Transcriptional regulation of Sis1 promotes fitness but not feedback in the heat shock response

  1. Rania Garde
  2. Abhyudai Singh
  3. Asif Ali
  4. David Pincus

  • Curated by eLife

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    Evaluation Summary:

    In this paper the authors report an updated theoretical model describing in mathematical terms how the Hsf1 transcription factor is activated in yeast in response to heat shock, and demonstrate that rather than denatured mature proteins, Hsf1 activation involves newly synthesized proteins that sequester the Hsp70 chaperone away from the inactive Hsp70/Hsf1 complex, releasing active Hsf1. They also describe a general role for the Sis1 co-chaperone in maintaining the fitness of yeast cells under stress conditions, such as heat shock, that is independent of regulation of Hsf1.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

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Abstract

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

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  1. Version published to 10.7554/elife.79444 on eLife
  2. eLife

    Author Response

    Reviewer #1 (Public Review):

    This refinement of their model, coupled with the demonstration that the Sis1 J protein chaperone does not appear to play a direct role in the inactivation phase of the HSR, provide a significant advance over their earlier work.

    We are pleased that the reviewer is satisfied that our new results represent a significant advance.

    A main weakness is that while the evidence that Sis1 is important for fitness of heat-stressed yeast cells is reasonable, exactly how Sis1 achieves this is not clear. In a single sentence the authors suggest that Sis1 might be an orphan ribosome chaperone, partly based on its nucleolar localization, but provide no evidence for this. If this were true, then one might expect a reduction in ribosome content under stress conditions (because there are more ORPS to take care of because of translation stalling?) and a decreased rate of protein synthesis (yes, this happens, how much this is due to overall translation suppression vs there being less ribosomes to translation things, is unknown and hard to test), which could be tested. Some further insights into this more general role of Sis1 would strengthen the authors' conclusions.

    We would like to make a distinction between the important biochemical roles for Sis1 in the cellular response to heat shock – which we explore elsewhere – and the role we are investigating here for the regulation of Sis1 expression by Hsf1. For new insights into the functional role of Sis1 as a chaperone for orphan ribosomal proteins, please see our recent preprint (Ali et al., https://www.biorxiv.org/content/10.1101/2022.11.09.515856v1). Here, we have focused on how Sis1 transcriptional regulation promotes fitness. Please see above for the description of the new mechanistic insight we have into the role of Sis1 expression tuning in controlling stress granules.

    Moreover, whether Sis1 plays a general role in the fitness of cells under stress has not been firmly established, i.e., is its mechanistic role the same in heat shock conditions and under nutrient stress conditions? Without knowing the mechanistic basis for how Sis1 maintains the fitness of heat-stressed cells, it is not possible to conclude that the same mechanism is at play in cells grown on a non-preferred carbon source.

    As described above, we have now provided evidence that the inability to properly tune Sis1 expression levels in the 2xSUP35-SIS1 strain results in disrupted stress granule homeostasis, linking a known function of Sis1 to a known process driven by nutrient stress.

    Figure 4: This is an ingenious experiment to study the subcellular localization of newly synthesized Sis1 in response to heat shock, compared to that of the heat-shock inducible Hsp70 Ssa1. However, based on the images presented in panel B it is hard to know how discrete the subnuclear distributions of Sis1 and Ssa1 really are, and ideally what is needed is to be able to analyze their localizations when both tagged proteins are expressed in the same cell, although this would obviously not be possible using the halo-tagged protein system. In addition, one would like to know the localization of Hsf1 in the cell at the same time. As it stands, these data seem overinterpreted, and it remains possible that some other event such as an inactivating post-translational modification of Sis1 under heat shock conditions might be involved in inactivating its function.

    To address this concern, we constructed two new imaging strains expressing Hsf1-mVenus/Halo-Sis1 and Hsf1-mVenus/Halo-Ssa1 (Hsp70) and used pulse-labeling followed by live lattice light sheet 3D imaging to resolve the subcellar localization of newly synthesized Sis1 and Hsp70 with respect to Hsf1 over a heat shock time course. Unfortunately, we cannot monitor newly induced Sis1 and newly induced Hsp70 simultaneously in the same cells with the HaloTag pulse labeling system. We found that a significantly greater fraction of newly synthesized Hsp70 colocalizes with Hsf1 than new Sis1. Thus, while we cannot directly image new Sis1 and Hsp70 in the same cell, we clearly observe a differential localization pattern with respect to Hsf1. These data are included in the revised Figure 4.

    One way to establish whether Sis1 nucleolar sequestration prevents it from acting on Hsf1 during the inactivation phase of the HSR would be to selectively disrupt its nucleolar localization signal eliminated while retaining its nuclear localization and determine how expression of such a mutant perturbed the inactivation kinetics of the HSR.

    Unfortunately, there is no known Sis1 nucleolar localization signal that we could use in the experiment you propose. In the preprint described above, we show that direct interactions with oRPs recruit Sis1 to the nucleolar periphery, but we do not yet know binding to oRPs is competitive with binding to Hsf1.

