Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response

Domain: biology
Published: 2025-12-13
Editorial outcome: minor_revision

Abstract
Cells must respond rapidly to heat stress by activating multiple signaling pathways that preserve proteostasis. In budding yeast, this includes induction of Hsf1 and Msn2/4-mediated transcription, cell integrity signaling, stress-triggered phase separation of proteins, and inhibition of translation. How these pathways are so rapidly activated and coordinated remains unclear. We show that the mechanosensor Mid2 senses heat-induced membrane stretch and leads to rapid phosphorylation of the cytosolic Hsp70 Ssa1 at a well-conserved threonine (T492). Phosphorylation of T492 leads to epichaperome rearrangement promoting fine-tuning of multiple cellular processes including translational pausing, HSF activity, MAPK signaling and stress granule resolution. Taken together, these results provide a comprehensive, unified theory of the global yeast heat shock response mediated by the Hsp70 chaperone code.

Introduction
The heat shock protein 70 (Hsp70) family represents a well-conserved yet functionally diverse group crucial for various cellular housekeeping functions1,2. The Hsp70 proteins are integral to the Heat Shock Response (HSR) because they bind and stabilize unfolded or misfolded proteins, facilitating their refolding or degradation. The yeast cytosolic Hsp70 family consists of the Ssa1-4 isoforms, with Ssa1 and Ssa2 constitutively expressed and Ssa3 and Ssa4 stress-inducible3. Despite their high sequence similarity, these isoforms exhibit distinct expression patterns and functions under stress conditions3. Hsp70s are composed of three main domains: the N-terminal ATPase domain, the substrate-binding domain (SBD), and the C-terminal “lid”. Hydrolysis of ATP to ADP in the N-terminal domain drives conformational changes that regulate substrate binding, folding and release. This allosteric mechanism is essential for the chaperone activity of Hsp70, allowing it to transiently bind to a wide array of client proteins, thereby preventing aggregation and assisting in proper folding4,5. Chaperones exist as part of a wider Environmental Stress Response (ESR), a highly conserved mechanism that allows cells to adapt to various stress conditions, including heat, osmotic shock, oxidative stress, and nutrient deprivation. Upon exposure to these stressors, yeast cells globally downregulate genes involved in growth and proliferation while upregulating a suite of stress-responsive genes6. These genes encode proteins that aid in detoxification, repair damaged macromolecules, and maintain cellular integrity5. The Heat Shock Response (HSR), an evolutionarily conserved protective mechanism from yeast to humans, promotes protein homeostasis and cellular fitness during proteotoxic stress. As a component of the broader environmental stress response (ESR), HSR operates through two primary transcription factors, Hsf1 and Msn2/47. The heat shock response (HSR) is a subset of the ESR and is fundamental for survival at high temperatures. This response is characterized by the rapid induction of heat shock proteins (HSPs) that include Hsp70 and Hsp908,9. The chaperone titration model suggests that Hsf1 is kept inactive during basal conditions through interactions with Hsp70. Upon heat shock, the accumulation of misfolded proteins titrates Hsp70 away from Hsf1, allowing Hsf1 to bind Heat Shock Elements (HSEs), initiating the transcription of HSP genes10. This model, however, does not explain the rapid Hsf1 activation observed in response to heat shock, which occurs faster than the time required for titration to occur. The interaction between Hsp70 and Hsf1 occurs via the SBD domain of Hsp70 and two sites on Hsf1, the CE2 site in the C-terminal activation domain (C-AD) and a recently identified site in the N-terminal activation domain (N-AD)9,11. Studies have shown that Hsf1 activation can be regulated by accelerating the substrate release kinetics of Hsp70 in the nuclear compartment11. Other proposed HSR mechanisms include the direct sensing of heat by Hsf112, hyperphosphorylation13, and condensation of Hsf110,14.

In parallel with the HSR, heat shock activates the Cell Wall Integrity (CWI) pathway, which is crucial for maintaining the structure and function of the cell wall under various stress conditions15. This pathway is primarily activated by environmental stresses such as heat shock, osmotic stress, and mechanical damage. At the core of the CWI pathway are membrane-bound mechanosensors like Wsc1 and Mid2, which detect cell wall stress and initiate a signaling cascade. These sensors activate Protein Kinase C (Pkc1), which then triggers a MAP kinase cascade involving Bck1, Mkk1/2, and Mpk1. This cascade leads to the activation of transcription factors such as Rlm1 and Swi4/Swi6, which promote gene expression in cell wall synthesis and remodeling16. Although activation of Mpk1 occurs relatively fast (approximately 15 min), Pkc1 pathway-mediated transcription occurs over much longer time periods, with genes such as PRM5 and FKS2 being maximally induced 8h after the initial stress17,18. In contrast to metazoans, yeast only has one PKC isoform, Pkc1, which is essential for cell survival. This essential nature derives from the inability of pkc1Δ cells to maintain their cell wall structure and osmotic balance. Correspondingly, pkc1Δ cells can be kept viable on media supplemented with osmotic stabilizers such as sorbitol18. Although a large number of Pkc1-mediated effects occur through MAP kinase components such as Mpk1, it is now clear that 19 Pkc1 regulates numerous other targets20. Although different PKC isoforms display slightly varied substrate sequence specificity, it is commonly accepted that the general motif comprises basic amino acids (Arg or Lys) at positions −2 and +2 relative to the phosphorylated residue20–22.

While the HSR and CWI pathways have been long established as key for the response to heat, more recently, the importance of protective protein phase separation has been highlighted. Heat causes protein misfolding and aggregation and, in eukaryotic cells, triggers the aggregation of proteins and RNA into biomolecular condensates23. Biomolecular condensates are adaptive, membrane-less structures of concentrated biomolecules that differ from protein-misfolded aggregates. These include stress granules, which contain ribosome-associated mRNAs and translation machinery components, and processing bodies (P-bodies), which accumulate translationally inactive mRNAs lacking initiation factors24–27. P-bodies and stress granules (SGs) are distinct organelles that are thought to be functionally linked. Recent evidence suggests that pre-existing P-bodies may serve as nucleation sites for SG assembly28. SGs maintain a dynamic equilibrium with polysomes and show sensitivity to translation inhibitors, functioning as crucial triage centers that direct mRNAs toward translation, storage, or degradation29. Poly-A-binding protein (Pab1) is a defining marker of stress granules30. Stress granules are endogenous protein aggregates that are disassembled without degradation during recovery. In addition to this relatively slow transcriptional response (30+ mins), recent studies have demonstrated the rapid formation of reversible quinary protein aggregates in response to acute heat exposure that occurs after only a few minutes31. The second type of biomolecular condensate-P-bodies specifically function as concentration hubs for translationally inactive mRNPs, housing machinery for both translation suppression and RNA degradation, and contain a fundamental set of conserved proteins26. Currently, the molecular mechanisms for this rapid response and the effect of heat shock on Hsp70 post-translational modification remain unknown.

A final universal paradoxical feature of the heat shock response is that while heat induces the expression of heat shock proteins, it overall leads to a global cessation of translation. This translational arrest is a protective mechanism that conserves energy and resources while the cell deals with the immediate threat posed by elevated temperatures. During heat shock, the dissociation of Hsp70 from ribosomal components plays a pivotal role in halting translation. This dissociation causes a pause in translational elongation, preventing the accumulation of misfolded proteins that could overwhelm the chaperone machinery32. In addition, specific kinases, such as Gcn2, are activated by stress and phosphorylate the alpha subunit of eukaryotic initiation factor 2 (eIF2α), further inhibiting translation initiation33. Heat shock has also been recognized to impair protein synthesis, affecting the elongation phase34–36.

As proteomic technologies have improved, a large number of post-translational modifications (PTMs) have been discovered on molecular chaperones, collectively known as the Chaperone Code. This code fine-tunes chaperone functions by altering their interactions with client and co-chaperone proteins. Although a number of Hsp70 PTMs have been described, less than ten have been fully defined in terms of regulation and physiological relevance1. In this study, we seek to understand the interplay between the Hsp70 chaperone code and proteostasis. We identify a rapidly heat-induced phosphorylation site on yeast Hsp70 at T492 that Pkc1 mediates during heat shock. Phosphorylation of T492 promotes epichaperome remodeling, which drives the dissociation of Hsp70-Hsf1 and the activation of HSR. T492 phosphorylation also alters Hsp70-ribosome interactions, explaining the long-established observation of heat-induced translational pausing. Finally, we identify the CWI protein Bck1 as a bona fide client of yeast Hsp70 whose activity is dependent on T492 status. Together, our data provide a paradigm for how cells can coordinate activation of diverse signaling pathways that respond to heat through modulation of the Hsp70 chaperone code.

Results
Heat shock induces rapid phosphorylation of Ssa1 T492 in yeast
In an effort to understand how heat shock alters Ssa1 phosphorylation, yeast expressing FLAG-tagged Ssa1 were grown to the exponential phase and then treated for 1 h at 39 ˚C. Ssa1 was purified and subjected to mass spectrometry analysis. We identified a single site, threonine T492 (T492) that was induced upon heat shock. This residue is located in the substrate-binding domain of Ssa1 in close proximity to the region responsible for client binding (Fig. 1a). T492 is surprisingly well-conserved, indicative of this residue being important for chaperone function (Fig. 1b). To validate our mass spectrometry data, we generated a custom phospho-specific antibody for the T492 site and analyzed phosphorylation of T492 in yeast exposed to heat shock over a period of 4 h. To remove the confounding effects of the highly similar Ssa2, 3, and 4 proteins, we used the well-established MH272 ssa1-4 strain, in which all four SSA genes have been deleted and functionally complemented by SSA1 expressed from a URA3-marked centromeric plasmid37. The strain was transformed with a LEU2-marked plasmid bearing either wild-type FLAG-tagged Ssa1 or the non-phosphorylatable mutant ssa1-T492A. The URA3 plasmid was evicted on 5-fluoro-orotic acid (5-FOA) media to yield strains expressing wild-type Ssa1, Ssa1-T492A as the sole Ssa protein in the cell, hereafter referred to as FLAG-WT or T492A cells. FLAG-WT or T492A cells were grown to mid-log phase and then exposed to 39 ˚C. Aliquots of cells were taken after 0, 1, 2 and 4 h, at which T492 phosphorylation was assessed via Western Blotting. Ssa1 T492 phosphorylation was minimal in unstressed cells, but was observed in cells heat-stressed for 1 h (Fig. 1c). Validating the specificity of the phospho T492 antibody, no bands were seen in T492A lysates (Fig. 1c). To examine whether T492 phosphorylation occurs at shorter time scales, we examined T492 phosphorylation at sub-60 min time points. A robust increase in T492 phosphorylation was observed even after 1 min, suggesting this response represents an early stage of the heat shock response (Fig. 1d and Supplementary Fig. 1).

The Ssa isoforms are also phosphorylated in response to thermal stress
The four cytosolic Hsp70s in yeast Ssa1, 2, 3 and 4 are highly similar in amino acid sequence and appear to have at least some functional overlap3,38. Given that the T492 site is conserved in Ssa1-4, we queried whether T492 is phosphorylated in all Ssa1, 2, 3 and 4. We first generated yeast expressing either Ssa1, 2, 3, 4 on constitutive promoters as the sole Ssa in the cell and assessed T492 phosphorylation in these cells after 1 h of heat shock at 39 ˚C. Excitingly, heat-induced T492 phosphorylation was observed in Ssa1-4 (Fig. 1e). This identifies a conservation of a phosphorylation site between yeast Hsp70 paralogs.

Heat-shock induced phosphorylation of T492 is independent of protein unfolding
Heat elicits a variety of effects on cells, including protein denaturation. To determine whether heat-induced phosphorylation of Ssa1 was promoted by protein misfolding, we utilized Azetidine-2-carboxylic acid (AZC), a proline analog that is incorporated in proteins during protein synthesis and triggers protein unfolding39. Treatment of yeast with AZC did not promote Ssa1 T492 phosphorylation even over extended time periods (up to 4 h), suggesting that protein unfolding is not the primary stimulus for T492 activation (Fig. 1f).

Heat-induced T492 phosphorylation is dependent on the Mid2 mechanosensor
Heat triggers activation of the yeast cell wall integrity MAPK pathway (Fig. 2a)16. To determine whether activation of Ssa1 phosphorylation was dependent on CWI signaling, we assessed whether T492 phosphorylation was impacted by loss of the Wsc1 or Mid2 mechanosensors39,40. Although heat-stimulated Ssa1 T492 phosphorylation was unchanged in a wsc1Δ strain, cells lacking Mid2 were unable to induce T492 phosphorylation (Fig. 2b). This result is consistent with those in Fig. 1f, as AZC does not activate Mid2 or the rest of the cell integrity pathway40. Heat activates the CWI by causing stretching of the plasma membrane41,42. To determine if heat-activated phosphorylation of Ssa1 T492 was caused through a similar mechanism, we treated yeast with the membrane stretching agent chlorpromazine (CPZ) and assessed T492 status (Fig. 2c). Robust T492 phosphorylation was observed after 1 h of CPZ treatment (Fig. 2c). Taken together, these data suggest that T492 phosphorylation is not mediated by protein misfolding, but by membrane stretch, detected through the Mid2 mechanosensor.

Yeast Protein Kinase C (Pkc1) directly phosphorylates Ssa1 T492
The requirement of Mid2 for T492 phosphorylation suggested the potential involvement of CWI kinases Pkc1, Bck1, Mkk1/2 and Mpk1 in phosphorylating Ssa1 (Fig. 2a). Several indirect lines of evidence implicated Pkc1 as the potential kinase for Ssa1, including the observation that overexpression of a hyperactive mutant of Pkc1 resulted in an increase in T492 phosphorylation20,21,43. In addition, Ssa1 has an arginine at the -3 position and a basic residue at the +2 position relative to the T492 site, which is the preferred substrate sequence for PKC proteins. To examine whether T492 phosphorylation was catalyzed by Pkc1, we decided to assess T492 phosphorylation when Pkc1 function was ablated. As the Pkc1 function is essential for cell viability, we utilized the auxin-inducible degron (AID) system44. After genomically tagging Pkc1 on the C-terminus with AID, we checked the system’s functionality by plating the WT and Pkc1-AID cells on media containing or lacking auxin. Although both strains grew on YPD, only WT was able to grow on media containing auxin. The Pkc1-AID strain was, however, able to grow on auxin media when osmotic support was provided in the form of sorbitol (Supplementary Fig. 2a). These results were consistent with Pkc1 being degraded in the presence of auxin. We grew the Pkc1-AID strain to mid-log phase at 25 ˚C and then added auxin to ablate Pkc1 function. For each indicated time point, yeast were shifted to 39 ˚C for 30 min, and then T492 status was assessed via Western Blotting. Robust T492 phosphorylation was observed post-heat shock in WT and Pkc1-AID pre-auxin addition (Fig. 2d). Post-auxin addition, the ability of heat shock to stimulate T492 phosphorylation diminished over time, being barely observable 4 hrs after auxin treatment (Fig. 2d). This loss of T492 phosphorylation corresponded with a decrease of Pkc1-AID abundance (Supplementary Fig. 2b). Pkc1 is part of a kinase cascade consisting of Pkc1, Bck1, Mkk1/2 and Mpk1 (Fig. 2a). To rule out the possibility that Pkc1-dependence of T492 phosphorylation was via these other kinases, we assessed T492 phosphorylation in cells lacking either Bck1, Mkk1/2 or Mpk1 (Fig. 2e). Cells lacking these kinases were still able to activate T492 phosphorylation in response to heat stress, indicating that Pkc1 is directly phosphorylating this site. To complement these results, we performed an in vitro kinase assay with purified Pkc1 and recombinant Ssa1. Purified Pkc1 was able to phosphorylate Ssa1 on T492 (Fig. 2f). Taken together, these data suggest that Pkc1 is directly able to phosphorylate Ssa1 on T492 in response to heat shock.

