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Most eukaryotic genes encode polypeptides that are either obligate members of hetero-stoichiometric complexes or clients of organelle-targeting pathways. Proteins in these classes can be released from the ribosome as “orphans”—newly synthesized proteins not associated with their stoichiometric binding partner(s) and/or not targeted to their destination organelle. Here we integrate recent findings suggesting that although cells selectively degrade orphan proteins under homeostatic conditions, they can preserve them in chaperone-regulated biomolecular condensates during stress. These orphan protein condensates activate the heat shock response (HSR) and represent subcellular sites where the chaperones induced by the HSR execute their functions. Reversible condensation of orphan proteins may broadly safeguard labile precursors during stress. 

Cells maintain homeostasis via dynamic regulation of stress response pathways. Stress pathways transiently induce response regulons via negative feedback loops, but the extent to which individual genes provide feedback has not been comprehensively measured for any pathway. Here, we disrupted the induction of each gene in the Saccharomyces cerevisiae heat shock response (HSR) and quantified cell growth and HSR dynamics following heat shock. The screen revealed a core feedback loop governing the expression of the chaperone Hsp70 reinforced by an auxiliary feedback loop controlling Hsp70 subcellular localization. Mathematical modeling and live imaging demonstrated that multiple HSR targets converge to promote Hsp70 nuclear localization via its release from cytosolic condensates. Following ethanol stress, a distinct set of factors similarly converged on Hsp70, suggesting that nonredundant subsets of the HSR regulon confer feedback under different conditions. Flexible spatiotemporal feedback loops may broadly organize stress response regulons and expand their adaptive capacity. 

The heat shock response (HSR) controls expression of molecular chaperones to maintain protein homeostasis. Previously, we proposed a feedback loop model of the HSR in which heat-denatured proteins sequester the chaperone Hsp70 to activate the HSR, and subsequent induction of Hsp70 deactivates the HSR (Krakowiak et al., 2018; Zheng et al., 2016). However, recent work has implicated newly synthesized proteins (NSPs) – rather than unfolded mature proteins – and the Hsp70 co-chaperone Sis1 in HSR regulation, yet their contributions to HSR dynamics have not been determined. Here, we generate a new mathematical model that incorporates NSPs and Sis1 into the HSR activation mechanism, and we perform genetic decoupling and pulse-labeling experiments to demonstrate that Sis1 induction is dispensable for HSR deactivation. Rather than providing negative feedback to the HSR, transcriptional regulation of Sis1 by Hsf1 promotes fitness by coordinating stress granules and carbon metabolism. These results support an overall model in which NSPs signal the HSR by sequestering Sis1 and Hsp70, while induction of Hsp70 – but not Sis1 – attenuates the response.