    Reviewer #2 (Public Review):

    This study aims to provide a needed update and validation of a previously outlined mathematical model that describes HSR/Hsf1 regulation. The purpose of the update is to incorporate the impact of newly translated proteins as negative regulators of Hsf1 following heat shock. A requirement for ongoing translation to mount the HSR and activate Hsf1 has been described in several recent studies. Moreover, the study addresses the role of the Hsp70 cochaperone Sis1 in HSR regulation, including its potential function in negative feedback regulation following heat-shock.

    The main strength of the study is that it combines quantitative modeling with a well-defined experimental system to generate data. Overall, the model appears to accurately reflect the behavior of HSR under the employed experimental conditions and provides and elegant example of a formalized model for this simple regulatory circuit. Another strength of the study is that it addresses the functional involvement of Sis1 in HSR/Hsf1 regulatory mechanisms and rules out Sis1 involvement in negative feedback regulation of Hsf1 following heat shock. This finding is of importance in light of the complexity of Sis1 involvement in HSR/Hsf1 regulation suggested by the literature. The authors also document a need for endogenous SIS1 promoter regulation during growth on non-fermentable carbon sources.

    The study is important for the advancement of Hsf1 research and it may provide inspiration for the study of other chaperone-titrated transcriptional mechanisms such as the UPR or bacterial stress sigma factors.

    We thank the reviewer for the generous evaluation.

    Reviewer #3 (Public Review):

    This paper follows other excellent work from the Pincus laboratory detailing the molecular mechanisms of Hsf1 regulation and extending experimental observations into predictive mathematical models. Overall, the work is top-quality, however, the findings are incremental in nature with respect to our understanding of the HSR and refine existing models rather than break new experimental or conceptual ground. Additionally, the relevance of the non-fermentable carbon source growth phenotype for the 2XSUP35pr-SIS1 strain is unclear with respect to HSR regulation.

    We thank the reviewer for this fair assessment of the work.

  3. eLife

    Evaluation Summary:

    In this paper the authors report an updated theoretical model describing in mathematical terms how the Hsf1 transcription factor is activated in yeast in response to heat shock, and demonstrate that rather than denatured mature proteins, Hsf1 activation involves newly synthesized proteins that sequester the Hsp70 chaperone away from the inactive Hsp70/Hsf1 complex, releasing active Hsf1. They also describe a general role for the Sis1 co-chaperone in maintaining the fitness of yeast cells under stress conditions, such as heat shock, that is independent of regulation of Hsf1.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)

  4. eLife

    Reviewer #1 (Public Review):

    In this Research Advance, the authors build on two earlier eLife papers that described and experimentally validated a mathematical model of the transcriptional response of yeast to heat shock in which unfolded proteins sequester Hsp70 away from Hsf1 promoting an Hsf1-driven transcriptional program, and report two new findings. First, they provide evidence that upon heat shock it is newly synthesized proteins rather than denatured mature proteins that sequester the Hsp70 chaperone away from Hsf1 permitting Hsf1 to bind to target genes and drive the heat shock-induced gene transcription program during the heat shock response (HSR), and, second, by analyzing the role of the Sis1 Hsp70 co-chaperone in the HSR they showed that Sis1 does not have a direct negative role in the HSR, but rather is needed for fitness during prolonged stress.

    Because recent studies using cycloheximide to block protein synthesis have suggested that it is newly synthesized proteins in the process of folding rather than denatured mature proteins that are the clients for Hsp70 responsible the HSR, the authors reconfigured their model by assuming that heat shock slows the folding of newly synthesized proteins and adding the rate of translation as a new input function. They validated their new model using a yeast strain that has an HSE-YFP reporter gene as an HSR readout, and showed that rapamycin treatment, which reduces the rate of translation, resulted in a decrease in the HSR, that is predicted with kinetics predicted by their new model. In addition, based on their own recent work showing that the Sis1, a J-protein chaperone, regulates the HSR by promoting Hsf1-Hsp70 association in the nucleus to repress Hsf1 activity under non-heat shock conditions, they also incorporated Sis1, a Hsp70 co-chaperone, as a new component of their model circuitry. By experimentally induced eviction of Sis1 from the nucleus, they observed reduced Hsf1 activity towards the HSE-YFP reporter in the absence of a temperature shift, as predicted by the model. The new model also accounted for the rapid initial and then subsequent slowing kinetics of the HSR as it reached a maximum, as well as the different levels of HSR induction at increasing temperatures above 35oC. Moreover, even though the SIS1 promoter has an HSE and its basal transcription is driven by Hsf1, the elimination of this regulatory step experimentally showed that Hsf1-driven Sis1 transcription was not required for temperature shift-induced HSR output, implying, as the model predicted, that increased Sis1 expression is not important and not needed for negative feedback inactivation of Hsf1. This was tested directly by generating a strain in which the SIS1 promoter was replaced with two copies of the SUP35 promoter to maintain the basal expression level of Sis1, which showed normal kinetics of HSR inactivation under several experimental conditions. Using a Halo-tag pulse protocol, they demonstrated that heat shock induction of newly synthesized Sis1-halo was delayed and that the new Sis1 protein was preferentially localized around the nucleolus away from Hsf1, as determined using an Nsr1-mScarlet nucleolar marker, and thus Sis1 would presumably not be in a position to promote Hsp70/Hsf1 interaction and repression of Hsf1 activity. Finally. to investigate what role Sis1 plays in heat-stressed cells, they showed that the 2xSUP35pr-SIS1 yeast strain had reduced fitness compared to the other strains after 4 hours at 37ºC, suggesting that Sis1 has an undefined role in maintaining fitness in heat-stressed cells. Consistent with this, they showed that Sis1 also has a role in maintaining fitness in yeast cells growing on a non-preferred carbon source.