Phosphorylation of T492 fine-tunes the Hsp70 epichaperome
Interactions of chaperones (epichaperome) are highly dynamic and vary depending on internal and external cues all of which modulate the chaperone code1,45–50. We considered the possibility that phosphorylation of Ssa1 at T492 may dictate the breadth and specificity of the Ssa1 epichaperome. We purified the Ssa1 complexes from heat-shocked WT and T492A cells and then compared their composition using mass spectrometry (Fig. 3a). The interacting proteins that are significantly downregulated were defined as p<0.05 and have a log2 fold change < -1. For the significantly upregulated proteins, they have p<0.05 and a log2 fold change > 1. We identified 647 proteins (1% FDR) and quantified 380 of those, which were significant. Of the total interactors identified, 24% displayed a preference for binding to WT Ssa1, 23% displayed a preference for binding to T492A samples, and 54% showed an equal affinity for both versions of Ssa1 (Fig. 3b). To determine the nature of the protein interactions altered by T492 phosphorylation we performed Gene ontology (GO) analysis. GO analysis revealed significant enrichment of multiple cellular functions, several of which were unique to either WT or T492A samples (Fig. 3c). The WT Ssa1 interactome displayed a preference for proteins involved in ribosome translation, protein trafficking and cytoskeletal organization (Fig. 3c). In contrast, the T492A mutant showed a preference for proteins involved in proteostasis and metabolism (Fig. 3c). Protein interactions can be dictated by protein localization. GO analysis of interactor localization revealed subtle changes between WT and T492A samples. WT Ssa1 displayed a binding preference for proteins in the endomembrane system and cell cortex. In contrast, the T492A mutant selectively preferred proteins found in the extracellular region and cell wall (Fig. 3d). Both yeast Hsp70 and Hsp90 bind intrinsically-disordered regions (IDRs) on proteins47,51. To understand whether T492 phosphorylation may impact the ability of Ssa1 to bind IDRs, we batch analyzed clients for the percentage of intrinsically disordered amino acids via MobiDB. There were roughly threefold fewer disordered proteins in T492-dependent interactors (Fig. 3e). The activity and specificity of Ssa1 is impacted through interaction with co-chaperone proteins that include Sis1 and Ydj152–58. We considered the possibility that the altered clientome of Ssa1 in T492A may also be an effect of differential co-chaperone/chaperone binding. In our proteomics study, mutation of T492 impacted the association of nine key chaperone and co-chaperone proteins (Fig. 3f). These included the major Hsp40-Ydj1, nucleotide exchange factors-Sse2, Fes1, and Lhs1. Interestingly, the T492A mutant displayed enhanced binding with the ribosomal-associated-Ssz1, the Hsp90 paralog Hsp82, and its co-chaperones Sgt2 and Sti1 (Fig. 3f). Despite this major remodeling of the Ssa1 interactome upon mutation of T492, Hsp40-Sis1, other Hsp70 paralogs-Ssb1, Ssc1, Kar2, and the mitochondrial chaperone Hsp60 showed equal interaction with WT and mutant under heat shock (Fig. 3f).

Phosphorylation of Ssa1 T492 is critical for correct activation of the Heat Shock Response
Molecular chaperones are critical for the refolding of heat-denatured proteins and thus survival at high temperatures. Although viable at 30 ˚C, T492A cells were unable to grow on solid media at 37 ˚C, suggesting a possible defect in the heat shock response (Fig. 4a). To investigate this possibility, we assessed the expression of an HSE-driven destabilized luciferase reporter11 in WT and T492A cells. WT cells exposed to 39 ˚C mounted a rapid and robust HSR within minutes, reaching a maximum after 30 minutes of heat exposure. In contrast, T492A cells did not induce significant expression of the HSE-luciferase construct (Fig. 4b). To confirm that the loss of HSE-reporter activity in T492A was due to transcriptional rather than translational effects, we measured the transcription of the luciferase gene in these cells under unstressed and heat-shocked conditions via RT-PCR. As expected, HSE-mediated luciferase transcription was compromised in the T492A mutant strain (Fig. 4c). We also determined the expression of HSP104 and BTN2, genes whose transcription are highly-dependent on Hsf1 activity59. In agreement with our previous data, the heat-induced transcription of these genes were abolished in T492A cells (Fig. 4c). To complement our transcriptional data, we assessed induction of the well-characterized heat-inducible protein Hsp26 in WT and T492 cells. Hsp26 levels did not respond to exposure to 39 ˚C in T492A cells, confirming that T492 phosphorylation is critical for the heat shock response (Fig. 4d).

T492 phosphorylation controls the interaction between Ssa1 and Hsf1
The lack of heat-induced transcriptional response in T492A cells suggested a defect in Hsf1 regulation. Ssa1 regulates the response to heat shock through its interaction with Heat Shock Factor, Hsf19–11,60–64. To understand whether Ssa1-Hsf1 interaction was impacted by the T492A mutation, we measured Ssa1-Hsf1 affinity in WT and T492A cells under unstressed and heat shock-treated conditions via co-immunoprecipitation. As expected, in WT cells the interaction between Ssa1 and Hsf1 was disrupted by heat shock (Fig. 4e). In contrast, the Ssa1-Hsf1 interaction remained unchanged in T492A heat-shocked cells, providing a rationale for the defective HSR observed (Fig. 4e). Together with our proteomics showing unchanged Sis1 binding to Ssa1‑T492A, these data indicate that Sis1‑dependent assembly of the Hsf1–Hsp70 complex is intact, whereas Pkc1‑driven Ssa1‑T492 phosphorylation is required for rapid heat‑induced release of Hsf1.

Accelerating Hsp70 substrate release suppresses the temperature sensitivity of T492 cells
Release of Hsp70 from Hsf1 can be accelerated by overexpression of the nuclear-targeted Sse1 co-chaperone protein12. Our data suggested the primary cause of heat-sensitivity of T492 cells may be an inability for Ssa1 to release Hsf1. We expressed either WT Sse1 or Sse1-NLS in WT or T492 yeast and examined the ability of transformants to grow at elevated temperatures (Fig. 4f). In agreement with our hypothesis, the temperature-sensitivity of T492 cells was suppressed by overexpression of Sse1-NLS but not WT Sse1 (Fig. 4f).

Elevated Ssa1 abundance in T492A cells is driven by altered transcriptional programs
The data from 4a-f clearly suggested a defect in Hsf1-driven transcription in the T492A mutant, potentially leading to decreased Ssa protein abundance. Paradoxically, throughout our experiments, we noticed that the steady-state level of the T492A mutant was substantially higher than that of WT (Figs. 1c, 2c and 4e). To determine whether the increased abundance of the T492A mutant could be attributed to transcriptional effects, we created yeast strains expressing FLAG-Ssa1 and FLAG-T492A from the constitutive GPD promoter. In these strains, FLAG-Ssa1 and FLAG-T492A were expressed at equal levels (Supplementary Fig. 3a). To ensure the abundance of T492A in Fig. 4a was not the primary driver of the ts-sensitive phenotype seen in Fig. 4a, we repeated the serial dilutions using our newly generated GPD-Flag-Ssa1 and GPD-Flag-T492A strains. The GPD-Flag-T492A was as sensitive to 37 ˚C as the SSA promoter equivalent, confirming that the phenotype observed was promoter-independent (Supplementary Fig. 3b). To confirm that transcription of T492A was elevated in our native promoter strains, we performed RT-PCR. In accordance with our results, T492A transcription was elevated compared to WT in native promoter strains, but not in strains expressing FLAG-Ssa1 from the GPD promoter (Supplementary Fig. 3c and d). While physiologically relevant, we considered the possibility that the reinforced interaction between Hsf1 and Ssa1 in T492A may be driven by increased Ssa1 abundance rather than enhanced Ssa1-Hsf1 binding affinity. We revisited the IP experiment in Fig. 4e using the newly generated GPD-FLAG-Ssa1 strains. Although the abundance of WT Ssa1 and T492A was equalized in these strains, Hsf1 displayed a substantially enhanced interaction with T492A compared to WT (Supplementary Fig. 3e). Previous studies have shown that a subset of heat-induced genes are controlled by Msn2/4 and that inhibiting Hsf1 activity can induce Msn2/4-mediated transcription65. To query whether T492 status may also impact Msn2/4 activity, we examined the levels of Hsp12 (a reporter for Msn2/4-mediated transcription) in WT and T492A cells (Fig. 4g). T492A cells displayed a constitutive activity of Msn2/4 compared to WT, potentially explaining the increased transcription of SSA1 in the SSA-driven T492A strain (Fig. 4g). Taken together, these data demonstrate that T492 phosphorylation is important for Ssa1-Hsf1 dissociation upon heat shock and appropriate activation of heat shock-induced transcription (Fig. 4h).

T492 phosphorylation fine-tunes Ssa1 ribosome engagement and translation fidelity during heat shock
Several of the Ssa1 interactors altered by T492 phosphorylation are important for protein translation, ribosome assembly, and translation initiation (Fig. 5a and b). Heat shock remodels Hsp70-ribosome interactions, and both transient dissociation and persistent over-association of Hsp70 have been reported to impede elongation, thereby contributing to global translational pausing32. Phosphorylation of Hsp70 on T495 (equivalent to T492 in yeast) by the Legionella Legk4 kinase during infection can trigger a similar effect to heat shock66. To determine whether the T492A mutation in yeast may alter translation, we quantified the extent of global translational repression during temperature stress in WT and T492A yeast using the ratio of polysome to monosome signal in polysome profiles in a manner similar to ref. 14. Both strains showed a similar decrease in translation upon a 10-minute stress at 39 ˚C, and both strains recovered to nearly unstressed levels after 30 minutes at 25 ˚C (see Fig. 5c). In order to track chaperone association with translating ribosomes during stress, we purified proteins from the polysome fractions and quantified the abundance of chaperones by western blotting. While at 25 ˚C, chaperone association with polysomes was similar across strains, after 10 minutes at 39 ˚C, levels of polysome-bound Ssa1-T492A increased while levels of polysome-bound WT Ssa1 and other chaperones remained steady (Fig. 5d). In order to quantify overall protein synthesis, we measured incorporation of the methionine analog L-homopropargylglycine (HPG) into newly synthesized proteins over the course of 30 minutes at 25 ˚C or 39 ˚C (see Fig. 5e)68. Control cells were additionally incubated with cycloheximide to prevent translation. At the specified time points, cells were fixed with ethanol, and then the HPG was modified with a fluorescent dye using a copper-chelating click reaction67. The fluorescence intensity of each cell was then measured by flow cytometry. At both temperatures, the Ssa1-T942A strain had lower translation rates than WT as measured by HPG incorporation (p=0.0005 by an analysis of covariance test). If T492 phosphorylation is required to fine-tune Ssa1-ribosomal protein interactions during heat stress, we considered the possibility that the T492A mutant may disrupt translational fidelity. We assessed translational fidelity in WT and T492A mutant cells using a well-established dual luciferase assay. In this system, Renilla luciferase (Rluc) and firefly luciferase (Fluc) are under the control of separate constitutive promoters51,68. The Fluc mRNA has a non-traditional start codon (UUG), which is only used when there is a loss of translational fidelity51. The Rluc mRNA has a classic AUG start codon and therefore acts as a control for alterations in overall translation. Using this system, we observed an approximately threefold increase in utilization of the non-canonical start codon in T492A cells (Fig. 5f). Taken together, these data suggest that loss of T492 phosphorylation causes persistent Ssa1 association with elongating ribosomes and a measurable drop in codon-specific translation fidelity.

T492 phosphorylation is involved in P-body disassembly
Our epichaperome analysis revealed several protein interactors that comprise stress granules, P-bodies or both (Figs. 3 and 6a). Both of these types of cytoplasmic RNA-protein granules are induced under heat stress23,31,69–71. We queried whether the T492 phosphorylation state may impact stress granule and P-body formation by tracking the localization of Edc3-mCherry (P-body marker) and Pab1-GFP in WT and T492A cells. Despite previous observations that the resolution of Pab1 requires Ssa1 and associated co-chaperones, the formation and resolution of Pab1 foci were not dependent on T492 phosphorylation (Fig. 6a–e). In contrast, while Edc3 foci formation was unaffected by T492 status, the kinetics of Edc3 foci disassembly were impaired in the T492A mutant during 60–120 min recovery (Fig. 6a–e).

Heat-induced phosphorylation of yeast Hsp70 promotes CWI signal amplification
To understand the physiological impact of T492 phosphorylation, we screened WT and T492A cells for their resistance to a range of cellular stresses (Fig. 7a). While T492A cells grew at nearly WT rates at 30 ˚C, they were nevertheless compromised for their resistance to temperature, caffeine, calcofluor white (CFW) and hydroxyurea (HU). These stresses are well-known activators of the Cell Wall Integrity (CWI) pathway, and consequently, we considered the possibility that T492 phosphorylation may also govern cell integrity signaling. We assessed activity of the CWI pathway in WT and T492 cells via PRM5 and FKS2 promoter-lacZ reporter assays as in refs. 17, 73. T492A cells were unable to recapitulate WT levels of heat-induced FKS2 and PRM5 transcription (Fig. 6b, c). While both PRM5 and FKS2 expression are dependent on cell integrity, their modes of activation differ. PRM5 expression is dependent on Rlm1 phosphorylation, catalyzed by the Mpk1 kinase17,73. In contrast, FKS2 expression requires dual phosphorylation of Mpk1, but is independent of Mpk1 catalytic activity72,74. Consistent with our promoter-reporter results, T492A cells were defective in Mpk1 dual phosphorylation, but not Mpk1 stability (Fig. 7d). The upstream semi-redundant kinases Mkk1 and Mkk2 were not affected by the mutation of T492, so we focused our attention further upstream on the Bck1 kinase (Fig. 7e, f, respectively). To determine whether the point of failure in the cell integrity of T492A cells was at Bck1, we assessed Bck1-HA levels (driven by a constitutive promoter) in WT and T492A cells (Fig. 4g). Strikingly, the abundance of Bck1 was substantially lowered in T492A cells (Fig. 4g). Bck1 is a 147 kDa protein that consists of a kinase domain and a large intrinsically disordered domain of unknown function75,76. To determine if T492 phosphorylation preferentially impacted a particular domain of Bck1, we separately expressed the N- and C-terminal domains of Bck1 (amino acids 1–738 and 739–1478) each with an HA tag in WT and T492A cells (Fig. 7g). Although the abundance of Bck1 (1–738) was not impacted by T492 status, we observed a decrease in the steady-state levels of Bck1 (739–1478) in T492A cells upon heat shock (Fig. 7g). To determine whether Bck1 may be a client of Ssa1, we performed directed co-immunoprecipitation experiments of FLAG-Ssa1 against Bck1-HA (both fragments) in WT and T492A yeast. We observed a robust interaction of Ssa1 with both Bck1 fragments, independent of heat shock (Fig. 7h, i). Overall, these data identify a requirement for Ssa1 T492 phosphorylation in promoting CWI signaling through support of Bck1.

Methods
Reagents and resources
Details on all reagents and resources (yeast strains and plasmids), primer sequences are provided in Supplementary Data 1–5.

Yeast Strains and growth conditions
Yeast cultures were grown in either YPD (1% yeast extract, 2% glucose, 2% peptone) or grown in SD (0.67% yeast nitrogen base without amino acids and carbohydrates, 2% glucose) supplemented with the appropriate nutrients to select for plasmids and tagged genes. Escherichia coli DH5α was used to propagate all plasmids. E. coli cells were cultured in Luria broth medium (1% Bacto tryptone, 0.5% Bacto yeast extract, 1% NaCl) and transformed to ampicillin or kanamycin resistance by standard methods. To construct the T492A yeast strain, we used a classic plasmid swap method37. The T492A mutation was engineered by Genscript into plasmid pC210, which drives SSA1 expression from the constitutive SSA2 promoter102. For FLAG-tagged versions of Ssa1 (WT and T492A) were created via Genscript by insertion of a N-terminal FLAG-tag into Ssa1-pC210. Full sequences of all these plasmids are available in Supplementary Data 5. WT or T492A Ssa1 constructs (either untagged or FLAG-tagged) were transformed into yeast strain 37 ssa1–4Δ using PEG/lithium acetate. After restreaking onto media lacking leucine and 5-fluoro-orotic acid (5-FOA), resulting in yeast that expressed WT or T492A Ssa1 (tagged or untagged) as the sole cytoplasmic Hsp70 in the cell.

For epitope tagging the genomic copy of HSP12 with a GFP epitope tag at the carboxy terminus, the pFA6a-GFP-His3MX6 plasmid was used. For epitope tagging the genomic copy of MKK1, and MKK2 with an HA epitope tag at the carboxy terminus, the pFA6a-3HA-His3MX6 plasmid was used. Primer sequences are available in Supplementary Data 4. Genotypes were confirmed by PCR analysis.

