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  1. Reviewer #3 (Public Review):

    In this manuscript, Takaine et. al. leveraged their QUEEN ATP biosensor to ask an interesting and important question: how and why cells maintain high and stable ATP concentrations in Saccharomyces cerevisiae.

    The strength of their approach is to obtain single-cell quantification of ATP concentration over time. They use the technology to demonstrate the importance of the AMP kinase, and two other proteins involved in ATP synthesis/homeostasis (the adenylate kinase, ADK1, and the transcription factor, BAS1) in the maintenance of stable and high levels of ATP.

    The main novelty of their findings with respect to ATP homeostasis is the detection of sudden, transient decreases in ATP concentration in mutants. The main claim in the title and abstract of the paper is that "High and stable ATP levels prevent aberrant intracellular protein aggregation". In our opinion, the data do not yet support this claim.

    Essential issues:

    1. The most important missing experiment, which would be required to support the title, is to image both ATP levels and protein aggregation events in the same cell. The current dataset shows that the mutants under study have both decreased ATP levels and suggest that these levels are less stable, and finally that complete ATP depletion leads to protein aggregation, but it is not possible to extrapolate these observations to the current conclusions.

    2. The second most important issue is a lack of statistics with respect to spontaneous drops in ATP concentration. A couple of examples are shown, but it should be possible to obtain data for hundreds of cells. Do the examples in figure 2 represent 90% of cells? 1% of cells? 1/1000? We need to be given a more complete sense of the penetrance of these effects.

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  2. Reviewer #2 (Public Review):

    Takaine et al., use a fluorescent reporter to quantify ATP levels within single yeast cells with high temporal resolution. With this approach, they aim to understand the molecular components required to maintain cytoplasmic ATP levels at a constant 4 mM concentration. They identify two enzymes (ADK, AMPK) and one transcription factor (Bas1) that cooperate in buffering cellular ATP levels. Without these proteins, yeast cells experience transient depletions of ATP, which the authors term "ATP catastrophes". These stochastic events are sometimes reversed, but sometimes not, leading to death of the cell. Such ATP catastrophes also make the cell prone to aggregation of neuropathic peptides, which could explain why protein aggregates occur in aging neurons (which experience declines in ATP levels). Their experiments provide strong in vivo evidence that cells maintain high levels of ATP to keep proteins soluble in a crowded cytoplasm.

    Strengths:

    1. This work moves the field forward by providing a single-cell approach. Previous studies of ATP levels analyzed extracts taken from cell populations, which could hide cell-to-cell variability. Indeed, using their ATP reporter, Takaine et al. demonstrate how ATP levels are dynamic, different between cells, and can even undergo dramatic stochastic changes.

    2. The authors use a variety of orthogonal approaches to test their hypotheses. They use the ATP probe QUEEN as their primary approach, but back it up with biochemical analysis of ATP levels in cell populations. Furthermore, they use genetic knockouts, acute insults (chemicals to deplete ATP), and rescue experiments to corroborate their results.

    3. The paper is well written and the logic is easy to follow.

    Weaknesses/Criticisms:

    1. Possible indirect effects due to knock outs of AMPK, ADK, and Bas1. These proteins are involved in many biochemical pathways, including lipid homeostasis, mitophagy, and ATP regulation. How do we know that snf1 KO (AMPK knock out) directly effects ATP levels? Also, it is possible that these yeast have acquired suppressor mutations that let them survive at reduced ATP levels, which could confound interpretation of the results.

    2. Lack of wild-type controls in Figure 2. The authors do quote their previous paper, but I want to see the controls done the exact same way. I need to know that transient changes in ATP levels are due to the mutations and not to user error or a different microscope setup. This is really important, since observation of the "ATP catastrophe" is a major finding of this paper.

    3. Insufficient quantification of the ATP catastrophe phenotype. Figure 2 shows only two cells, so I'm not sure how representative these data are. This is an important discovery, so it deserves better quantification and characterization. It would be important to quantify: a) how many cells in a population experience ATP catastrophe, b) the average time interval of depressed ATP levels before restoration, c) frequency of ATP catastrophes in a single cell, and d) how long can ATP levels be suppressed before the cell dies.

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  3. Reviewer #1 (Public Review):

    For bacteria, yeast and mammalian cells, energy depletion has been linked to a vitrification of cytosol and protein aggregation. Previous studies have postulated this is in part due to acidification and the shift in pH to match a large set of proteins pIs resulting in large-scale protein aggregation as well as changes in crowding of the cytosol. Additionally, a more direct role for ATP in protein aggregation has been proposed through its chemical properties as a hydrotrope. The appeal of this hypothesis is that the steady-state levels of ATP far exceed the Kd of most enzymes pointing to a potential non-enzymatic role for the high levels.

    In this study, the authors take advantage of a FRET-reporter for ATP that they developed previously called "Queen". They then manipulate ATP levels using mutants in AMP kinase(Snf1) or Adenyl kinase (Adk1) and find null mutants indeed have lower concentrations of ATP and experience sudden drops in ATP levels which the authors term ATP catastrophe. These mutants also show genetic interactions with protein folding/glycosylation pathways and are sensitive to conditions that generate proteotoxic stress. Hsp104 forms foci in the genetically induced lowered ATP levels as well as exogenous ectopic aggregation prone proteins such as alpha-synuclein. The authors attempt to show that the cause of aggregation is due to limiting ATP directly by adding excess adenosine to the media and showing this diminished the formation of foci, potentially due to the ability of increased exogenous to raise ATP levels according to previous reports.

    The issue of whether ATP levels play a direct or indirect role in preventing protein aggregation is extraordinarily challenging to address. While ATP can act as a hydrotrope, the formation of aggregates could be due to limitations of the activity of chaperones and helicases which would not be surprising role for ATP in the cell. While the experiments are carefully performed, well analyzed and fairly interpreted; questions still remain about the impact of these experiments on understanding how ATP impacts cytosol.

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  4. Evaluation Summary:

    Over the past decade, the role of ATP levels in the material properties of cells has gathered substantial interest in part because of the potential role of ATP in solubilizing biomolecular condensates. This study uses a quantitative imaging-based measurement of ATP levels in live cells to assess the impact of mutants in ATP homeostasis on ATP levels and protein aggregation. The strength of this paper is the quantitative, single cell analysis, and the manipulation of ATP using native control pathways. The authors suggest that fluctuations in ATP concentrations can lead to protein aggregation, which would be of broad interest to many fields, including cell biology, aging and neurodegeneration.

    (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 #3 agreed to share their name with the authors.)

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