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Transient intracellular acidification regulates the core transcriptional heat shock response

Catherine G Triandafillou, Christopher D Katanski, Aaron R Dinner, D. Allan Drummond

Preprint posted on September 12, 2018 https://www.biorxiv.org/content/early/2018/09/12/414706

A short-lived decrease in the pH of the cellular interior is critical to mount a response to environmental stresses such as increased temperature.

Selected by Srivats Venkataramanan

Context:

In 1987, over three decades ago, a group led by Dr. Ludger Rensing at the University of Bremen leveraged pH-dependent chemical shifts in intra-cellular inorganic phosphate using [31P] NMR spectra and discovered that the cytoplasm of budding yeast cells acidifies when the yeast are exposed to a variety of stresses [1]. They also noted that this drop in intracellular pH is correlated with increased expression of heat shock proteins but were unable to identify any causal relationships between the two [1]. In subsequent studies, this stress-induced intracellular acidification has been shown to be a common feature amongst most eukaryotes [2, 3]. However, opinions have fluctuated between whether this phenomenon is a cytotoxic consequence of stress-induced damage, or a cytoprotective aspect of the cellular stress response [4].

In this preprint, the authors develop and utilize a system that simultaneously manipulates and monitors intracellular pH in budding yeast and can uncouple it from the heat shock stress response. This system reveals that a transient acidification of the cytoplasm upon stress – and subsequent restoration of the pH to physiological levels – functions as one input of an ‘AND’ gate (with the other input being the stress itself) to activate the transcriptional heat shock stress response. The data allow the authors to persuasively argue that intracellular acidification is an adaptive response to stress.

 

Key Findings:

The authors have developed a two-color system to monitor intracellular pH and the cellular response to stress simultaneously using flow cytometry. A pH-sensitive fluorescent protein reports on the intracellular pH, and a different non-overlapping fluorescent protein fused to a canonical heat shock response protein informs on the dynamics of the stress response. Though typically found in an acidic environment (~ pH 4.0), yeast maintain a slightly basic intracellular pH of ~7.5. Using their newly developed system, the authors show that when the yeast are subjected to short periods of elevated temperature, their pH drops rapidly to ~ 6.8.  When the temperature stress is relieved, normal intracellular pH is restored within a few minutes (Figure 1 – Figure 1A of the manuscript) and the reporter for the heat shock response rises to detectable levels (a delay that is likely due to the kinetics of protein maturation).

Figure 1: intracellular pH during heat shock and recovery

Next, the authors treat cells with nigericin, a chemical that blocks regulation of intracellular pH, therefore rendering it completely dependent on the media in which cells are grown. Cells subjected to a pH range of 5.0 – 7.5 during the heat stress (and recovery in normal media at ~ pH 4.0 after stress), show robust induction of the heat shock response (albeit at varied kinetics – dependent on the rate of post-stress recovery to resting pH). Remarkably, cells that were never allowed to acidify their cytosol (by growing them in media at pH 7.5 both during stress and recovery phases) never induce the heat shock protein. This indicates that:

  • Cytosolic acidification is critical for the induction of the heat shock response
  • Cytosolic acidification can be temporally decoupled from the heat shock itself, and still produce a response.
  • The stress response depends on the recovery from acidification to a resting pH.

Further, cells subjected to Nigericin treatment without heat shock don’t induce the heat shock protein regardless of the pH to which they are coerced, indicating that:

  • Cytosolic acidification is not sufficient for induction of the stress response.

Using RNA-sequencing, the authors discover that the cytosolic acidification is required for induction of the subset of stress-response genes whose transcription is dependent on Hsf1, a key transcription factor that regulates the yeast stress response, indicating that transient cytosolic acidification is required for Hsf1 activation.

 

Open questions, future directions, and why I chose this preprint:

            Hsf1 is an interesting protein. The canonical understanding is that when the cell is growing under conducive conditions, Hsf1 is sequestered by its binding to Hsp70. Under heat stress, Hsp70 is titrated away by exposed hydrophobic patches caused by heat-induced misfolding, releasing Hsf1 [5]. The primary question the manuscript raises is fairly obvious: “how does transient cellular acidification lead to Hsf1 activation?” The authors speculate on multiple hypotheses within the manuscript and favor a model wherein the Hsp70 substrate that titrates it away from Hsf1 is sub-toxic, stress-triggered protein aggregates that depend on cytosolic acidification. These aggregates would be comprised of “sensor” proteins, which would “demix” or separate from the cytosol in a biphasic manner, dependent on both temperature (the primary stressor) and the acidification of the cytosol. Previous work from the authors provides an example of how this might work. Poly(A)-binding protein, or Pab1, forms cytosolic aggregates in response to stress, in a fashion that is coordinately regulated by temperature and pH. The authors propose a model in which proteins like Pab1 synthesize and integrate multiple signals, such as the primary stress itself, and subsequent acidification – with the nucleation of Pab1 aggregates being dependent on thermal misfolding, and their subsequent growth into robust Hsp70 substrates being dependent on a drop in cytosolic pH – which is in fact exactly how Pab1 aggregates behave [6]. It is an elegant model, and satisfyingly explains the observations within the paper – but remains untested.

The proton transport mechanism for the intracellular acidification also remains undetermined, to the best of my knowledge. The author’s data indicates that the acidification is a consequence of proton influx from the growth medium, but the nature of the transporter remains unknown. The system the authors have developed provides a platform for both targeted and unbiased screens to identify the proton transporter, as well as potential sensor proteins (although functional redundancy of the latter category might complicate matters).

I was excited to read this paper as I felt it provided and interesting perspective on gene regulation under conditions of stress. The ubiquitous nature of temperature stress, both in terms of the breadth of organisms (and the cells that make up those organisms) that experience temperature stress, and the diversity of contexts under which the elevated temperature is experienced, raises the intriguing possibility that the cytoplasmic acidification provides an additional logic layer to correspondingly diversify the cellular response. For instance, elevated temperature in an acidic environment (permitting acidification) could elicit different gene regulatory responses than in a basic environment (restricting acidification). Furthermore, the original discovery by the Rensing group also reports similar drops in intracellular pH occurs in a response to a variety of different (but not all) stressors [1], begging the question whether other stress responses are also contextually interpreted based on environmental pH. The possibilities are endless, and endlessly intriguing.

 

References:

  1. Weitzel, G., U. Pilatus, and L. Rensing, The cytoplasmic pH, ATP content and total protein synthesis rate during heat-shock protein inducing treatments in yeast. Exp Cell Res, 1987. 170(1): p. 64-79.
  2. Bright, C.M. and D. Ellis, Intracellular pH changes induced by hypoxia and anoxia in isolated sheep heart Purkinje fibres. Exp Physiol, 1992. 77(1): p. 165-75.
  3. Zhong, M., S.J. Kim, and C. Wu, Sensitivity of Drosophila heat shock transcription factor to low pH. J Biol Chem, 1999. 274(5): p. 3135-40.
  4. Tombaugh, G.C. and R.M. Sapolsky, Evolving concepts about the role of acidosis in ischemic neuropathology. J Neurochem, 1993. 61(3): p. 793-803.
  5. Krakowiak, J., et al., Hsf1 and Hsp70 constitute a two-component feedback loop that regulates the yeast heat shock response. Elife, 2018. 7.
  6. Riback, J.A., et al., Stress-Triggered Phase Separation Is an Adaptive, Evolutionarily Tuned Response. Cell, 2017. 168(6): p. 1028-1040 e19.

Tags: heat, stress response, yeast

Posted on: 25th September 2018

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  • Author's response

    Catherine G Triandafillou shared

    Thanks for featuring our work in this preLight. On a personal note, I’m really excited about this project because it gave me the opportunity to use quantitative methods to investigate a biological phenomenon that I was drawn to out of pure curiosity. We noticed that acidification accompanied many stresses in budding yeast and other organisms, and wondered if it might be a regulated part of the response. I remember when I first realized we were on to something. I had finally ironed out all the wrinkles in the assay and measured induction after stress at different pHs, and when I analyzed the data I was amazed to see a huge difference between the response of acidified and non-acidified cells. The only problem: I was doing this analysis after going home for winter break, so I had to wait three weeks before I could get back into lab to verify the result!

    A lot of exciting future questions come out of this study, many of which are touched on in this preLight. Of particular interest, of course, is now to determine the mechanism by which pH change regulates Hsf1 activation. The identity of the putative pH-sensitive sensor protein(s) remains to be determined, as do the mechanisms of cellular pH regulation during stress. Moreover, this study ties into ideas that have their genesis in earlier work from the Drummond lab: a shift from thinking of elevated temperature as creating catastrophic events in the cell, to an environmental signal which the cell senses and interprets as a cue to shift from one adaptive program to another.

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