To generate a strain for the inducible degradation of Pkc1, the OsTir1-Myc tagged gene was first integrated into the yeast genome at the URA3 locus from ADHIp-OsTIR1-9Myc plasmid via PCR-mediated integrated gene tagging. Correct integration was confirmed via PCR. PKC1 was tagged with the degron domain via PCR-mediated integrated gene tagging. The C-terminal degron cassette was generated by amplifying it from the plasmid pMK43 (IAA17–KanMX4). This cassette was then transformed into yeast strains expressing the Oryza sativa F-box protein, OsTIR1. Cells that grew on media supplemented with G418 (Kanamycin) were restreaked and checked via PCR to confirm degron tag integration. Functionality of the Pkc1-AID system was verified by examining cell viability on media containing or lacking auxin. Details of the yeast strains used are provided in Supplementary Data 2.

Plasmid construction
Plasmids for the expression of Bck1 and yeast Hsp70 were synthesized by Vectorbuilder. Mutants of Ssa1 were created by Genscript. Full sequences of these plasmids are available in Supplementary Data 5. Details of the plasmids used are provided in Supplementary Data 2.

Growth assays
For serial dilutions, cells were grown to mid-log phase, 10-fold serially diluted and then plated onto appropriate media using a 48-pin replica-plating tool. Images of plates were taken after 3 days at 30 ˚C unless specified.

Pkc1-AID experiments
The Pkc1-AID strain was grown to the mid-log phase at 25 ˚C. 1 mM auxin was added to ablate the Pkc1 function for the indicated time points, and then the yeast were shifted to 39 ˚C for 30 min.

Luciferase assay
For the real-time luciferase activity assay, cells expressing the pHSE-lucCP+ plasmid11 were grown to mid-log phase at 25 ˚C. Activity of Hsf1 was determined by adding luciferin (final concentration 0.5 mM) and distributing 150 μl aliquots of the cultures into a white 96-well plate. Cells were incubated in a Synergy MX Microplate reader (Biotek Instruments) at 39 ˚C for 200 min, and luminescence was read every 5 min. The graph was prepared using GraphPad Prism 7. All experiments were conducted with at least 5 biological replicates.

Immunoprecipitation
For FLAG IP, cells were harvested, and FLAG-tagged proteins were isolated as follows: Protein was extracted via bead beating in 500 μl binding buffer (50 mM Na-phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20). 200 μg of protein extract was incubated with 30 μl anti-Flag M2 magnetic beads (Sigma) at 4 ˚C overnight. Anti-FLAG M2 beads were collected by magnet then washed 5 times with 500 μl binding buffer. After the final wash, the buffer was aspirated, and beads were incubated with 65 μl Elution buffer (binding buffer supplemented with 10 μg/ml 3X FLAG peptide (ApexBio) for 1 h at room temperature, then beads were collected via magnet. The supernatant containing purified FLAG-protein was transferred to a fresh tube, 25 μl of 4X SDS-PAGE sample buffer was added and the sample was denatured for 5 min at 95 ˚C. The eluates were separated by SDS-PAGE (7.5–10%) and visualized by immunoblotting.

For HA IP, cells were harvested, and HA-tagged proteins were isolated as follows: Protein was extracted via bead beating in 500 μl binding buffer (50 mM Na-phosphate pH 8.0, 300 mM NaCl, 0.01% Tween-20). 200 μg of protein extract was incubated with 30 μl anti-HA magnetic beads (Thermo Fisher Scientific) at 4 ˚C for 30 min. Anti-HA beads were collected by magnet then washed 5 times with 500 μl binding buffer. After the final wash, the buffer was aspirated, and beads were incubated with 65 μl Elution buffer and 15 μl of 4X loading dye and boiled at 100 ˚C for 10 min, then beads were collected via magnet. The supernatant containing purified HA-protein was transferred to a fresh tube, 25 μl of 4X SDS-PAGE sample buffer was added and the sample was denatured for 5 min at 95 ˚C. The eluates were separated by SDS-PAGE (7.5–10%) and visualized by immunoblotting.

For GFP IP, cells were harvested, and GFP-tagged proteins were isolated as follows: Protein was extracted via bead beating in 500 μl binding buffer (50 mM Na-phosphate, pH 8.0, 300 mM NaCl, 0.01% Tween-20). 200 μg of protein extract was incubated with 30 μl anti-GFP magnetic beads at 4 ˚C overnight. Anti-GFP beads were collected by magnet, then washed 5 times with 500 μl binding buffer. After the final wash, the buffer was aspirated, and beads were incubated with 65 μl Elution buffer and 15 μl of 4X loading dye and boiled at 100 ˚C for 10 min, then beads were collected via magnet. The supernatant containing purified HA-protein was transferred to a fresh tube, 25 μl of 5x SDS-PAGE sample buffer was added, and the sample was denatured for 5 min at 95 ˚C. The eluates were separated by SDS-PAGE (7.5–10%) and visualized by immunoblotting.

Immunoblotting
Protein extracts were made as described41. 15 μg of protein was separated by 4%–12% NuPAGE SDS-PAGE (Thermo). Proteins were detected using the following antibodies; Anti-Phospho T492 (21st-century biochemicals), Anti-PGK1 (Thermo #22C5D8), Anti FLAG (Sigma, #F1365), Anti-His (QIAGEN #34670), Anti-GAPDH (Thermo MA5-15738), Anti-Hsc70 (Rockland immunochemicals #200301F64), Anti-HA (Thermo #26183), Anti-Ydj1 (StressMarq #SMC-166D), Anti-Sis1 (Gift from Dr. E Craig), Anti-Hsp26 (Gift from Dr. J Buchner), Anti-GFP (Roche #1814460), Anti-Mpk1 (SCBT #133189), Anti-mAID (Proteintech #28209-1-AP) and Anti-phospho Mpk1 (Cell-signaling #4695). Blots were imaged on a ChemiDoc MP imaging system (Bio-Rad) after being treated with SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo). For multiplex blots, fluorescent secondary antibodies (Bio-Rad Starbright #12004159 and #12005867) were added to the blots and imaged on the ChemiDoc MP imaging system (Bio-Rad) using multiplex settings. The blots were then subsequently stripped and re-probed with relevant antibodies using a mild or harsh stripping buffer (glycine, SDS, Tween 20, pH 2.2, or 10% SDS, 0.5 M Tris HCl, pH 6.8 with β-mercaptoethanol, respectively).

In vitro kinase assays
Wild-type FLAG-Ssa1 were transformed with a plasmid expressing HA-Pkc1 under a galactose promoter41. Cells were grown in SD-URA Galactose at 25 ˚C overnight, and grown to the mid-log phase and heat shocked for 30 min. Protein was extracted via bead beating. For HA pulldown, anti-HA antibody-conjugated beads (Thermo Fisher Scientific) were washed 3 times with kinase buffer (100 mM Tris HCl (pH 8), 100 mM NaCl, 10 mM MgCl 2 and 20% glycerol). 100 μg of protein extract was added to the beads, which were incubated at 4 ˚C for 1 h. The supernatant was removed, and the beads were supplemented with 40 μl of kinase buffer along with 10 μg of purified full-length His-Ssa1 (gift from Dr. C Prodromou). After substrate addition, 100 μM of ATP was added to initiate the kinase reaction. The reaction was incubated for 1 h at room temperature. The reaction was terminated by adding 60 μl of 5x SDS-PAGE sample buffer (loading buffer), followed by boiling for 10–15 minutes. Samples were then separated using 4–12% SDS-PAGE gel and immunoblotted using phospho T492 antibody, His antibody, and HA antibody.

Pab1 and Edc3 foci quantification
Yeast cells from cultures grown to log phase (OD600≈0.5) were mounted in selective growth medium (C-URA), and 3D image stacks were collected at 0.3 µm z increments on a DeltaVision Elite Workstation (Cytiva) based on an inverted microscope (IX-70; Olympus) using a 100× 1.4 NA oil immersion lens. Images were captured at 22 ˚C with a 12-bit charge-coupled device camera (CoolSnap HQ; Photometrics) and deconvolved using the iterative-constrained algorithm and the measured point spread function. Image analysis and preparation was done using Softworx 6.5 (Cytivia) and FIJI ImageJ103. For heat shock experiments, cells were incubated in growth media in a 39 ˚C water bath (New Brunswick Scientific) for 15 min, 30 min, 1 hour, or 2 h, then immediately mounted on slides for imaging. To quantify the presence of Pab1 and Edc3 foci, a minimum of 100 cells were analyzed for each timepoint. Wildtype or T492A cells were visually scored for the presence of internal fluorescent foci from 3D projection images. Timepoints for the presence of foci were compared via single-factor ANOVA analysis. All experimental conditions were repeated in biological triplicate.

Polysome profiling
To grow cells for polysome profiling, 200 mL of yeast were grown in SCD-Leu overnight with shaking at 25 ˚C until they reached an OD600=0.4. Cells were collected onto a 0.45 µM filter (Cytiva #60173) using vacuum filtration (Sigma #Z290432) and immediately transferred to 100 mL of SCD-Leu prewarmed to 25 ˚C or 39 ˚C. After 10 minutes of incubation with periodic swirling the cells were either cooled back to 25 ˚C in a water bath and then transferred to a shaking incubator at 25 ˚C or harvested immediately via vacuum filtration onto a filter, followed by scraping the cells (Fisher #8100241) and plunging directly into liquid N2. The frozen cells were transferred to a pre-chilled 2 mL tube (Eppendorf #022363352). Cells were lysed with a pre-chilled 7 mm stainless steel ball (Qiagen #69990) using four 90 sec, 30 Hz pulses in a Retsch MM 400 mixer mill, chilling in liquid N2 between pulses. Sample was resuspended in 900 µL polysome lysis buffer (20 mM HEPES-KOH pH 7.4 (Sigma #H4034), 100 mM KCl (Fisher #BP366), 5 mM MgCl2 (Sigma #M2670), 200 μg/mL heparin (Calbiochem #375095), 1% triton X-100 (Sigma #T8787), 0.5 mM TCEP (Goldbio #TCEP25), 100 μg/mL cycloheximide (Thermo #J67043AD), 40 U/ml RNase inhibitor (NEB #M0314L), 1:100 EDTA-free Halt protease inhibitor (Thermo #PI78437). The lysate was clarified by centrifugation at 3000xg for 2 min, and the clarified lysate was transferred to a new tube. The OD260 of each lysate was measured using an LVis plate on a CLARIOstar Plus (BMG), and the concentrations were normalized across samples using lysis buffer. Finally, aliquots were flash frozen in liquid N2, and total samples were collected by mixing 20 µL of sample with 2x LDS buffer (500 mM Tris pH 8.5, 4% lithium dodecyl sulfate, 20% glycerol, 1 mM EDTA, 0.2% (w/v) orange G, 2.5% 2-Mercaptoethanol). A 10–50% continuous sucrose gradient in polysome gradient buffer (5 mM HEPES-KOH pH 7.4, 140 mM KCl, 5 mM MgCl 2, 100 μg/ml cycloheximide, 0.5 mM TCEP) was prepared in SW41 Ti tubes (Seton #7030) using a Biocomp Gradient Master with the 10–50% short sucrose program and cooled to 4 ˚C. Clarified lysate (200 µL) was loaded on top of the gradient, and gradients were spun in a SW41 Ti rotor at 281000xg for 105 min at 4 ˚C. Gradients were fractionated into 0.8 mL fractions using a Biocomp Piston Gradient Fractionator with a Triax flow cell monitoring at 260 nm. Polysome to monosome ratios were calculated using background-subtracted values. Proteins were precipitated from fractions using Trichloroacetic acid (TCA). First, 5 µL of 1 mg/mL BSA was added to each fraction as carrier protein. Then TCA was added to a final concentration of 10%, samples were vortexed and frozen at −20 ˚C for at least one hour. Samples were then centrifuged at 4 ˚C, 21,000xg for 15 min, and the supernatant was removed. Fractions were combined together by centrifugation in the same tube before a final wash with 10% TCA. Each tube was then washed twice with chilled acetone, dried for 5 min at 70 ˚C before being resuspended in 2x LDS sample buffer (500 mM Tris pH 8.5, 4% lithium dodecyl sulfate, 20% glycerol, 1 mM EDTA, 0.2% (w/v) orange G, 2.5% 2-Mercaptoethanol), incubated with shaking at 70 ˚C for 10 minutes, vortexed and finally centrifuged at 21,000 × g for 1 min. Samples were then run on a Bis-Tris, 4–12% gel with MES buffer (Genscript #M00654), transferred to PVDF membrane (Millipore Sigma #IPFL85R) using a Trans-blot turbo transfer system (Bio-rad #1704150) in transfer buffer (0.3 M glycine, 0.3 M Tris, 0.1% SDS, 20% methanol). Membranes were blocked with 5% milk for 1 hr before incubating with the specified primary antibodies overnight. Westerns were then visualized using donkey anti-mouse 800CW (LI-COR #925-32212) and goat anti-rabbit 680 (LI-COR #925-68071) on a LI-COR Odyssey M imager.

Translation activity via HPG incorporation
Translation was measured via HPG incorporation, based off the method used in refs. 68, 104, 105 Yeast cells were grown to OD600=0.3 in SCD-leu medium (Sunrise #1304-030) at 25 ˚C. Cells were transferred to a 2 mL deep well plate (VWR #75870-792), pelleted by centrifugation at 2000xg for 5 min and resuspended in SCD-met (Sunrise #1343-030). For cycloheximide (CHX) treated samples, CHX (Thermo Scientific #357420010) was also included at 100 µg/mL. Cells were pelleted again and resuspended in 900 µL pre-warmed SC-met containing 50 µM of homopropargylglycine (HPG, Vector Laboratories #CCT-1067-25) and CHX for controls or in SC-met with no HPG. Cells were incubated at the indicated temperature (25 ˚C or 39 ˚C) for the specified time points (10, 20, 30 min) in a water bath. Protein synthesis was halted by the addition of 100 µL of 1 mg/mL CHX. Cells were then pelleted, resuspended with 300 µL of ice-cold PBS + 100 µg/mL CHX, and fixed by the addition of 700 µL of ice-cold ethanol. Cells were fixed for 1 hr at RT or overnight at 4 ˚C. They were then washed with PBS + 3% BSA (GoldBio #A-420-100), and resuspended in click chemistry reaction buffer (50 mM Tris pH 7.5, 150 mM NaCl, 2.5 mM THPTA (Combi-blocks #QH-3278-1g), 1 mM CuSO 4 (Sigma #C1297100G), 2.8 mM fresh sodium ascorbate, and 5 µM AFDye 647 Picolyl Azide (Vector #CCT13001)). After 30 minutes, cells were pelleted, washed once with 3% BSA in PBS, once with wash buffer (0.5 mM EDTA, 2 mM NaN 3), and finally resuspended in PBS. Samples were measured using a Cytek Aurora 4 flow cytometer at the Texas A&M University Flow Cytometry Facility. The data were then analyzed in R using custom code utilizing the flowCore (https://doi.org/10.1186/1471-2105-10-106), tidyverse (https://doi.org/10.21105/joss.01686), and sf package (https://doi.org/10.32614/RJ-2018-009). Singlets were selected based on FSC.A, SSC.A and FSC.H values. The brightest 647 channel (R2) was then normalized to the SSC.A channel for every cell to control for cell size. Cells were filtered for those with higher intensities than the no HPG control. The mean normalized 647 intensity of the population was then calculated for every sample. Finally, this mean intensity was normalized to the mean of the WT samples at 10 minutes at a given temperature. At least 1000 cells were quantified in every sample.

Mass spectrometry
Six immunoprecipitations (three biological replicates of wild type Ssa1 controls and three biological replicates of Ssa1-T492A phosphodeficient mutants) were eluted from beads using 8 M urea, 10 mM DTT in 50 mM Tris pH 8.5 for 45 min at room temperature with mixing at 600 rpm before alkylation with 50 mM IAA for 30 min in the dark. Samples were diluted sixfold with 50 mM Tris pH 8.5 to reach <2 M Urea concentration and digested with 0.4 µg of trypsin-LysC mix (Promega) overnight at 37 ˚C. Tryptic peptides were desalted with Pierce C18 Desalting Spin Columns (Thermo Fisher Scientific) according to the manufacturer’s protocol, dried down on SpeedVac, and resuspended in mobile phase A (0.2% formic acid in water) immediately prior to mass spectrometric analysis.

