Heat Shock: When pH comes to the rescue

In starving yeast exposed to thermal stress, a transient drop in intracellular pH helps to trigger the heat shock response.
  1. Davi Gonçalves
  2. Alec Santiago
  3. Kevin A Morano  Is a corresponding author
  1. Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, United States
  2. MD Anderson UTHealth Graduate School of Biomedical Sciences, University of Texas, United States

Who has never had a stressful day at work? In crisis mode, we typically ensure professional survival by dropping everything and redirecting all our resources to the most important tasks, even enlisting specialized support staff to get the job done. From yeast to humans, most eukaryotic cells adopt the same strategy. When exposed to physiological stressors that may prevent their proteins from folding correctly, they call on molecular chaperones that can recognize damaged or misfolded proteins and assist in their removal or repair. In particular, events such as extreme heat, starvation or toxic substances can switch on Hsf1, the transcription factor which controls the genetic program that coordinates the creation of chaperones (Verghese et al., 2012).

This program, called the heat shock response, is shut down in the absence of stress. But how do cells then ‘know’ when to induce it? Three decades ago Betty Craig and Carol Gross of the University of Wisconsin-Madison proposed that, directly or indirectly, the trigger would involve misfolded proteins interacting with chaperones, in particular one known as Hsp70 (Craig and Gross, 1991). Recent work has largely validated this hypothesis: Hsp70 binds to Hsf1 to block its activity, until the concentration of misfolded proteins rises so much that they pull Hsp70 away from Hsf1 (Zheng et al., 2016; Krakowiak et al., 2018; Peffer et al., 2019). In this process, juvenile proteins that are just being translated act as the main Hsp70 trigger, as they are exquisitely sensitive to environmental changes and tend to misfold easily (Masser et al., 2019). Yet, starving cells – in which translation is strongly reduced – can still mount a modest heat shock response, suggesting that this neat and tidy model is actually incomplete. Now, in eLife, Allan Drummond and colleagues at the University of Chicago – including Catherine Triandafillou as first author – report a new, translation-independent pathway that triggers the heat shock response in the yeast species Saccharomyces cerevisiae (Triandafillou et al., 2020).

This pathway relies on the fact that elevated temperatures lead to a temporary drop in the pH of cells (Weitzel et al., 1985). To investigate how acidification is linked to the heat shock response, the team developed sophisticated and sensitive methods for monitoring ‘live’ both the pH and products of the heat shock response inside individual yeast cells. This was achieved by harnessing fluorescence readouts reported by flow cytometry (Franzmann and Alberti, 2019). In addition, the pH of the cells was ‘clamped’ at specific values.

Together, these approaches confirmed that when yeast cells are suddenly exposed to a temperature of 42 °C, their internal pH drops (from approximately 7.5 to 6.8) and their heat shock response is switched on. Under normal nutrient conditions, preventing acidification had little effect on the heat shock response. However, doing so when yeast lacked glucose all but abolished the response, and the same effect was observed in cells in which translation was blocked. Crucially, RNA sequencing experiments showed that a lack of acidification only stopped the heat shock response, in particular shutting down genes controlled by Hsf1; global transcription was not affected, and neither was a parallel stress response pathway governed by the Msn2/4 transcription factors (Verghese et al., 2012). Cellular acidification did not need to take place at the same time as the thermal stress, as a post-stress pH reduction rescued the potency of the heat shock response. In addition, pH levels needed to return to their pre-stress levels for the heat shock response to be optimal, suggesting that long-term acidification may be detrimental. Finally, the team explored whether acidification was required for cell survival. Cells that could not adjust their pH during heat shock survived, but competitive growth assays showed that they entered the cell cycle more slowly, and that they were ultimately out-competed by cells that could acidify.

The work by Triandafillou et al. uncovers a mechanism that allows cells which are not actively translating to respond to thermal stress, and to persist in a population (Figure 1). While the combination of heat shock and starvation is rare in the laboratory, it is likely common for wild yeast. These organisms lie mostly dormant and starving on the surface of fruit through the day, while they are exposed to extreme swings in ambient temperature. This ‘secondary’ way to induce the heat shock response may also help yeast face the heat and starvation they encounter when accidentally ingested by fruit-eating birds. This would enable the cells to spread to new geographic areas, providing yet another means of evolutionary competitiveness for the species.

