Engineering ATP Import in Yeast Uncovers a Synthetic Route to Extend Cellular Lifespan

Reviewer #1 (Public review):

Summary:

The authors aim to engineer a synthetic system for manipulating ATP homeostasis in budding yeast by expressing the microsporidian nucleotide transporter NTT1, thereby enabling ATP import from the extracellular environment. Using this system, they attempt to test whether intracellular ATP abundance causally regulates replicative lifespan and whether extracellular ATP sensing contributes independently to longevity pathways. The manuscript presents data from ATP biosensing, transcriptomics, mitochondrial perturbations, and microfluidic aging assays to build a dual-mechanism model linking ATP availability, MAPK signaling, mitochondrial function, and aging trajectories.

Strengths:

A major strength of the study is its creative application of xenotopic synthetic biology to directly manipulate ATP homeostasis-an ambitious approach that addresses an important and difficult question in aging biology. The use of complementary methods, including single-cell ATP reporters, microfluidic lifespan measurements, and RNA-seq, generates a rich experimental dataset with the potential to reveal multiple layers of ATP-dependent physiological regulation. The manuscript also raises interesting hypotheses regarding extracellular nucleotide sensing and HOG/MAPK pathway involvement, opening conceptual space for future exploration of ATP-based signaling in yeast.

Weaknesses:

Despite these strengths, the manuscript suffers from several critical weaknesses that undermine the central conclusions. Foremost, the intracellular ATP measurements contradict key interpretations: NTT1 expression lowers ATP levels, yet multiple sections assert or assume that NTT1 increases intracellular ATP via import. This unresolved contradiction propagates throughout the mechanistic model. The authors do not consider or experimentally address the more parsimonious explanation that NTT1 may be a bidirectional ATP transporter, which would unify many perplexing results. Several important analyses are missing (e.g., transcriptomic comparison of NTT1 cells with vs. without ATP), and key signaling claims lack proper validation (e.g., Hog1 quantification, AMPK controls). Additionally, inconsistencies in figures-such as incorrect scale bars, mismatched ATP measurements, and a conceptual model contradicted by the data-further detract from clarity. As a result, the manuscript does not yet convincingly achieve its stated aims, and the current evidence does not adequately support the proposed causal relationships between ATP homeostasis and lifespan.

Reviewer #2 (Public review):

Summary:

This work presents interesting findings where the addition of exogenous ATP extends the replicative lifespan of yeast cells in a way that seems uncorrelated with actual increased intracellular ATP levels or mitochondria. To be clear, the addition of ATP to yeast growth media increases the number of cell divisions per cell in yeast. Expression of the NTT1 ATP transporter gene increases intracellular ATP levels according to LCMS analysis, but the effect on replicative lifespan works without the NTT1 gene and without an intracellular increase in ATP (possibly with a decrease in intracellular ATP), so the effect appears to be independent of the effect on intracellular ATP levels or mitochondria, as mitochondria-less R0 yeast cells also have increased numbers of cell division when grown with extracellular ATP. The plots in Figure 5 make it seem like exogenous ATP addition lowers intracellular ATP for both the NTT1 cells and the wild-type cells, and that is not what the data in Figure 2d with LCMS shows.

As an aside, this seems like a better model for increased tumor cell growth in the presence of increased extracellular ATP, which happens in some cancers.

Restated, the data suggest they were successful in increasing intracellular ATP by LCMS, but not by queen reporter, and that the seemingly likely increased intracellular ATP was not causative, as cells that did not have an increase in intracellular ATP, but had the same exogenous ATP addition, also gained an increase in replicative lifespan. There could also be two distinct mechanisms extending replicative lifespan to the same degree in these two different strains. More measurements, controls, and analyses are needed to accurately determine what is happening with intracellular ATP levels with age. It is currently unknown if there is any correlation between ATP levels and replicative aging (with properly controlled longitudinal measurements).

Strengths:

Longitudinal imaging of single cells. Analyzed ATP levels with two approaches. Creative approach to use NTT1 transporter to increase intracellular ATP levels. Solid replicative lifespan data.

Weaknesses:

Mostly unclear about ATP levels with age and the relationship, or lack thereo,f between intracellular ATP levels and replicative lifespan. No idea what this effect depends on, but some ideas what it does not depend on (mitochondria or increased intracellular ATP). Experiments seem to lack biological controls (cells without gfp) for age related changes in autofluorescence (and pH that can affect gfp signal) for the fluorescent microscopy quantifying ATP with age using the QUEEN reporter (seems that way as written); conflicting evidence on ATP levels; lack of LC-MS measurements in old cells; no apparent correlation between ATP levels and replicative lifespan, but that could be wrong - just not apparent from the longitudinal data plots. The LCMS data seems better than the microscopy data on ATP because the microscopy approach seems to lack proper biological controls, and the selection of only the top 40% of pixels to quantify signal seems unjustified as written, and possibly prone to technical artifacts. Figure 2 B&C plots of ATP levels should show what the cells were normalized to. The figures also seem too diluted and should probably be combined or put in the supplements (hog1 western) if they do not relate to the lifespan effect. There seem to be some technical scientific editorial errors, like in Figure 7.

Author response:

Thank you for considering our manuscript, “Engineering ATP Import in Yeast Uncovers a Synthetic Route to Extend Cellular Lifespan” (eLife-RP-RA-2025-109761) for publication in eLife. We appreciate the time and effort invested by the reviewers and editors.

We have carefully read the eLife assessment and both public reviews. After thorough evaluation, we believe there is a significant factual misunderstanding that has propagated through both reviews and fundamentally affected the interpretation of our central findings and the overall evaluation.