Liquid chromatography–tandem mass spectrometry peptide analysis
Resuspended peptides were separated by nanoflow reversed-phase liquid chromatography (LC). An Ultimate 3000 UHPLC (Thermo Scientific) was used to load ~1 µg of peptides onto the column and separate them at a flow rate of 300 nL/min. Each sample was injected twice to allow for the use of different fragmentation techniques. The column was a 15 cm long EASY-Spray C18 (packed with 2 µm PepMap C18 particles, 75 µm internal diameter, Thermo Scientific). The analytical gradient was performed by increasing the relative concentration of mobile phase B (0.2% formic acid, 4.8% water in acetonitrile) the following steps: from 2% to 30% in 32 min, from 30% to 50% in 5 min, and from 50 to 85% in 5 min (for washing the column). The 4 min wash at high organic concentration was followed by moving to 15% in 2 minutes, increasing to 70% in 1 min for a secondary wash before re-equilibration of the column at 2% B for 7.5 min, for a total runtime of 68 min. A 2.2 kV potential was applied to the column outlet using an in-house nano ESI source based on the University of Washington design for generating nano-electrospray.

All mass spectrometry (MS) measurements were performed on a tribrid Orbitrap Eclipse (Thermo Scientific). Broadband mass spectra (MS1) were recorded in the Orbitrap over a 375–1500 m/z window, using a resolving power of 120,000 (at 200 m/z) and an automatic gain control (AGC) target of 4e5 charges (maximum injection time: 50 ms). Precursor ions were quadrupole selected (isolation window: 1.6 m/z) based on a data-dependent logic, using a maximum duty cycle time of 3 s. Monoisotopic precursor selection and dynamic exclusion (60 s) were applied. Peptides were filtered by intensity and charge state, allowing the fragmentation only of precursors from 2+ to 7+. Tandem mass spectrometry (MS2) was performed by fragmenting each precursor passing the selection criteria using both higher energy collisional dissociation (HCD; first injection) with normalized collision energy (NCE) set at 30% and electron transfer dissociation–higher energy collisional dissociation (EThcD; second injection), with ETD reagent target set at 5e5, reaction time calculated on the basis of a calibration curve and supplemental collisional activation set at NCE = 10%. The AGC target for both HCD and EThcD MS2 was set at 8e4 (maximum injection time: 55 ms), and spectra were recorded at 15,000 resolving power. A list of all interactors identified is provided in Supplementary Table 3.

MS Data analysis
For general identification of all proteins included in the samples, HCD fragmentation data were processed with Protein Discoverer 2.4, utilizing Sequest HT and MS Amanda search engines. For both, Precursor Mass Tolerance was 10 ppm, and Fragment Mass Tolerance was 0.02 Da. Carbamidomethylation (C) was allowed as a static modification, and dynamic modifications were as follows: Oxidation (M), Acetyl (protein N-term). Identified peptides were validated using Percolator, and the target FDR value was set to 0.01 (strict) and 0.05 (relaxed). Identified peptides were validated using Percolator, and the target FDR value was set to 0.01 (strict) and 0.05 (relaxed). Changes in protein abundance between the two strains were statistically tested by ANOVA using the built-in function within Proteome Discoverer. 3 replicates were used for each strain. The data was normalized on Ssa1.

Gene ontology enrichment analysis
Gene ontology analysis of Ssa1 immunoprecipitated complexes were accomplished using Yeast GO Slim.

Calculation of protein intrinsically-disordered content
The percentage of intrinsically disordered residues for interactors that are preferentially bound to WT or T492 Ssa1 were obtained via the MobiDB database (https://mobidb.org/). Proteins were considered disordered if they contained >40% disordered residues.

General data Analyses. Data processing and analyses were performed using GraphPad Prism (version 7).