Two parallel pathways can induce the heat shock response in yeast.

The heat shock response is controlled by the heat shock transcription factor Hsf1; it is repressed (HSRoff) when conditions are stable, but rapidly induced (HSRon) by a high temperature. In ‘nutrient-rich’ cells (top), translation is robust and thermal stress (flame) causes a subset of nascent polypeptides to misfold and aggregate, ultimately activating Hsf1 and the heat shock response. When cells are starving (and have therefore stopped translation), the heat shock response is still induced; the trigger presumably involves mature, folded proteins assembling into phase-separated structures that can recruit chaperones. Thermal stress leads to a drop in pH (blue arrow) in all cells, but only starving cells require acidification to trigger the heat shock response.

Ironically, this discovery brings the field back full circle, to the same type of question posed over 30 years ago: what triggers this translation-independent pathway? The answer may lie in the phenomenon of phase separation, in which compartments that are not enclosed within a membrane can form inside cells to host specific biological processes. Recent work has shown that many cytoplasmic proteins can undergo phase separation to form transient assemblies that are different from the aggregates normally created by misfolding proteins (Wallace et al., 2015; Riback et al., 2017; Franzmann and Alberti, 2019). In particular, temperature and pH can control the formation of these structures. It is thus tempting to speculate that one or more such proteins, or perhaps the assemblies themselves, are recognized by Hsp70 to trigger the heat shock response. These substrates now await identification.

References

Article and author information

Author details

  1. Davi Gonçalves

    Davi Gonçalves is in the Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, United States

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-4646-5706
  2. Alec Santiago

    Alec Santiago is in the Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, and the MD Anderson UTHealth Graduate School of Biomedical Sciences, University of Texas, Houston, United States

    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-7794-477X
  3. Kevin A Morano

    Kevin A Morano is in the Department of Microbiology and Molecular Genetics, McGovern Medical School at UTHealth, Houston, United States

    For correspondence
    Kevin.A.Morano@uth.tmc.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-5992-0253

Publication history

  1. Version of Record published: September 11, 2020 (version 1)

Copyright

© 2020, Gonçalves et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

Metrics

  • 1,807
    Page views
  • 147
    Downloads
  • 1
    Citations

Article citation count generated by polling the highest count across the following sources: Crossref, PubMed Central, Scopus.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Davi Gonçalves
  2. Alec Santiago
  3. Kevin A Morano
(2020)
Heat Shock: When pH comes to the rescue
eLife 9:e62022.
https://doi.org/10.7554/eLife.62022
  1. Further reading

Further reading

    1. Biochemistry and Chemical Biology
    2. Cell Biology
    Kristian Davidsen, Jonathan S Marvin ... Lucas B Sullivan
    Research Article

    Intracellular levels of the amino acid aspartate are responsive to changes in metabolism in mammalian cells and can correspondingly alter cell function, highlighting the need for robust tools to measure aspartate abundance. However, comprehensive understanding of aspartate metabolism has been limited by the throughput, cost, and static nature of the mass spectrometry (MS)-based measurements that are typically employed to measure aspartate levels. To address these issues, we have developed a green fluorescent protein (GFP)-based sensor of aspartate (jAspSnFR3), where the fluorescence intensity corresponds to aspartate concentration. As a purified protein, the sensor has a 20-fold increase in fluorescence upon aspartate saturation, with dose-dependent fluorescence changes covering a physiologically relevant aspartate concentration range and no significant off target binding. Expressed in mammalian cell lines, sensor intensity correlated with aspartate levels measured by MS and could resolve temporal changes in intracellular aspartate from genetic, pharmacological, and nutritional manipulations. These data demonstrate the utility of jAspSnFR3 and highlight the opportunities it provides for temporally resolved and high-throughput applications of variables that affect aspartate levels.

    1. Biochemistry and Chemical Biology
    Chi-Ning Chuang, Hou-Cheng Liu ... Ting-Fang Wang
    Research Article

    Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.