We must also express concern regarding the review process duration. We were informed that the manuscript experienced an extended review period (107 days) due to delay from a third reviewer. Ultimately, we received only two reviews.

The raised problem of our manuscript containing obvious internal contradictions or technical inconsistencies are not due to flawed data but due to a misinterpretation of measurement directionality.

We also acknowledge the fact that we should more explicitly describe the figure legend 5, and that the methods sections should include the experimental design that led to the reverse correlation of the AU units.

Together these facts led to the misinterpretation of the ATP measurements presented in Figure 5, specifically the directionality of the fluorescence-based ATP readout by both reviewers. In this essay, arbitrary units (AU) are reversely correlated with intracellular ATP abundance. Higher AU values correspond to lower ATP levels. This inverse relationship was clearly described in the Results section and figures marked with “Low versus High” of the manuscript, but it appears to have been overlooked. As a result, reviewers interpreted Figure 5 as contradicting Figure 2, when in fact the two datasets are fully consistent.

Because this misunderstanding affected interpretation of the foundational ATP data, it appears to have influenced evaluation of all downstream conclusions. For example, neither reviewer meaningfully engaged with:

- The identification of distinct cell death trajectories.

- The mitochondrial dependency of NTT1-associated toxicity.

- The integration of ATP depletion with mitochondrial function.

- The distinction between intracellular ATP manipulation and extracellular ATP sensing mechanisms.

We fully understand that when foundational data appears contradictory, reviewers naturally deprioritize downstream conclusions. However, in this case, the foundational contradiction does not exist it arises from a misreading of the reporter’s scale.

From the Results section of the manuscript:

“Our analysis of ATP abundance throughout the yeast lifespan showed that yeast cells are born with low ATP levels, which gradually increase during their lifespan. Some cells completed their lifespan without any observable reduction in ATP abundance, while others showed a drastic decrease in ATP levels during late life (Fig. 5A–D, Supplementary File S3), consistent with previous observations supporting two modes of yeast lifespan, mediated by mitochondrial and/or SIR2 function (42,46–49). Consistent with our data presented in Figure 2, we also observed significantly lower ATP abundance in NTT1-expressing cells throughout their entire lifespan compared to Wt control cells (Fig. 5A–C). Furthermore, these cells displayed significantly reduced mean and maximum replicative lifespan (RLS), directly indicating that intracellular ATP depletion shortens lifespan (Fig. 5D). Next, we assessed RLS and age-associated ATP changes under ATP supplementation. We found that exposing NTT1 cells to medium supplemented with 10 µM ATP restored intracellular ATP levels (Fig. 5A–C) and significantly (p = 4.03E-18) increased both mean and maximum RLS to levels comparable to WT cells (Fig. 5D).”

This section explicitly explains that Figure 5 is consistent with Figure 2. LC-MS data (Figure 2) show intracellular ATP depletion in NTT1 cells under baseline conditions and restoration upon extracellular ATP supplementation. Figure 5 shows the same pattern longitudinally. The apparent contradiction raised by both reviewers stems entirely from misreading the directionality of the AU scale.

In the public assessment,

Concerns are raised about:

- “Internally inconsistent, particularly regarding intracellular ATP measurements”

- “Mismatched ATP measurements”

- “Conceptual model contradicted by the data”

- “The plots in Figure 5 make it seem like exogenous ATP addition lowers intracellular ATP…”

These statements arise directly from the reversed interpretation of the AU scale. If the inverse relationship had been recognized, these perceived inconsistencies would not exist. Unfortunately, this misunderstanding then influenced broader interpretations, including the conclusion that the fundamental NTT1 model is internally contradictory.

Similarly, Reviewer #2 states that LC-MS and QUEEN reporter data conflict and that ATP supplementation appears to lower intracellular ATP. This again reflects the same directional misunderstanding. There is no conflict between Figure 2 and Figure 5. Both show reduced ATP in NTT1 cells and restoration upon ATP supplementation.

A second major point concerns the bidirectional transporter hypothesis. Reviewer #1 suggests that NTT1 may be bidirectional. However, NTT1 is well-characterized in the literature as a nucleotide transporter that exchanges extracellular ATP for intracellular ADP. We clearly described this in Figure 1C and cited the appropriate primary literature. The suggestion that we failed to consider directionality appears to stem from the same misinterpretation of intracellular ATP levels. We agree that clarifying the role of ADP/AMP depletion in NTT1-expressing cells would strengthen the manuscript, and we are prepared to revise the text to more explicitly describe how intracellular nucleotide exchange dynamics contribute to ATP depletion under baseline conditions.

We also note that several criticisms, such as:

-“Incorrect scale bars”

- “Figure 5C does not match 5AB”

- “Conceptual model contradicted by the data”

- “No apparent correlation between ATP levels and lifespan”

Are all rooted in this central misunderstanding of how ATP abundance is represented in the fluorescence measurements.

To address this constructively during the next revision, we are willing to:

(1) Revise all relevant figure legends to explicitly state that AU values are inversely correlated with ATP abundance. We will expand materials and methods section for clarifying reverse correlation and/or will generate new figures to minimize the confusion.

(2) Add clarifying annotations directly onto the figures.

(3) Include new figures for further validation of observed nucleotide changes.

(4) We will expand our RNAseq data analyses.

(5) Expand discussion of nucleotide exchange dynamics and transporter directionality

(6) Adress remaining concerns with additional analyses, experiments and clarification throughout the manuscript.