Quantitative RT-PCR
Quantitation of yeast SSA1 and Luciferase transcription was carried out as in ref. 106. Briefly, yeast cells were grown overnight at 30 ˚C, re-inoculated at OD600 of 0.2–0.4 and then grown for a further 4 h at 25 ˚C. Cells were heat shocked at 39 ˚C for 30 min, and total RNA was extracted from cells using a Gene Jet RNA extraction kit. As a control, untreated cells were left at 25 ˚C. Total RNA (1 μg) was treated with 10 units of RNase-free DNase I (Thermo) for 30 min at 37 ˚C to remove contaminating DNA. DNAse I activity was stopped by adding 1 μL of 50 mM EDTA and incubating at 65 ˚C for 10 min. cDNA synthesis was carried out by iScript reverse transcriptase (BioRad) on aliquots of 1 μg RNA. The single-stranded cDNA products were used in qPCR on an ABI Fast 2000 real-time PCR detection system based on SYBR Green fluorescence. Sequences of oligo pairs are listed in Supplementary Data 4. Expression was normalized against that of ACT1 in each strain, and the resulting ratios in WT cells were defined as one-fold.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In the manuscript by Omkar et al., the main claim is that heat shock mediates the phosphorylation of Hsp70 at T492 and this phosphorylation is responsible for a wide variety of effects of the heat shock response. They demonstrate that heat shock induces rapid phosphorylation of Ssa1T492. Interestingly, this effect is independent of protein unfolding but dependent on the mechanosensor Mid2 and is exerted directly by the protein kinase C (Pkc1). They show that T492 is critical for the activation of the heat shock response and claim a role in the phosphorylation of this residue for HSF1-Ssa1 dissociation, protein translation, ribosome association, P-body disassembly, and CWI signaling. However, as detailed below, there are many flaws in the design, interpretation, and conclusions of the results that make this manuscript unsuitable for publication (the comments are ordered according to the order of results in the text):\n1) The authors show that T492 phosphorylation of Ssa1 by heat shock depends on Mid2 but not Wsc1. Previous reports on the CWI sensors required for Mpk1 activation under heat shock conditions showed that both proteins (Mid2 and Wsc1) sense heat shock (Verna et a, PNAS, 1997; Gray et al, EMBO J, 1997; Ketela et al., J. Bacteriology, 1999). How do explain the authors these differences?\n2) Regarding the experiments showing that Pkc1 mediates phosphorylation of Ssa1 at T492:\na) Figure S1 shows the cells' phenotype as validation of the Pkc1-AID strain, but levels of Pkc1 parallel to Phospho T492 (Figure 2d) need to be shown.\nb) In the in vitro kinase assay (Figure 2f), Pkc1 was purified from cells growing at 25ºC, but the effect of temperature (39ºC) also needed to be assayed.\n3) Regarding the Mass spec experiments comparing heat-shocked WT and T492A cells, I have several questions and comments (see below), but my main criticism is that it does not contribute much to the characterization of the functional role of Ssa1 T492 phosphorylation.\na) Although the objective is to compare and quantify differences in protein interactors of both proteins, using controls of untagged strains should avoid non-specific interactions.\nb) Details about how quantification was measured are missing: How many replicates were analyzed? What kind of normalization was applied? Moreover, no experimental validation of any of the proteins identified was carried out. This is absolutely necessary, particularly in relation to some of the proteins whose functional relationship with Ssa1 phosphorylation is discussed throughout the work.\nc) ln 784: fragment mass tolerance (0.2 Da) is too high (an adequate setting should be 0.02).\nd) 647 proteins are identified and 560 quantified, but only 380 are included in the Excel file. Why?\ne) The text through lines 256-262 has several errors: the mutation does not impact the association of fifteen chaperones and co-chaperones, but only nine. The other six do not show differences.\nf) One of the conclusions of the proteomic approach is that there are differences in the sets of client proteins bound to Ssa1 WT and T492A regarding the IDR composition of interactors. However, the authors do not clarify much about this conclusion. In line 251, they say that there are subtle differences; in the discussion (line 490), they say that it is possible that phosphorylation of T492 under heat shock promotes reinforced interaction IDP proteins, but in the abstract (ln 31), they assert that the phosphorylation of T492 leads altered preference for clients with IDRs. Furthermore, there is no statistical analysis of the data shown in Figure 3e, which would be necessary in order to draw valid conclusions. In addition, the figure legend does not provide a clear description of what exactly is represented on the graph (mean value, etc.). Additionally, it appears that there are some negative values for the percentage of IDR in the client proteins; how can this be possible?\n4) From the results of experiments described in Figure 4d, the authors claim that T492 phosphorylation is important for Ssa1-Hsf1 dissociation upon heat shock. This is a critical point in explaining the mechanism by which phosphorylation of Hsp70 by Pkc1 would trigger Hsf1-Hsp70 dissociation, leading to the activation of the HSF response. However, I do not think the results properly support this conclusion derived from the fact that mutation of T492 leads to the overexpression of Ssa1. Apparently, there are no differences in the amount of Ssa1 or Ssa1-T492A co-immunoprecipitated with Hsf1, but we can not exclude that this is due to the high amount of Ssa1 T492A and not to differences in the affinity between Hsf1 and Ssa1 as a consequence of the mutation. Experiments to measure differences in the affinity of Ssa1 variants for Hsf1 are needed. Additionally, reducing the amount of extract in the Co-IP and co-IP experiments in the opposite direction (immunoprecipitation with anti-Flag) could help. Moreover, Co-IP experiments should include a control of cells expressing only GFP to exclude possible nonspecific binding. Additionally, experiments showing that mutation of Thr 492 to Glu or Asp reverses the loss of dissociation should be appropriate.\nContrary to Figure 4d's conclusion, the MS interactors results provided in Table S3 show that Hsf1 did not display a preference for Ssa1 T492A (Log2(WT/T492A)=-0.075), but this is not discussed.\n5) The authors argue (ln 300-306) that overexpression of Ssa1 T492A is due to an activation of a Msn2/4 transcriptional response, but from my point of view, this is an overestimated conclusion. The authors show that levels of Hsp12 are elevated in these circumstances, but no additional proof (i.e mRNA levels of SSA1 in a msn2/4 mutant strain; activation of Msn2/4; nuclear translocation) is shown. Moreover, the authors claim that their data demonstrate that T492 phosphorylation is important for the activation of both Hsf1and Msn2/4-mediated heat shock-induced transcriptional response. However, levels of Hsp12 in the T492A strain are induced at 25ºC, suggesting that Msn2/4 activation in this strain, if it exists, is not mediated by heat shock but induced constitutively.\nIt is also important to mention here that, in contrast to these results, previous works (Beltrao et al, Cell. 2012 Jul 20;150(2):413-25) did not report overexpression of Ssa1 in a T492A S495A strain. This is note even commented in the manuscript.\n6) Regarding the results relating Hsp70 phosphorylation and ribosome association:\na) Conclusions from quantifying data from the Western blots in Figure 5d require further explanations about the number of replicates and statistical analysis of the differences. Otherwise, conclusions should be overestimated and not supported by the data.\nb) As stated by the authors (ln 310), Hsp70 dissociation from the ribosomes during heat shock promotes translational pausing. However, the author's results do not fit with this argument since they observe that, while polysome-bound Ssa1-T492A increases with respect to the WT, the T492A strain displays lower translation rates than the WT. Moreover, other publications studying the effect of phosphorylation of this residue report opposite conclusions. Moss et al. (Cell Host Microbe, 2019) reported that phosphorylation of the human Hsp70 in the equivalent residue (T495) increases its binding to polysomes, leading to translation inhibition. Additionally, Beltrao et al (2012), showed that the mutant T492A S495A was defective in polysome binding.\nc) Looking at the graphics in Figure 5f, it seems that the threefold increase in the mutant referenced in the text (ln 339) is overestimated. Additionally, at what temperature were these experiments performed? Is this effect a consequence of heat shock?\nd) In the legend of Figure 5f, they mention that statistical significance was calculated via ANOVA. However, they only have two groups, so this is not an appropriate analysis.\n7) Regarding the experiments of P-body formation upon heat shock and disassembly:\na) The description of these results is not acceptable. What was the heat shock temperature used in these experiments?. Looking at the legend of Figure 6, the experiments were carried out at 46ºC for 15 min and 30 min. However, looking at the methods section they say that these experiments were carried out at 39ºC. Moreover, heat shock recovery in Figure 6 is indicated at 30ºC during 60 and 120 min, but in methods, they describe 1 or 2 hours of heat shock at 39ºC. Stress granules in response to heat shock (46ºC) have been described in several reports but at shorter times (10 min). Since heat shock experiments carried out by the authors throughout the whole paper have been developed at 39ºC, these experiments should be also done at 39ºC (15, 30 min) and 46ºC (10 min)\nb) In addition to % of cells containing P-bodies, the number of P-bodies per 100 cells should be included.\nc) The effect of the T492A mutation in Pab1 foci disassembly shown in the graphics of Figure 6 does not correspond to the pictures: in the picture of Pab1/WT Ssa1 in recovery (30ºC) there are almost no foci.\nd) How do the authors explain that in the majority of the pictures stress granules (Pab1) co-localize with P-bodies (Edc3)?\n8) Regarding the relationship between heat-induced phosphorylation of Hsp70 and CWI pathway, the authors show that heat shock-mediated phosphorylation of Hsp70 in T492 via Pkc1 is absolutely necessary for the activation of Mpk1 and for the transcriptional response via the CWI pathway. This is quite interesting and innovative. However, the experiments developed to find out the mechanism responsible for this connection do not support the conclusions, and the mechanism is not characterized:\na) The authors focus on the possibility that the effect of Ssa1 T492A mutation on Mpk1 activation can be a consequence of differences in the levels of the protein kinases Mkk1, Mkk12, and Bck1, but there are many other possibilities.\nb) Even focusing only on this possibility, the results do not support the conclusions:\n•They work with two fragments of Bck1 instead of the full-length protein because they were unsuccessful in detecting Bck1 even in WT cells (ln 373). Even they argue explicitly (ln 375) that since Bck1 discovery, there are no reports containing Western blots of this protein. However, this is not true: to my knowledge at least in two publications full-length versions of Bck1 fused to Myc (Hruby et al, J Cell Sci, 2011, 124 (1):35-46; García et al, J Cell Sci,2016, 129: 1649-1660) have been reported, so all the experiments using the Bck1 protein fragmented in two pieces (Figure 7 g, h, i) are not justified.\n•Moreover, the authors describe in line 238 that co-IP experiments were done in WT and T492A strains but no CoIP of the mutant is shown in Figure 7\n•In any case, the decrease in the levels reported in for the carboxy fragment can not account for the complete blocking of Mpk1 activation and lack of induction of FKS2 and PRM5.\n9) There are many errors and an absence of information in the majority of the figure legends\n-Figure 1a: The predicted structure of Ssa1 is shown, but no reference is provided for how or where this prediction was obtained.\n-Figure 1d: The Western blot only shows WT cells, despite the figure legend mentioning both WT and T492A cells.\n-Figure 1e: The legend states that heat shock was performed at the indicated time points, but it was only performed at a single time point (1 h), as mentioned in the Results section.\n-Figure 2d: The legend states that cells were treated with auxin and incubated at 39°C for different times. However, the actual experiment involved auxin treatment for the indicated times, followed by a 30-minute incubation at 39°C for all samples.\n-Figure 2f: the legend mentions the use of antisera to FLAG epitope, but the epitope shown in the figure is HIS.\n-Figure 3e: In line 864, a verb is missing, making the conclusion unclear.\n-Figure 4d: The legend does not mention the antisera used for the PGK1 epitope.\n-Figure 4f: The legend mentions the use of antisera to PGK1 epitope, but the epitope shown in the figure is GAPDH.\n-Figure 5f: The temperature at which the experiment was performed is not indicated. Additionally, the legend mentions an ANOVA analysis, which is not appropriate since only two groups are being compared.\n-Figure 7i: The legend states that \"the C-terminus of Bck1 interacts with Bck1,\" but it should be \"the C-terminus of Bck1 interacts with Ssa1.\"\n-Figure S1: It needs further explanation\n10) Regarding the “Materials and Methods” section, I found several inconsistencies and a lack of information necessary for experimental reproducibility.\n-It is stated that reagents are provided in the supplementary tables, but this table is missing.\n-The description of plasmid construction for the expression of Flag-Ssa2, Flag-Ssa3, Flag-Ssa4, and the HA-tagged Bck1 fragments lacks necessary details.\n-The luciferase assay is described twice in this section, with inconsistencies between the two versions.\n•In the first description (line 579), cells were grown at 25°C, while in the second (line 648), they were grown at 30°C.\n•The first version states that incubation lasted 90 minutes with measurements taken every 5 minutes, whereas the second version states that incubation lasted 90 minutes with readings every 3 minutes.\n•Both descriptions mention incubation at 37°C, but the correct temperature is 39°C according to the graph.\n-In vitro kinase assay: The composition of the kinase buffer and loading buffer is not specified.\n-Pab1 and Edc1 foci quantification. According to the figure, cells were heat-shocked at 46°C for 15 and 30 minutes, followed by recovery at 30°C for 1 and 2 hours. However, the Materials and Methods section states that cells were incubated at 39°C for 15, 30, 60, or 120 minutes, which does not match the figure.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This manuscript investigates the role of threonine 492 phosphorylation of yeast Hsp70 (Ssa1) during heat stress. The authors demonstrate that heat shock induces Ssa1 T492 phosphorylation, dependent on the cell wall sensor Mid2 and the Pkc1 kinase of the cell wall integrity (CWI) pathway. This phosphorylation is shown to be essential for Hsf1 release and proper activation of the heat shock response (HSR), potentially contributing to its rapid kinetics. Furthermore, T492 phosphorylation influences Ssa1’s ribosome association and translation fidelity during heat stress, and regulates P-body disassembly during recovery. Notably, the CWI pathway is not activated in T492A mutant cells during heat shock, though a direct mechanistic link remains to be established.\nThis study demonstrates a novel and extremely intriguing connection between cell-wall integrity pathway-mediated Hsp70 phosphorylation and regulation of the heat shock response. It elucidates how environmental stress signals are transmitted from cell surface sensors to the nucleus, activating the stress response, thereby contributing to our understanding of the chaperone code. Overall, much of the data are very compelling and the quality of the results is high. However, open questions remain, and some of the results require additional support, as detailed below.\n1. Please specify the heat shock duration for all the related Figures or at least mention it in the figure legends.\n2. Figures 1c, 1d, 1e and 2e indicate a low level of T492 phosphorylation without heat shock. This should be explicitly recognized in the text. The authors should also discuss potential kinases and the functional implications of this basal phosphorylation.\n3. In Figure 2b, residual T492 phosphorylation is observed in the absence of Mid2. To ensure reproducibility of this finding, the authors should repeat this experiment a minimum of three independent times and present the average signal, similar to that in Figures 4c and 4f, or clarify if this was indeed done. Additionally, given the potential for delayed responses, this experiment should be conducted using the 4-hour timeline employed in Figure 1c to definitively rule out any delayed phosphorylation kinetics. The authors should also discuss why in the absence of Mid2, Wsc1 could not activate Pkc1 to phosphorylate Ssa1. It is unclear what significance should be attached to the apparent Mid2 specificity, if any.\n4. In Figure 2d, the authors should examine Pkc1 protein levels upon auxin induction at the indicated time points to strengthen the correlation between T492 phosphorylation reduction and Pkc1 levels. This could be achieved by using anti AID tag antibody or tagging Pkc1 with additional small epitope tag, such as FLAG or HA. Furthermore, if generating a pkc1 null mutant in sorbitol-containing media is feasible, utilizing this mutant directly would provide a more straightforward and definitive assessment of Pkc1's role in T492 phosphorylation.\n5. While Figure 2f demonstrates that Pkc1 can phosphorylate Ssa1 in vitro, given that T492 is within the preferred PKC substrate motif, in vivo validation of this interaction is important. To confirm this interaction within cells, a crosslinking and pulldown experiment using Pkc1 as bait to detect Ssa1 association and T492 phosphorylation should be considered. Notably, Pkc1 was absent from the Ssa1 interactors identified by mass spectrometry (MS) (Supplementary table S1). This discrepancy requires discussion within the text.\n6. The authors noted that Ssa1 T492A exhibits significantly higher expression (at least five-fold) compared to Ssa1 WT (Figures 1c, 2c, 4d). In Figure 3, where MS data and changes in Ssa1 interactors are presented, it is unclear whether the levels of detected interactors were normalized to the respective levels of Ssa1 WT and Ssa1 T492A. If normalization was not performed, the observed preferential binding of interactors requires re-evaluation to account for the differential expression of Ssa1 variants.\n7. The observed growth defect of the T492A mutant at 37 °C (Figure 4a) and the corresponding lack of Hsf1-mediated heat shock response (Figures 4b, c) are very intriguing. However, given the significantly higher expression of T492A compared to WT (at least five-fold), it is possible that the observed Hsf1 trapping is an artifact of T492A overexpression, rather than a direct effect of the T492A mutation itself. To address this, we recommend reducing T492A expression using a weaker heterologous promoter and examining whether Hsf1 still fails to be released under heat shock condition. Alternatively, a phosphorylation mimetic mutation (T492D) could be introduced to assess whether it retains the ability to bind Hsf1 upon heat shock.\n8. Additionally, regarding Figure 4A, the observed temperature sensitivity at 37˚C appears to be significantly remediated by the addition of sorbitol, an osmotic stabilizer. This finding suggests that the growth defect is not due to a reduced heat shock response, as mentioned in the text, but mostly due to the clear defects in CWI signaling shown in Figure 7. This in some way alters the interpretation of the effects of T492 phosphorylation in both the results and discussion and should be clearly explained.\n9. In Figure 4d, the signal for WT Ssa1 appears relatively weak. To ensure the reliability of the observed reduction in Ssa1-Hsf1 binding upon heat shock, we recommend repeating this experiment at least two additional times and quantifying the average signal. Furthermore, overexpressing WT Ssa1 may enhance the signal and provide a more robust demonstration of the reduction in Hsf1 binding during heat shock.\n10. The observation of Msn2/4 activation in the T492A mutant under normal conditions (Figure 4f) suggests a complete blockage of Hsf1 activity. This raises crucial questions regarding the mechanism of T492A's effect. Specifically, it is necessary to determine whether T492A expression is upregulated by the activated Msn2/4 pathway or the T492A mutation stabilizes the Ssa1 structure, thereby enhancing its apparent Hsf1 binding affinity.\n11. In Figure 6e, the authors must rule out whether the observed slow clearance of P-bodies in T492A cells is a consequence of the increased abundance of Ssa1 T492A. This could be done using a lower expression construct as described above.\n12. The observed inactivation of the CWI pathway in the T492A mutant (Figure 7a-c) is an interesting finding. However, the in vivo interaction of Ssa1 with Bck1 fragments (Figure 7h, i) raises a significant discrepancy, as Bck1 was not identified as an Ssa1 interactor in the mass spectrometry data (Supplementary table S1). The authors must address this discrepancy and clarify whether the Bck1 fragments represent the native state of Bck1 or potentially misfolded states that attract Ssa1 binding, taking into consideration the challenges inherent with working with the full-length protein. Furthermore, the phosphorylation status of Mkk1/Mkk2 in T492A cells under heat shock condition should be evaluated to further elucidate the CWI pathway inactivation.