hsf1转录活性的全局紊乱，若同时破坏两个调控位点，则会对基因表达与细胞适合度产生协同效应。最后，本研究发现，在亲缘关系相近的拉克兰谢酵母（Lachancea kluyveri）中，Hsp70同样可与Hsf1的两个转录激活结构域结合，这提示该调控机制在进化上具有保守性，以维持细胞蛋白质稳态。实验整体设计：针对4种不同酵母菌株，在30℃条件下设置3次生物学重复（biological replicates）进行分析；同时针对1株野生型菌株，在37℃条件下处理15分钟后开展分析。