    The updated model of the HSR, which still retains the two-component feedback loop consisting of the chaperone Hsp70 and the transcription factor Hsf1 of the original model but replaces the unfolded protein activation step with an equivalent step involving unfolded newly synthesized proteins, appears to be able to model cellar responses to heat shock quite accurately. This refinement of their model, coupled with the demonstration that the Sis1 J protein chaperone does not appear to play a direct role in the inactivation phase of the HSR, provide a significant advance over their earlier work.

    A main weakness is that while the evidence that Sis1 is important for fitness of heat-stressed yeast cells is reasonable, exactly how Sis1 achieves this is not clear. In a single sentence the authors suggest that Sis1 might be an orphan ribosome chaperone, partly based on its nucleolar localization, but provide no evidence for this. If this were true, then one might expect a reduction in ribosome content under stress conditions and a decreased rate of protein synthesis, which could be tested. Some further insights into this more general role of Sis1 would strengthen the authors' conclusions.

    Moreover, whether Sis1 plays a general role in the fitness of cells under stress has not been firmly established, i.e., is its mechanistic role the same in heat shock conditions and under nutrient stress conditions? Without knowing the mechanistic basis for how Sis1 maintains the fitness of heat-stressed cells, it is not possible to conclude that the same mechanism is at play in cells grown on a non-preferred carbon source.

    Figure 4: This is an ingenious experiment to study the subcellular localization of newly synthesized Sis1 in response to heat shock, compared to that of the heat-shock inducible Hsp70 Ssa1. However, based on the images presented in panel B it is hard to know how discrete the subnuclear distributions of Sis1 and Ssa1 really are, and ideally what is needed is to be able to analyze their localizations when both tagged proteins are expressed in the same cell, although this would obviously not be possible using the halo-tagged protein system. In addition, one would like to know the localization of Hsf1 in the cell at the same time. As it stands, these data seem overinterpreted, and it remains possible that dome other event such as an inactivating post-translational modification of Sis1 under heat shock conditions might be involved in inactivating its function.

    One way to establish whether Sis1 nucleolar sequestration prevents it from acting on Hsf1 during the inactivation phase of the HSR would be to selectively disrupt its nucleolar localization signal eliminated while retaining its nuclear localization and determine how expression of such a mutant perturbed the inactivation kinetics of the HSR.

  5. eLife

    Reviewer #2 (Public Review):

    This study aims to provide a needed update and validation of a previously outlined mathematical model that describes HSR/Hsf1 regulation. The purpose of the update is to incorporate the impact of newly translated proteins as negative regulators of Hsf1 following heat shock. A requirement for ongoing translation to mount the HSR and activate Hsf1 has been described in several recent studies. Moreover, the study addresses the role of the Hsp70 cochaperone Sis1 in HSR regulation, including its potential function in negative feedback regulation following heat-shock.

    The main strength of the study is that it combines quantitative modeling with a well-defined experimental system to generate data. Overall, the model appears to accurately reflect the behavior of HSR under the employed experimental conditions and provides and elegant example of a formalized model for this simple regulatory circuit. Another strength of the study is that it addresses the functional involvement of Sis1 in HSR/Hsf1 regulatory mechanisms and rules out Sis1 involvement in negative feedback regulation of Hsf1 following heat shock. This finding is of importance in light of the complexity of Sis1 involvement in HSR/Hsf1 regulation suggested by the literature. The authors also document a need for endogenous SIS1 promoter regulation during growth on non-fermentable carbon sources.

    The study is important for the advancement of Hsf1 research and it may provide inspiration for the study of other chaperone-titrated transcriptional mechanisms such as the UPR or bacterial stress sigma factors.

  6. eLife

    Reviewer #3 (Public Review):

    This paper follows other excellent work from the Pincus laboratory detailing the molecular mechanisms of Hsf1 regulation and extending experimental observations into predictive mathematical models. Overall, the work is top-quality, however, the findings are incremental in nature with respect to our understanding of the HSR and refine existing models rather than break new experimental or conceptual ground. Additionally, the relevance of the non-fermentable carbon source growth phenotype for the 2XSUP35pr-SIS1 strain is unclear with respect to HSR regulation.

  7. Version published to 10.1101/2022.04.27.489698v1 on bioRxiv