\nMinor comments:\n13. Introduce the full terms for AZC and CPZ upon their first mention in the text.\n14. Add periods at the end of sentences on lines 99, 167, 467, and 716.\n15. Use subscript for numerical values in chemical formulas (e.g., NaN₃, N₂, CuSO₄, MgCl₂).\n16. Common chemical names, such as 'glycerol,' should not be capitalized unless they appear at the beginning of a sentence.\n17. Line 685: Remove the extra \")\".\n18. Line 308: The title 'Hsp70 phosphorylation is critical for protein translation and ribosome association' should be revised. The polysome fractions are normal, and the data show that T492A only increases its presence at the ribosome and affects translation fidelity, while translation itself continues. Please revise the title to accurately reflect these findings.\n19. Line 403: change \"Notably, T492 occurs rapidly\" to \"Notably, T492 phosphorylation occurs rapidly.\"\n20. Line 448: replace \"Sis\" with \"Sis1.\"\n21. Line 747: revise the sentence \"Samples were diluted 6x with 50 mM tris pH 8.5 to reach <\" to ensure it is grammatically correct.\n22. Line 754: change \"~1 ug\" to \"~1  g.\"\n23. Line 756: define \"i.d.\" in the context of the PepMap C18 particles.\n24. Line 483: change \"The yeast cell integrity pathway\" to \"The yeast cell wall integrity pathway.\"\n25. Line 860: capitalize \"V\" in \"vocalno\" to \"Volcano.\"\n26. Line 863-864: Add a verb to the sentence \"T492 phosphorylation does not the binding of intrinsically-disordered proteins.\"\n27. Add the hour unit to the time of the AZC treatment in Figure 1f.\n28. Revise \"Translational pausing\" to \"Translational fidelity\" in Figure 8.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "In this interesting multidisciplinary study, Truman and colleagues make important contributions to our understanding of how distinct features of the budding yeast heat shock response, a longstanding model for the conserved eukaryotic HSR, are integrated. Among the notable observations is that phosphorylation of Ssa1 T492 is required for full Hsf1 activation (however, see below). If confirmed, this process, operating alongside the classic chaperone titration mechanism, can provide an explanation for the rapidity of Hsf1 activation which is detected within seconds – and as pointed out by the authors, too rapid to be explained by chaperone titration mediated by unfolded nucleoplasmic/cytosolic proteins alone. Additional strengths of the study are the multiple approaches used, the very high quality of data and the thoughtful writing (although see below).\nMy enthusiasm is tempered by several concerns that I believe need to be addressed before the manuscript is suitable for publication in Nat. Commun.\n1. A key claim is that Hsf1 transcriptional activation is functionally linked to the phosphorylation status of Ssa1 T492. This is based on experiments conducted using a specialized strain in which the genes encoding all 4 cytosolic Hsp70’s – SSA1, SSA2, SSA3, SSA4 – have been deleted and WT and mutant Ssa1 are expressed from multicopy centromeric plasmids. My concern is that the authors use these strains to assay Hsf1 activity indirectly, using either a luciferase reporter (Fig. 4b) or Western blot of a representative Hsf1 target gene (Fig. 4c). As later shown (Fig. 5d, e), the T492A mutation in Ssa1 strongly inhibits global translation (although unaddressed is the possibility that translation of Hsf1 target mRNAs is spared). Thus, the assays used for Hsf1 activation are potentially confounded by suppressed translation of HSP mRNAs (including the HSE-driven mRNA encoding the luciferase reporter) elicited by the same point mutation that obviates release of Hsf1 +HS (a striking observation shown in Fig. 4d). The authors need to use a more direct assay of Hsf1 activation (such as RT-qPCR or Pol II ChIP) to substantiate this key point.\n2. As alluded above, the authors surmise that kinetics of phosphorylation of T492 are sufficiently rapid to explain the rapidity with which Hsf1 itself is activated in heat shocked yeast, a phenomenon that occurs within ~1 min (see, for example, PMID 28970326), too rapid to be explained by the classic chaperone titration model. If this is in fact the mechanism, then to prove it the authors need to fine-tune their kinetics analysis beyond what is presented in Fig 1d to include pertinent early time points.\n3. A potential limitation is that Ssa1 T492A is expressed at a substantially higher level than WT Ssa1 (this needs to be quantified) in the experiments that employ the MH272 strain, which appear to be the vast majority. At the very least, the authors need to acknowledge this potential confounding aspect of the study.\n4. A paradox is that interaction of Sis1 with Ssa1 is not affected by the T492A mutation, yet Sis1 is required to maintain Hsf1 in a transcriptionally inactive state (PMID 33326013). The authors need to address this.\n5. The Introduction and (especially) Discussion need to be tightened up. At places, these sections read like review article rather than a research paper.\nMinor points\n1. The key study identifying Hsp70 as the principal chaperone that represses yeast Hsf1 is ref. 9. This, and a companion study (PMID 29393852), should be clearly acknowledged throughout the ms. (e.g., on lines 68, 279, 392). Likewise, the initial study identifying Hsp70 as a principal repressor of mammalian HSF1 is ref. 59. It too needs to be more clearly acknowledged.\n2. Misinterpretation of data: lines 157-158 (“not detected” is inconsistent with data presented in Figs. 1c, d, e; 2e).\n3. Criteria for identifying the presence of foci in Fig. 6 needs to be clearly stated. Pab1-GFP foci are visible in T492A cells under recovery conditions, yet this is not evident from the bar graph.\n4. Figures 1a and 5c could be clearer through labelling of each of the three Hsp70 domains and the free, monosome and polysome peaks.\n5. The authors are inconsistent in their reference to the role played by Msn2 in the yeast transcriptional response to stress. In places, they lump Msn2 with Hsf1 as intrinsic to the HSR; in other places they characterize Msn2 as driving a separate environmental stress response. Just be consistent.\n6. Writing is for the part clear; however, there are multiple places where either punctuation or words are missing (and/or word choice is poor/misleading). For example: lines 99, 105, 141, 143, 154, 179, 190, 258, 382, 403, 471, 504, 864, 870, 877, 879 (antagonizes not “mediates”), 920, 949-950.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "We offer below our point-by-point responses to queries and comments on our submitted manuscript titled, “Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response”. We would like to thank the reviewers for your constructive comments and very positive feedback. We believe that the revised manuscript and associated new data provide an improved and compelling story for publication in Nature Communications.\nResponse to Reviewer #1\nWe would like to thank the reviewer and trainee for their thoughtful review of our manuscript. We have performed additional experiments that clarify your queries and in doing so we believe the manuscript has been significantly improved.\n1) The authors show that T492 phosphorylation of Ssa1 by heat shock depends on Mid2 but not Wsc1. Previous reports on the CWI sensors required for Mpk1 activation under heat shock conditions showed that both proteins (Mid2 and Wsc1) sense heat shock (Verna et a, PNAS, 1997; Gray et al, EMBO J, 1997; Ketela et al., J. Bacteriology, 1999). How do explain the authors these differences?\nDespite extensive research, the cellular rationale for the presence of multiple upstream receptors in the CWI pathway remains unresolved. Nevertheless, the studies cited by the reviewer clearly demonstrate that deletion of MID2 results in temperature-sensitive phenotypes and impaired Mpk1 phosphorylation. In this context, the mid2Δ strain phenocopies the ssa1-T492A mutant, underscoring the functional relevance of this signaling axis. While deletion of WSC1 also confers temperature sensitivity, this observation is not contradictory but instead supports the notion of parallel or partially redundant signaling branches within the pathway. We have revised the Discussion section to clarify and incorporate these interpretations.\n2) Regarding the experiments showing that Pkc1 mediates phosphorylation of Ssa1 at T492:\na) Figure S1 shows the cells' phenotype as validation of the Pkc1-AID strain, but levels of Pkc1 parallel to Phospho T492 (Figure 2d) need to be shown.\nb) In the in vitro kinase assay (Figure 2f), Pkc1 was purified from cells growing at 25ºC, but the effect of temperature (39ºC) also needed to be assayed.\nTo address point a) We have now included a figure showing the levels of Pkc1-AID as requested using an AID antibody. This figure can be seen as Fig.S2b. For point b) the experiment was performed using Pkc1 purified from heat-treated cells (as is the standard protocol in the field). Our apologies for this error; we have now revised the text.\n3) Regarding the Mass spec experiments comparing heat-shocked WT and T492A cells, I have several questions and comments (see below), but my main criticism is that it does not contribute much to the characterization of the functional role of Ssa1 T492 phosphorylation.\na) Although the objective is to compare and quantify differences in protein interactors of both proteins, using controls of untagged strains should avoid non-specific interactions.\nWe appreciate the reviewers comment about untagged strains. Over time, proteomic technologies, our experimental methodologies and our analysis methods have improved to the degree that we don’t typically need to use an untagged control anymore. This has been our standard practice for all our publications on chaperone interactions from PLOS Genetics to Cell.\nb) Details about how quantification was measured are missing: How many replicates were analyzed? What kind of normalization was applied?\nWe have addressed this in the manuscript.\nMoreover, no experimental validation of any of the proteins identified was carried out. This is absolutely necessary, particularly in relation to some of the proteins whose functional relationship with Ssa1 phosphorylation is discussed throughout the work.\nProteomic technologies have advanced substantially over time to the degree that they are much more accurate than older methods like Western Blots. This together with the large number of interactions observed make it impractical to validate interactions using other methods. Some of the substantially changed interactions will be the focus of our group’s future studies, but we believe this is beyond the scope of the current study.\nc) ln 784: fragment mass tolerance (0.2 Da) is too high (an adequate setting should be 0.02).\nYes, this is a typographical error, and we have now corrected it in the revised manuscript.\nd) 647 proteins are identified and 560 quantified, but only 380 are included in the Excel file. Why?\nThere was a text error; we have revised the manuscript.\ne) The text through lines 256-262 has several errors: the mutation does not impact the association of fifteen chaperones and co-chaperones, but only nine. The other six do not show differences.\nWe have now addressed the errors and revised the manuscript.\nf) One of the conclusions of the proteomic approach is that there are differences in the sets of client proteins bound to Ssa1 WT and T492A regarding the IDR composition of interactors.\nHowever, the authors do not clarify much about this conclusion.\nWe appreciate that the data were not clear for the IDPs. We have revised our analysis and show that the majority of Ssa1-IDP interactions are actually T492 phosphorylation state-independent.\n4) From the results of experiments described in Figure 4d, the authors claim that T492 phosphorylation is important for Ssa1-Hsf1 dissociation upon heat shock. This is a critical point in explaining the mechanism by which phosphorylation of Hsp70 by Pkc1 would trigger Hsf1-Hsp70 dissociation, leading to the activation of the HSF response. However, I do not think the results properly support this conclusion derived from the fact that mutation of T492 leads to the overexpression of Ssa1. Apparently, there are no differences in the amount of Ssa1 or Ssa1-T492A co-immunoprecipitated with Hsf1, but we can not exclude that this is due to the high amount of Ssa1 T492A and not to differences in the affinity between Hsf1 and Ssa1 as a consequence of the mutation. Experiments to measure differences in the affinity of Ssa1 variants for Hsf1 are needed. Additionally, reducing the amount of extract in the Co-IP and co-IP experiments in the opposite direction (immunoprecipitation with anti-Flag) could help. Moreover, Co-IP experiments should include a control of cells expressing only GFP to exclude possible nonspecific binding. Additionally, experiments showing that mutation of Thr 492 to Glu or Asp reverses the loss of dissociation should be appropriate.\nIn this study, we used native SSA promoters on our constructs to keep the conditions as physiologically relevant as possible. As the reviewer noted, we observe higher Ssa1 expression in the T492A expression compared to WT, which we believed to be due to overcompensation by Msn2/4 signaling. To address issues potentially caused by this overexpression, we reconstructed the WT and T492A strain using plasmids with constitutive GPD promoters. In these strains, Ssa1 levels are equal, confirming that the expression change previously seen was promoter-driven. Encouragingly, the GPD-driven Ssa1 strains display the same phenotypes as SSA-driven ones (see Fig. S3b). We repeated the Hsf1-Ssa1 interaction experiment using the new strains and we see an even clearer T492-driven Hsf1 interaction difference. This data can be found as Fig. S3e.\nThe suggested experiment using the phosphomimic is challenging due to the inviability of the T392E mutant and there is no guarantee that T492E would mimic a truly phosphorylated state.\nContrary to Figure 4d's conclusion, the MS interactors results provided in Table S3 show that Hsf1 did not display a preference for Ssa1 T492A (Log2(WT/T492A)=-0.075), but this is not discussed.\nIt should be noted that Hsf1 has a very low cellular abundance (less than 2000 molecules per cell) and is typically hard to quantify by MS. We believe our revised experiments provide enough data to confidently say that T492 alters binding with Hsf1.\n5) The authors argue (ln 300-306) that overexpression of Ssa1 T492A is due to an activation of a Msn2/4 transcriptional response, but from my point of view, this is an overestimated conclusion. The authors show that levels of Hsp12 are elevated in these circumstances, but no additional proof (i.e mRNA levels of SSA1 in a msn2/4 mutant strain; activation of Msn2/4; nuclear translocation) is shown. Moreover, the authors claim that their data demonstrate that T492 phosphorylation is important for the activation of both Hsf1and Msn2/4-mediated heat shock-induced transcriptional response. However, levels of Hsp12 in the T492A strain are induced at 25ºC, suggesting that Msn2/4 activation in this strain, if it exists, is not mediated by heat shock but induced constitutively.\nAs noted above, when we replace the promoter of our SSA promoter-driven Ssa1 construct with a constitutive GPD promoter, the levels of Ssa1 T492A are expressed at equivalent levels to WT. This suggests the effect seen is transcriptional rather than degradation of the protein. In our T492A mutant, Hsp12 levels are constitutively high suggesting overactivation of the Msn2/4 pathway. The reviewer is correct that this response is not impacted by heat and this is because in the T492A mutant, heat-induced T492 phosphorylation cannot occur. We attempted to delete Msn2/4 in our T492A mutant background, but could not generate the strain. This is probably because loss of Hsf1 function in an Msn2/4 background is lethal (Mühlhofer et al., 2024). We have instead revised the text to take into account the reviewer’s point.\nIt is also important to mention here that, in contrast to these results, previous works (Beltrao et al, Cell. 2012 Jul 20;150(2):413-25) did not report overexpression of Ssa1 in a T492A S495A strain. This is note even commented in the manuscript.\nIt should be noted that in the excellent Beltrao study all constructs are expressed via constitutive GPD promoters, whereas we used the SSA promoter to attempt to provide more native conditions. However as mentioned above, we have reconstructed the WT and T492A strains using the GPD promoter. In this strain, T492A has the same abundance as WT (while displaying all the same phenotypes). We have added this data to the revised manuscript and figures (Fig.S3a-b).\n6) Regarding the results relating Hsp70 phosphorylation and ribosome association:\na) Conclusions from quantifying data from the Western blots in Figure 5d require further explanations about the number of replicates and statistical analysis of the differences. Otherwise, conclusions should be overestimated and not supported by the data.\nWe agree and have made the requested changes throughout.\nb) As stated by the authors (ln 310), Hsp70 dissociation from the ribosomes during heat shock promotes translational pausing. However, the author's results do not fit with this argument since they observe that, while polysome-bound Ssa1-T492A increases with respect to the WT, the T492A strain displays lower translation rates than the WT. Moreover, other publications studying the effect of phosphorylation of this residue report opposite conclusions. Moss et al. (Cell Host Microbe, 2019) reported that phosphorylation of the human Hsp70 in the equivalent residue (T495) increases its binding to polysomes, leading to translation inhibition. Additionally, Beltrao et al (2012), showed that the mutant T492A S495A was defective in polysome binding.\nWe appreciate the reviewer’s careful reading and the opportunity to clarify the apparent discrepancy. Our original sentence implied that complete dissociation of Hsp70 from ribosomes is necessary for every instance of translational pausing. Recent work as well as our own data indicate a more nuanced model in which either excessive association or complete dissociation of cytosolic Hsp70 can slow translation, depending on the chaperone’s nucleotide state and the stage of elongation. We have therefore revised the text to read: “Heat shock remodels Hsp70-ribosome interactions, and both transient dissociation and persistent over-association of Hsp70 have been reported to impede elongation, thereby contributing to global translational pausing (Shalgi et al., 2013).” In addition, our data (Fig. 5d-f) show that the unphosphorylatable T492A variant remains locked on heavy polysomes during heat shock. This persistent binding is predicted to hinder elongation factor access, slow ribosome translocation and reduce methionine-analog (HPG) incorporation, exactly as we observe. Importantly, polysome accumulation in the mutant is accompanied by slower ribosome run-off in time-course profiles (new Supplementary Fig. S4c), indicating stalled, not active, ribosomes. Thus, higher polysome-bound Ssa1 does not equal higher productive translation.\nOur results differ slightly from Moss et al. 2019 and Beltrao et al (2012 probably due to the systems and experimental conditions tested. For example, Beltrao’s double (T492A/S495A) or triple mutants severely altered the substrate-binding groove geometry, likely compromising ribosome affinity altogether. In contrast, our single-site mutant preserves the binding interface but blocks the ATP-regulated release step, producing hyper-association. Both our study and Moss et al. examined chaperone behaviour under acute stress (heat or LegK4 effector), conditions under which stalled ribosomes accumulate. Beltrao et al. assayed cells in steady-state growth, where Hsp70 turnover on ribosomes is faster and phosphorylation may promote initial recruitment rather than release. Finally, Human Hsc70 lacks the yeast-specific co-chaperone RAC-Ssb system; nevertheless, the convergence between Moss et al. and our yeast data-increased polysome binding when T492/T495 cannot be dephosphorylated supports a conserved “release” function for this modification.\nc) Looking at the graphics in Figure 5f, it seems that the threefold increase in the mutant referenced in the text (ln 339) is overestimated. Additionally, at what temperature were these experiments performed? Is this effect a consequence of heat shock?\nWe have resolved this in the text.\nd) In the legend of Figure 5f, they mention that statistical significance was calculated via ANOVA. However, they only have two groups, so this is not an appropriate analysis.\nWe apologize. We have resolved this in the text.\n7) Regarding the experiments of P-body formation upon heat shock and disassembly:\na) The description of these results is not acceptable. What was the heat shock temperature used in these experiments?. Looking at the legend of Figure 6, the experiments were carried out at 46ºC for 15 min and 30 min. However, looking at the methods section they say that these experiments were carried out at 39ºC. Moreover, heat shock recovery in Figure 6 is indicated at 30ºC during 60 and 120 min, but in methods, they describe 1 or 2 hours of heat shock at 39ºC. Stress granules in response to heat shock (46ºC) have been described in several reports but at shorter times (10 min).\nWe apologize, this was a labeling error. We have corrected the labels for these figures.\na) The effect of the T492A mutation in Pab1 foci disassembly shown in the graphics of Figure 6 does not correspond to the pictures: in the picture of Pab1/WT Ssa1 in recovery (30ºC) there are almost no foci.\nb) How do the authors explain that in the majority of the pictures stress granules (Pab1) co-localize with P-bodies (Edc3)?\nWe agree that these proteins can be seen to colocalize and this reflects well-documented biology rather than a technical issue. P-Bodies are known to nucleate early and can seed stress granule assembly (Kedersha et al.2005 and Buchan et al. 2008).\n8) Regarding the relationship between heat-induced phosphorylation of Hsp70 and CWI pathway, the authors show that heat shock-mediated phosphorylation of Hsp70 in T492 via Pkc1 is absolutely necessary for the activation of Mpk1 and for the transcriptional response via the CWI pathway. This is quite interesting and innovative. However, the experiments developed to find out the mechanism responsible for this connection do not support the conclusions, and the mechanism is not characterized:\na) The authors focus on the possibility that the effect of Ssa1 T492A mutation on Mpk1 activation can be a consequence of differences in the levels of the protein kinases Mkk1, Mkk12, and Bck1, but there are many other possibilities.\nOur data strikingly show that Mpk1 phosphorylation is lost in T492A mutants. Given the role of chaperones in stabilizing client proteins, it is a logical assumption that a CWI protein is a client of Ssa1. Mkk1/2 abundance are not impacted in T492A, so we logically went upstream to Bck1. Our data clearly show that Ssa1 interacts with Bck1 and that Bck1 domains are destabilized in T492A.\nb) Even focusing only on this possibility, the results do not support the conclusions:\n•They work with two fragments of Bck1 instead of the full-length protein because they were unsuccessful in detecting Bck1 even in WT cells (ln 373). Even they argue explicitly (ln 375) that since Bck1 discovery, there are no reports containing Western blots of this protein. However, this is not true: to my knowledge at least in two publications full-length versions of Bck1 fused to Myc (Hruby et al, J Cell Sci, 2011, 124 (1):35-46; García et al, J Cell Sci,2016, 129: 1649-1660) have been reported, so all the experiments using the Bck1 protein fragmented in two pieces (Figure 7 g, h, i) are not justified.\nBck1 is an extremely challenging protein to work with. It is rather large, roughly 165KDa but with a corresponding low abundance of around 1200 molecules per cell. In Hruby et al, the authors also experienced the same issue as us and had to break Bck1 into fragments (1-800 aa, see Figure 2 of that paper). In Garcia et al, the authors claim that they natively tagged Bck1 and Pkc1 with a Myc tag. However if the reviewers look at Figure 3, Bck1 and Pkc1 are detected at the same MW-unusual given that these proteins have distinctly different molecular weights. In any case, we tried our best to study Bck1 as a full-length protein. As mentioned in the text, we attempted to tag it on the genome as in Garcia et al and did not detect the protein. We turned next to the ZZ-tagged collection and did not see any protein even under the galactose-driven promoter. We even fully sequenced and validated this plasmid to check that there were no frame shifts present. Fortunately, we were successful in detecting Bck1 when expressing domains as in Hruby et al. We have adjusted the text in the manuscript to reflect the limitations of our results.\n•Moreover, the authors describe in line 238 that co-IP experiments were done in WT and T492A strains but no CoIP of the mutant is shown in Figure 7\nResolved in the revised manuscript.\n•In any case, the decrease in the levels reported in for the carboxy fragment can not account for the complete blocking of Mpk1 activation and lack of induction of FKS2 and PRM5.\nIt is important to remember that for us to detect Bck1, we have substantially overexpressed the protein compared to native levels. While we agree this is not ideal, it was necessary for this experiment. Our working model is that the impact of T492A would be more substantial on the native protein. We have edited the manuscript to account for this.\n9) There are many errors and an absence of information in the majority of the figure legends\n-Figure 1a: The predicted structure of Ssa1 is shown, but no reference is provided for how or where this prediction was obtained.\nThis has been resolved in the manuscript.\n-Figure 1d: The Western blot only shows WT cells, despite the figure legend mentioning both WT and T492A cells.\nThis has been resolved in the manuscript.\n-Figure 1e: The legend states that heat shock was performed at the indicated time points, but it was only performed at a single time point (1 h), as mentioned in the Results section.\nThis has been resolved in the manuscript.\n-Figure 2d: The legend states that cells were treated with auxin and incubated at 39°C for different times. However, the actual experiment involved auxin treatment for the indicated times, followed by a 30-minute incubation at 39°C for all samples.\nThis has been resolved in the manuscript.\n-Figure 2f: the legend mentions the use of antisera to FLAG epitope, but the epitope shown in the figure is HIS.\nThis has been resolved in the manuscript.\n-Figure 3e: In line 864, a verb is missing, making the conclusion unclear.\nThis has been resolved in the manuscript.\n-Figure 4d: The legend does not mention the antisera used for the PGK1 epitope.\nThis has been resolved in the manuscript.\n-Figure 4f: The legend mentions the use of antisera to PGK1 epitope, but the epitope shown in the figure is GAPDH.\nThis has been resolved in the manuscript.\n-Figure 5f: The temperature at which the experiment was performed is not indicated.\nThis has been resolved in the manuscript.\nAdditionally, the legend mentions an ANOVA analysis, which is not appropriate since only two groups are being compared.\nThis has been resolved in the manuscript.\n-Figure 7i: The legend states that \"the C-terminus of Bck1 interacts with Bck1,\" but it should be \"the C-terminus of Bck1 interacts with Ssa1.\"\nThis has been resolved in the manuscript.\n-Figure S1: It needs further explanation\nThis has been addressed in the revision.\n10) Regarding the “Materials and Methods” section, I found several inconsistencies and a lack of information necessary for experimental reproducibility.\n-It is stated that reagents are provided in the supplementary tables, but this table is missing.\n-The description of plasmid construction for the expression of Flag-Ssa2, Flag-Ssa3, Flag-Ssa4, and the HA-tagged Bck1 fragments lacks necessary details.\nThese plasmids were constructed by Genscript via gene synthesis. We have now included the full sequences of all the plasmids generated in this study in the supplemental data.\n-The luciferase assay is described twice in this section, with inconsistencies between the two versions.\nThis has been resolved in the manuscript.\n•In the first description (line 579), cells were grown at 25°C, while in the second (line 648), they were grown at 30°C.\nThis has been resolved in the manuscript.\n•The first version states that incubation lasted 90 minutes with measurements taken every 5 minutes, whereas the second version states that incubation lasted 90 minutes with readings every 3 minutes.\nThis has been resolved in the manuscript.\n•Both descriptions mention incubation at 37°C, but the correct temperature is 39°C according to the graph.\nThis has been resolved in the manuscript.\n-In vitro kinase assay: The composition of the kinase buffer and loading buffer is not specified.\nThis has been resolved in the manuscript.\n-Pab1 and Edc1 foci quantification. According to the figure, cells were heat-shocked at 46°C for 15 and 30 minutes, followed by recovery at 30°C for 1 and 2 hours. However, the Materials and Methods section states that cells were incubated at 39°C for 15, 30, 60, or 120 minutes, which does not match the figure.\nThis has been resolved in the manuscript.\nReviewer #3 (Remarks to the Author)\nThis manuscript investigates the role of threonine 492 phosphorylation of yeast Hsp70 (Ssa1) during heat stress. The authors demonstrate that heat shock induces Ssa1 T492 phosphorylation, dependent on the cell wall sensor Mid2 and the Pkc1 kinase of the cell wall integrity (CWI) pathway. This phosphorylation is shown to be essential for Hsf1 release and proper activation of the heat shock response (HSR), potentially contributing to its rapid kinetics. Furthermore, T492 phosphorylation influences Ssa1’s ribosome association and translation fidelity during heat stress, and regulates P-body disassembly during recovery. Notably, the CWI pathway is not activated in T492A mutant cells during heat shock, though a direct mechanistic link remains to be established.\nThis study demonstrates a novel and extremely intriguing connection between cell-wall integrity pathway-mediated Hsp70 phosphorylation and regulation of the heat shock response. It elucidates how environmental stress signals are transmitted from cell surface sensors to the nucleus, activating the stress response, thereby contributing to our understanding of the chaperone code. Overall, much of the data are very compelling and the quality of the results is high. However, open questions remain, and some of the results require additional support, as detailed below.\n1. Please specify the heat shock duration for all the related Figures or at least mention it in the figure legends.\nThis has been resolved in the manuscript.\n2. Figures 1c, 1d, 1e and 2e indicate a low level of T492 phosphorylation without heat shock. This should be explicitly recognized in the text. The authors should also discuss potential kinases and the functional implications of this basal phosphorylation.\nThis basal level of Ssa1 phosphorylation is to be expected given that it is almost impossible to have a completely “unstressed” cell. The cell integrity pathway is always active at low levels even at 25˚C, which would explain the almost undetectable basal levels of T492 phosphorylation.\n3. In Figure 2b, residual T492 phosphorylation is observed in the absence of Mid2. To ensure reproducibility of this finding, the authors should repeat this experiment a minimum of three independent times and present the average signal, similar to that in Figures 4c and 4f, or clarify if this was indeed done. Additionally, given the potential for delayed responses, this experiment should be conducted using the 4-hour timeline employed in Figure 1c to definitively rule out any delayed phosphorylation kinetics. The authors should also discuss why in the absence of Mid2, Wsc1 could not activate Pkc1 to phosphorylate Ssa1. It is unclear what significance should be attached to the apparent Mid2 specificity, if any.\nWe have resolved this in the text.\n4. In Figure 2d, the authors should examine Pkc1 protein levels upon auxin induction at the indicated time points to strengthen the correlation between T492 phosphorylation reduction and Pkc1 levels. This could be achieved by using anti AID tag antibody or tagging Pkc1 with additional small epitope tag, such as FLAG or HA. Furthermore, if generating a pkc1 null mutant in sorbitol-containing media is feasible, utilizing this mutant directly would provide a more straightforward and definitive assessment of Pkc1's role in T492 phosphorylation.\nWe agree with the reviewer and have included a blot showing the level of Pkc1-AID (Fig. S2b). As expected, the degradation of Pkc1 tracks with loss of T492 phosphorylation.\n5. While Figure 2f demonstrates that Pkc1 can phosphorylate Ssa1 in vitro, given that T492 is within the preferred PKC substrate motif, in vivo validation of this interaction is important. To confirm this interaction within cells, a crosslinking and pulldown experiment using Pkc1 as bait to detect Ssa1 association and T492 phosphorylation should be considered. Notably, Pkc1 was absent from the Ssa1 interactors identified by mass spectrometry (MS) (Supplementary S1). This discrepancy requires discussion within the text.\nWe believe that our cellular experiments (Fig. 2d, e, f) demonstrate that Pkc1 function (but not Bck1, Mkk1/2 or Mpk1) is required for T492 phosphorylation. Given the additional evidence from the in vitro experiment we are confident that Pkc1 is the direct kinase for T492, especially given the substrate motif. It should be noted that kinase interactions are notoriously transient and that Pkc1 abundance in yeast is very low. Under these conditions, we would not expect to see Pkc1 as an interactor by MS.\n6. The authors noted that Ssa1 T492A exhibits significantly higher expression (at least five-fold) compared to Ssa1 WT (Figures 1c, 2c, 4d). In Figure 3, where MS data and changes in Ssa1 interactors are presented, it is unclear whether the levels of detected interactors were normalized to the respective levels of Ssa1 WT and Ssa1 T492A. If normalization was not performed, the observed preferential binding of interactors requires re-evaluation to account for the differential expression of Ssa1 variants.\nThis is a very important point and we thank the reviewer for bringing this up. We have over a decade of experience in characterizing the changing interactions of chaperones in response to stress and mutations1-8. In all of these studies (this one included), we normalized all the interactome data between the sets on the IP’s protein, in this case, Ssa1. We have clarified this in the text.\n7. The observed growth defect of the T492A mutant at 37 °C (Figure 4a) and the corresponding lack of Hsf1-mediated heat shock response (Figures 4b, c) are very intriguing. However, given the significantly higher expression of T492A compared to WT (at least five-fold), it is possible that the observed Hsf1 trapping is an artifact of T492A overexpression, rather than a direct effect of the T492A mutation itself. To address this, we recommend reducing T492A expression using a weaker heterologous promoter and examining whether Hsf1 still fails to be released under heat shock conditions.\nWe agree with the reviewer. We reconstructed the strains on a weaker but constitutive promoter (GPD). These new strains display the same phenotypes as before while being equalized for Ssa1 abundance (Fig.S3a-b). We repeated the Ssa1-Hsf1 IP with these new strains and observed an even clearer T492-mediated interaction change. This new data can be found as (Fig. S3e).\n8. Additionally, regarding Figure 4A, the observed temperature sensitivity at 37˚C appears to be significantly remediated by the addition of sorbitol, an osmotic stabilizer. This finding suggests that the growth defect is not due to a reduced heat shock response, as mentioned in the text, but mostly due to the clear defects in CWI signaling shown in Figure 7. This in some way alters the interpretation of the effects of T492 phosphorylation in both the results and discussion and should be clearly explained.\nThis is an excellent point and fortunately work we published over 20 years ago nicely explains this observation. Hsf1 controls the levels of several chaperones including Hsp90, a chaperone that stabilizes the Mpk1 kinase. Truncating Hsf1 (hsf1-583 mutant) in yeast results in lowered Hsp90 levels. Mpk1 is a client of the Hsp90 system and we found that in hsf1-583 Mpk1 is still dually phosphorylated but is unable to phosphorylate its downstream target Rlm1 (Truman et al. 2007). Because the terminal issue with the hsf1-583 mutant is loss of Mpk1 function, the ts nature of this mutant can be suppressed on media containing sorbitol. Our data shows that T492 phosphorylation regulates multiple components of the heat shock response cell integrity signaling, providing a novel paradigm tying these pathways together.\n9. In Figure 4d, the signal for WT Ssa1 appears relatively weak. To ensure the reliability of the observed reduction in Ssa1-Hsf1 binding upon heat shock, we recommend repeating this experiment at least two additional times and quantifying the average signal. Furthermore, overexpressing WT Ssa1 may enhance the signal and provide a more robust demonstration of the reduction in Hsf1 binding during heat shock.\nWe agree. We have the IP results and have provided new data with strains expressing Ssa1 at similar levels (see Fig. S3e).\n10. The observation of Msn2/4 activation in the T492A mutant under normal conditions (Figure 4f) suggests a complete blockage of Hsf1 activity. This raises crucial questions regarding the mechanism of T492A's effect. Specifically, it is necessary to determine whether T492A expression is upregulated by the activated Msn2/4 pathway or the T492A mutation stabilizes the Ssa1 structure, thereby enhancing its apparent Hsf1 binding affinity.\nWe agree and as mentioned above have created new strains that express Ssa1 on constitutive GPD promoters. In these strains, WT and T492A Ssa1 are expressed equally. In addition we have completed RT-PCR analyses demonstrating that the transcription of Ssa1 is upregulated in the T492A strain (Fig.3c-d). Taken together, this confirms that the difference between WT and T492A Ssa1 abundance in the original strains was caused by transcriptional changes.\n12. The observed inactivation of the CWI pathway in the T492A mutant (Figure 7a-c) is an interesting finding. However, the in vivo interaction of Ssa1 with Bck1 fragments (Figure 7h, i) raises a significant discrepancy, as Bck1 was not identified as an Ssa1 interactor in the mass spectrometry data (Supplementary table S1). The authors must address this discrepancy and clarify whether the Bck1 fragments represent the native state of Bck1 or potentially misfolded states that attract Ssa1 binding, taking into consideration the challenges inherent with working with the full-length protein. Furthermore, the phosphorylation status of Mkk1/Mkk2 in T492A cells under heat shock condition should be evaluated to further elucidate the CWI pathway inactivation.\nThe native level of Bck1 is extremely low (approx. 1000 molecules per cell compared to Ssa1) making it unlikely to be detectable by MS. We appreciate using domains of Bck1 are not ideal, but this was the only way we can detect this challenging protein. We have revised the manuscript text to discuss the challenges and pitfalls of our working model.\nMinor comments:\n13. Introduce the full terms for AZC and CPZ upon their first mention in the text.\nThis has been resolved in the manuscript.\n14. Add periods at the end of sentences on lines 99, 167, 467, and 716.\nThis has been resolved in the manuscript.\n15. Use subscript for numerical values in chemical formulas (e.g., NaN , N , CuSO , MgCl ).\n₃ ₂ ₄ ₂\nThis has been resolved in the manuscript.\n16. Common chemical names, such as 'glycerol,' should not be capitalized unless they appear at the beginning of a sentence.\nThis has been resolved in the manuscript.\n17. Line 685: Remove the extra \")\".\nThis has been resolved in the manuscript.\n18. Line 308: The title 'Hsp70 phosphorylation is critical for protein translation and ribosome association' should be revised. The polysome fractions are normal, and the data show that T492A only increases its presence at the ribosome and affects translation fidelity, while translation itself continues. Please revise the title to accurately reflect these findings.\nWe agree that the original subsection title overstated the impact on bulk protein synthesis and did not highlight the fidelity phenotype we uncovered. We have therefore replaced the title to “T492 phosphorylation fine-tunes Ssa1 ribosome engagement and translation fidelity during heat shock”.\n19. Line 403: change \"Notably, T492 occurs rapidly\" to \"Notably, T492 phosphorylation occurs rapidly.\"\nThis has been resolved in the manuscript.\n20. Line 448: replace \"Sis\" with \"Sis1.\"\nThis has been resolved in the manuscript.\n21. Line 747: revise the sentence \"Samples were diluted 6x with 50 mM tris pH 8.5 to reach <\" to ensure it is grammatically correct.\nThis has been resolved in the manuscript.\n22. Line 754: change \"~1 ug\" to \"~1 g.\"\nThis has been resolved in the manuscript.\n23. Line 756: define \"i.d..\" in the context of the PepMap C18 particles.\nDetails have been added.\n24. Line 483: change \"The yeast cell integrity pathway\" to \"The yeast cell wall integrity pathway.\"\nThis has been resolved in the manuscript.\n25. Line 860: capitalize \"V\" in \"vocalno\" to \"Volcano.\"\nThis has been resolved in the manuscript.\n26. Line 863-864: Add a verb to the sentence \"T492 phosphorylation does not the binding of intrinsically-disordered proteins.\"\nThis has been resolved in the manuscript.\n27. Add the hour unit to the time of the AZC treatment in Figure 1f.\nCompleted\n28. Revise \"Translational pausing\" to \"Translational fidelity\" in Figure 8.\nCompleted\nReviewer #5 (Remarks to the Author):\nIn this interesting multidisciplinary study, Truman and colleagues make important contributions to our understanding of how distinct features of the budding yeast heat shock response, a longstanding model for the conserved eukaryotic HSR, are integrated. Among the notable observations is that phosphorylation of Ssa1 T492 is required for full Hsf1 activation (however, see below). If confirmed, this process, operating alongside the classic chaperone titration mechanism, can provide an explanation for the rapidity of Hsf1 activation which is detected within seconds – and as pointed out by the authors, too rapid to be explained by chaperone titration mediated by unfolded nucleoplasmic/cytosolic proteins alone. Additional strengths of the study are the multiple approaches used, the very high quality of data and the thoughtful writing (although see below).\nMy enthusiasm is tempered by several concerns that I believe need to be addressed before the manuscript is suitable for publication in Nat. Commun.\n1. A key claim is that Hsf1 transcriptional activation is functionally linked to the phosphorylation status of Ssa1 T492. This is based on experiments conducted using a specialized strain in which the genes encoding all 4 cytosolic Hsp70’s – SSA1, SSA2, SSA3, SSA4 – have been deleted and WT and mutant Ssa1 are expressed from multicopy centromeric plasmids. My concern is that the authors use these strains to assay Hsf1 activity indirectly, using either a luciferase reporter (Fig. 4b) or Western blot of a representative Hsf1 target gene (Fig. 4c). As later shown (Fig. 5d, e), the T492A mutation in Ssa1 strongly inhibits global translation (although unaddressed is the possibility that translation of Hsf1 target mRNAs is spared). Thus, the assays used for Hsf1 activation are potentially confounded by suppressed translation of HSP mRNAs (including the HSE-driven mRNA encoding the luciferase reporter) elicited by the same point mutation that obviates release of Hsf1 +HS (a striking observation shown in Fig. 4d). The authors need to use a more direct assay of Hsf1 activation (such as RT-qPCR or Pol II ChIP) to substantiate this key point.\nWe understand the reviewer’s points that the altered luciferase activity seen in T492A may be a result of translational effects. We have completed the requested RT-PCR studies and these can be found in Fig. 4 and Fig. S3.\n2. As alluded above, the authors surmise that kinetics of phosphorylation of T492 are sufficiently rapid to explain the rapidity with which Hsf1 itself is activated in heat shocked yeast, a phenomenon that occurs within ~1 min (see, for example, PMID 28970326), too rapid to be explained by the classic chaperone titration model. If this is in fact the mechanism, then to prove it the authors need to fine-tune their kinetics analysis beyond what is presented in Fig 1d to include pertinent early time points.\nDespite the extreme technical challenges of this suggestion, we have now demonstrated that robust activation of this site occurs even 1 minute (!) of heat shock. This has now been added as Fig.S1.\n3. A potential limitation is that Ssa1 T492A is expressed at a substantially higher level than WT Ssa1 (this needs to be quantified) in the experiments that employ the MH272 strain, which appear to be the vast majority. At the very least, the authors need to acknowledge this potential confounding aspect of the study.\nIn this study, we used native SSA promoters on our constructs to keep the conditions as physiologically relevant as possible. As the reviewer noted, we observed a clear increase in Ssa1-T492A expression compared to WT, which we believed to be due to overcompensation by Msn2/4 signaling. To address issues potentially caused by this overexpression, we reconstructed the WT and T492A strain using plasmids with constitutive GPD promoters. In these strains, Ssa1 levels are equal, confirming that the expression change previously seen was promoter-driven. Encouragingly, the GPD-driven Ssa1 strains display the same phenotypes as SSA-driven ones (see Fig. S3b). We repeated the Hsf1-Ssa1 interaction experiment using the new strains, and we see an even clearer T492-driven Hsf1 interaction difference. This data can be found in Fig. S3e.\n4. A paradox is that interaction of Sis1 with Ssa1 is not affected by the T492A mutation, yet Sis1 is required to maintain Hsf1 in a transcriptionally inactive state (PMID 33326013). The authors need to address this.\nWe agree this is an important point, and our data resolve the apparent paradox by separating assembly of the Hsf1–Hsp70 repressor complex (which depends on Sis1) from the heat induced dissociation step (which depends on Ssa1 T492 phosphorylation and nucleotide exchange dynamics). Our epichaperome proteomics show that the T492A mutation diminishes Ydj1 binding but does not alter Sis1 association with Ssa1, indicating that Sis1-dependent recruitment/maintenance of the repressor complex remains intact in the mutant. Our Co IP experiments demonstrate that, in WT cells, heat shock triggers Ssa1–Hsf1 dissociation, whereas in T492A cells this dissociation fails—pinpointing the block at the release step rather than at Sis1 mediated assembly. Finally, overexpression of nuclear Sse1 (Sse1 NLS), which accelerates Hsp70 substrate release in the nucleus, suppresses the temperature sensitive phenotype of T492A, further supporting that T492 phosphorylation licenses efficient Hsf1 release rather than governing Sis1 dependent assembly. Overall, our results indicate that Pkc1 dependent phosphorylation of Ssa1 at T492 is required to trigger rapid release of Hsf1 on heat shock, a step that is mechanistically distinct from Sis1’s role. Consistent with this view, we note in the Discussion that T492 dependent remodeling preserves Sis1 interactions while altering others (e.g., Ydj1), reinforcing that the phosphorylation event fine tunes co chaperone usage without eliminating Sis1 function.\nMinor points\n1. The key study identifying Hsp70 as the principal chaperone that represses yeast Hsf1 is ref. 9. This, and a companion study (PMID 29393852), should be clearly acknowledged throughout the ms. (e.g., on lines 68, 279, 392). Likewise, the initial study identifying Hsp70 as a principal repressor of mammalian HSF1 is ref. 59. It too needs to be more clearly acknowledged.\nWe have added the appropriate references.\n2. Misinterpretation of data: lines 157-158 (“not detected” is inconsistent with data presented in Figs. 1c, d, e; 2e).\nWe agree and have edited the manuscript accordingly.\n3. The authors are inconsistent in their reference to the role played by Msn2 in the yeast transcriptional response to stress. In places, they lump Msn2 with Hsf1 as intrinsic to the HSR; in other places they characterize Msn2 as driving a separate environmental stress response. Just be consistent.\nWe have adjusted the text to state that activation of Hsf1 and Msn2/4 are overlapping but distinct parts of the heat shock response.\n4. Writing is for the most part clear; however, there are multiple places where either punctuation or words are missing (and/or word choice is poor/misleading). For example: lines 99, 105, 141, 143, 154, 179, 190, 258, 382, 403, 471, 504, 864, 870, 877, 879 (antagonizes not “mediates”), 920, 949-950.\nReviewer #6 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.\nThank you for taking time to review this manuscript, your thoughtful comments have helped us to improve the study.\nReferences\n1. Omkar, S. et al. Acetylation of the yeast Hsp40 chaperone protein Ydj1 fine-tunes proteostasis and translational fidelity. PLoS Genet.20, e1011338 (2024).\n2. Knighton, L. E., Nitika, Wolfgeher, D., Reitzel, A. M. & Truman, A. W. Dataset of Nematostella vectensis Hsp70 isoform interactomes upon heat shock. Data Brief27, 104580 (2019).\n3. Xu, L. et al. Rapid deacetylation of yeast Hsp70 mediates the cellular response to heat stress. Sci. Rep.9, 16260 (2019).\n4. Truman, A. W. et al. Quantitative proteomics of the yeast Hsp70/Hsp90 interactomes during DNA damage reveal chaperone-dependent regulation of ribonucleotide reductase. J. Proteomics 112, 285–300 (2015).\n5. Truman, A. W. et al. CDK-dependent Hsp70 Phosphorylation controls G1 cyclin abundance and cell-cycle progression. Cell151, 1308–1318 (2012).\n6. Dunn, D. M. et al. C-Abl mediated tyrosine phosphorylation of Aha1 activates its co-chaperone function in cancer cells. Cell Rep.12, 1006–1018 (2015).\n7. Woodford, M. R. et al. Mps1 mediated phosphorylation of Hsp90 confers renal cell carcinoma sensitivity and selectivity to Hsp90 inhibitors. Cell Rep.14, 872–884 (2016)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Although the authors addressed many of the points raised by this reviewer, there are still some of them that were not sufficiently or adequately approached:\nPoint 4: Regarding the mechanism by which phosphorylation of Hsp70 by Pkc1 would trigger Hsf1-Hsp70 dissociation, and in response to my criticism about the absence of differences in the amount of Ssa1 or Ssa1- T492A co-immunoprecipitated with Hsf1, this could be due to the high amount of Ssa1 T492A present and not to differences in the affinity between Hsf1 and Ssa1. The authors used WT and T492A strains with plasmids under constitutive GPD promoters. It is true that, as shown from the input, in these strains, Ssa1 levels are equal. The authors claim in the rebuttal letter, “We repeated the Hsf1-Ssa1 interaction experiment using the new strains, and we see an even clearer T492-driven Hsf1 interaction difference. This data can be found as Fig. S3e”. However, the data in Fig. S3e show: 1) If the amounts of immunoprecipitates loaded are equal in the WT and T492A strains, the amount of Ssa1 co-immunoprecipitated with Hsf1 is greater in the T492A than in the WT; and 2) Although a significant ammount of SSa1 remains bound to Hsf1 in the T492A strain at 39ºC, there is a decrease in the amount of Ssa1 co-immunoprecipitated with Hsf1 at this temperatura. Therefore, phosphorylation of T492 does not fully account for the dissociation. This needs to be explained and nuanced .\nPoint 8: Regarding the relationship between heat-induced phosphorylation of Hsp70 and the CWI pathway, I still believe that the conclusion about the role of Bck1 in the requirement for 408 Ssa1 T492 phosphorylation in promoting CWI signaling is overstated based on the results shown. The experiments should be conducted with the full-length Bck1. Surprisingly, the authors insist in the new version of the manuscript that no one has been able to express full-lenght Bck1, which is not true. In the rebuttal letter, they explain that they tried again to express the full-length Bck1 fused to Myc without success, and that Hruby et al experienced the same issue and also had to break Bck1 into two fragments. Nothing could be further from the truth. In the work by Hruby et al. (2011), the authors constructed several versions of the protein to analyze the role of the N-terminal regulatory domain, the C-terminal kinase domain, and residues 802-824 in various interactions, but they also expressed and analyzed a full-length C-terminally Myc-tagged version of Bck1-Myc (Supplementary Figure S1). Furthermore, they show that cooperation between the regulatory domain and the kinase region works together to achieve strong binding to Mkk1-Slt2, supporting the idea that full-length Bck1 is necessary to support these conclusions. This confirms the possibility of conducting these experiments. If the authors can not express full-length Bck1, they can request the strains that have already been published.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have done a solid job addressing the many points raised by all reviewers. Concerns expressed by the majority of the reviewers were remarkably similar and consistent and the responses, both written and experimental, satisfyingly addressed the major issues. Importantly, several claims were toned down to more appropriately reflect the data. Overall, I am satisfied with the resubmitted version of the manuscript and feel that it will be an important addition to the stress, signaling literature.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "The manuscript has been strengthened by inclusion of early time points in the Ssa1 phosphorylation analysis (lovely western blot in new Fig. S1) that provides compelling support for the authors’ argument that Hsf1 activation is too rapid to be explained by chaperone titration alone. Also strengthening the paper are experiments that express Ssa1(WT) and Ssa1(T492A) under control of the GPD promoter that result in equivalent Ssa1 expression levels (Fig. S3a,b,c,e), and inclusion of the RNA abundance measurement in Fig. 4c (note that for clarity, this graph should be relabeled as “Luciferase mRNA levels”).\nThe above notwithstanding, the new RNA measurements in Fig. S3d raise concern. It is very odd that the SSA1 RNA level is not increased in WT cells exposed to a 30 min 39°C heat shock given that multiple studies have observed a HS-induced increase in SSA1 RNA (e.g., nascent SSA1 RNA levels are induced ~20-fold in cells shifted from 30° to 39°C during the first 5 min of heat shock (PMID 30332327; Suppl. Table 1)). Equally puzzling is the >10-fold higher SSA1 mRNA levels in the mutant compared to the WT; these too show no increase following the 30 min HS. It is possible that this experiment was confounded by the fact that the SSA1(WT) and SSA1(T492A) genes were borne on multicopy CEN plasmids. (Were the WT and T492A genes regulated by the WT SSA1 promoter and if so, how much upstream region was present? The designation “SSA” is unclear.) The authors’ explanation that the enhanced SSA1 RNA expression reflects compensatory activation of Msn2/Msn4 (lines 322-329; 442-443) is inconsistent with experiments that show SSA1 is only weakly transcribed (5% of WT levels) in heat shocked cells in which nuclear Hsf1 has been depleted using anchor away (PMID 30332327; Suppl. Table 1). While measurement of luciferase mRNA levels arising from the integrated HSE-Luciferase reporter gene (Fig. 4c) are consistent with Hsf1 activity being inhibited in cells expressing Hsp70(T492A), RNA measurements of Hsf1-dependent genes located at their native chromosomal locus would be extremely helpful to clarify this important point (namely, that Hsf1 bound by Ssa1(T492A) is incapable of driving transcriptional activation of its natural targets). Candidates for such an analysis include SSA2, HSC82, HSP82 and BTN2 as these genes are strongly Hsf1-dependent with little or no Msn2/4 contribution (Solis et al, Mol. Cell 2016; Pincus et al, MBoC 2018). Clinching the idea would be an Hsf1 ChIP analysis that demonstrates equal occupancy of Hsf1 at one or more of these genes in WT and Ssa1(T492A) cells following an acute HS.\nAdditional points:\n1. Was NHS temperature in the experiments described above set at 25°C? This important detail was never made clear in any of the figures. It needs to be.\n2. Minor\na. Lines 1144-1145. Solis et al is incorrectly cited as Mol. Cell 2018. The year, volume and page numbers are incorrect.\nb. Details of the RT-qPCR assay are more appropriate for the M&Ms, not figure legends.\nc. Line 201: “arginine at the -3 position” is inconsistent with the sequences presented in Fig. 1b.\nd. Inaccurate wording, typos, punctuation issues still exist throughout the ms. For example: lines 130, 142, 190, 310, 905, 913, 915, 920.", "reviewer_6": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "Second Response to Reviewers\nWe offer below our point-by-point responses to reviewer queries and comments on our submitted manuscript titled, “Mechanosensor-mediated Hsp70 phosphorylation orchestrates the landscape of the heat shock response”. We would like to thank the reviewers for your constructive comments and positive feedback. We believe that this newly revised manuscript and additional clarifying data provide an improved and compelling story for publication in Nature Communications.\nResponse to Reviewer #1\n1) Although a significant amount of Ssa1 remains bound to Hsf1 in the T492A strain at 39ºC, there is a decrease in the amount of Ssa1 co-immunoprecipitated with Hsf1 at this temperature. Therefore, phosphorylation of T492 does not fully account for the dissociation. This needs to be explained and nuanced .\nWe agree and have edited the discussion appropriately.\n2) The experiments should be conducted with the full-length Bck1.\nWe agree and have now included a blot of full-length Bck1 levels in WT and T492A cells. In agreement with our previous data, levels of Bck1 are decreased in T492A cells (see Fig. 6g).\nResponse to Reviewer #3\n1) The authors have done a solid job addressing the many points raised by all reviewers. Concerns expressed by the majority of the reviewers were remarkably similar and consistent and the responses, both written and experimental, satisfyingly addressed the major issues. Importantly, several claims were toned down to more appropriately reflect the data. Overall, I am satisfied with the resubmitted version of the manuscript and feel that it will be an important addition to the stress, signaling literature.\nWe thank the reviewer for their kind comments.\nResponse to Reviewer #5\n1) The manuscript has been strengthened by inclusion of early time points in the Ssa1 phosphorylation analysis (lovely western blot in new Fig. S1) that provides compelling support for the authors’ argument that Hsf1 activation is too rapid to be explained by chaperone titration alone. Also strengthening the paper are experiments that express Ssa1(WT) and Ssa1(T492A) under control of the GPD promoter that result in equivalent Ssa1 expression levels (Fig. S3a,b,c,e), and inclusion of the RNA abundance measurement in Fig. 4c (note that for clarity, this graph should be relabeled as “Luciferase mRNA levels”).\nWe thank the reviewer for their kind comments. We have made the requested edits.\n2) It is very odd that the SSA1 RNA level is not increased in WT cells exposed to a 30 min 39°C heat shock given that multiple studies have observed a HS-induced increase in SSA1 RNA (e.g., nascent SSA1 RNA levels are induced ~20-fold in cells shifted from 30° to 39°C during the first 5 min of heat shock (PMID 30332327; Suppl. Table 1)). Equally puzzling is the >10-fold higher SSA1 mRNA levels in the mutant compared to the WT; these too show no increase following the 30 min HS. It is possible that this experiment was confounded by the fact that the SSA1(WT) and SSA1(T492A) genes were borne on multicopy CEN plasmids. (Were the WT and T492A genes regulated by the WT SSA1 promoter and if so, how much upstream region was present? The designation “SSA” is unclear.)\nRegarding the promoter, -554 to -1bp upstream of the gene was used and the full sequence of the plasmids are provided in the supplemental table. There are few factors that may account for the differences in the mentioned paper and our results. The strain backgrounds are very different-for example, our strain lacks Ssa2-4 and as the reviewer highlights the genes were borne on CEN plasmids. In addition, Our qPCR captures steady-state RNA at 30 min (not nascent RNA at 5 min) and therefore is not directly comparable to the early nascent-RNA measurements cited by the reviewer. It should be noted that our data are also consistent with our Western Blots and previously published literature where heat-induced changes in Ssa1 expression are not observed.\n3) The authors’ explanation that the enhanced SSA1 RNA expression reflects compensatory activation of Msn2/Msn4 (lines 322-329; 442-443) is inconsistent with experiments that show SSA1 is only weakly transcribed (5% of WT levels) in heat shocked cells in which nuclear Hsf1 has been depleted using anchor away (PMID 30332327; Suppl. Table 1).\nWhen both alleles are expressed from the constitutive GPD promoter, SSA1 transcript levels are equal in WT and T492A (no heat-induction expected), showing that the large difference arises upstream of coding sequence at the native SSA promoter and/or post-transcriptionally (Fig. S3c). The anchor away data the reviewer cites is hard to directly compare to ours because of the differences in the strains. In the anchor-away system, the addition of rapamycin triggers the export of Hsf1 from the nucleus. In our mutant T492A becomes locked onto Hsf1. This difference may be the source of altered SSA1 transcription.\n4) RNA measurements of Hsf1-dependent genes located at their native chromosomal locus would be extremely helpful to clarify this important point (namely, that Hsf1 bound by Ssa1(T492A) is incapable of driving transcriptional activation of its natural targets). Clinching the idea would be an Hsf1 ChIP analysis that demonstrates equal occupancy of Hsf1 at one or more of these genes in WT and Ssa1(T492A) cells following an acute HS.\nWe thank the reviewer for this suggestion. We have included additional RNA measurements of BTN2 and HSP104 in WT and T492A cells (Fig. 4c). In agreement with our previous data (Fig. 4c), expression of these Hsf-mediated genes are decreased in the T492A mutant. Due to constraints on time, expertise and resources, we are unable to carry out the suggested CHIP experiment. However, we are confident that our new and previously submitted data clearly demonstrate that the T492A mutant displays a defect in Hsf1 activity.\n5) Was NHS temperature in the experiments described above set at 25°C? This important detail was never made clear in any of the figures.\nWe apologize for this omission, yes, NHS was 25°C. We have updated the methods section to clarify this.\n6) Minor a. Lines 1144-1145. Solis et al is incorrectly cited as Mol. Cell 2018. The year, volume and page numbers are incorrect.\nWe have fixed this error.\n7) Details of the RT-qPCR assay are more appropriate for the M&Ms, not figure legends.\nWe agree and made appropriate changes to the figure legend.\n8) Line 201: “arginine at the -3 position” is inconsistent with the sequences presented in Fig. 1b.\nOur apologies for this error. The PKC phosphorylation substrate motif is typically (R/K)XpS/TX(R/K), where the basic amino acids (Arg or Lys) are at positions -2 and +2 relative to the phosphorylated residue. We have revised the text to reflect this with appropriate references.\n9) Inaccurate wording, typos, punctuation issues still exist throughout the ms. For example: lines 130, 142, 190, 310, 905, 913, 915, 920.\nWe have fixed the wording, typos, punctuation issues throughout the manuscript."}]

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