Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol

Reviewer #1 (Public review):

The manuscript by Bru et al. focuses on the role of vacuoles as a phosphate buffering system for yeast cells. The authors describe here the crosstalk between the vacuole and the cytosol using a combination of in vitro analyses of vacuoles and in vivo assays. They show that the luminal polyphosphatases of the vacuole can hydrolyze polyphosphates to generate inorganic phosphate, yet they are inhibited by high concentrations. This balances the synthesis of polyphosphates against the inorganic phosphate pool. Their data further show that the Pho91 transporter provides a valve for the cytosol as it gets activated by a decline in inositol pyrophosphate levels. The authors thus demonstrate how the vacuole functions as a phosphate buffering system to maintain a constant cytosolic inorganic phosphate pool.

This is a very consistent and well-written manuscript with a number of convincing experiments, where the authors use isolated vacuoles and cellular read-out systems to demonstrate the interplay of polyphosphate synthesis, hydrolysis, and release. The beauty of this system the authors present is the clear correlation between product inhibition and the role of Pho91 as a valve to release Pi to the cytosol to replenish the cytosolic pool. I find the paper overall an excellent fit and only have a few issues, including :

(1) Figure 3: The authors use in their assays 1 mM ZnCl2 or 1mM MgCl2. Is this concentration in the range of the vacuolar luminal ion concentration? Did they also test the effect of Ca2+, as this ion is also highly concentrated in the lumen?

(2) Regarding the concentration of 30 mM K-PI, did the authors also use higher and lower concentrations? I agree that there is inhibition by 30 mM, but they cannot derive conclusions on the luminal concentration if they use just one in their assay. A titration is necessary here.

(3) What are the consequences on vacuole morphology if the cells lack Pho91?

(4) Discussion: The authors do not refer to the effect of calcium, even though I would expect that the levels of the counterion should affect the phosphate metabolism. I would appreciate it if they would extend their discussion accordingly.

(5) I would appreciate a brief discussion on how phosphate sensing and control are done in human cells. Do they use a similar lysosomal buffer system?

Reviewer #2 (Public review):

Summary:

This manuscript presents a well-conceived and concise study that significantly advances our understanding of polyphosphate (polyP) metabolism and its role in cytosolic phosphate (Pi) homeostasis in a model unicellular eukaryote. The authors provide evidence that yeast vacuoles function as dynamic regulatory buffers for Pi homeostasis, integrating polyP synthesis, storage, and hydrolysis in response to cellular metabolic demands. The work is methodologically sound and offers valuable insights into the conserved mechanisms of phosphate regulation across eukaryotes.

Strengths:

The results demonstrate that the vacuolar transporter chaperone (VTC) complex, in conjunction with luminal polyphosphatases (Ppn1/Ppn2) and the Pi exporter Pho91, establishes a finely tuned feedback system that balances cytosolic Pi levels. Under Pi-replete conditions, inositol pyrophosphates (InsPPs) promote polyP synthesis and storage while inhibiting polyP hydrolysis, leading to vacuolar Pi accumulation.

Conversely, Pi scarcity triggers InsPP depletion, activating Pho91-mediated Pi export and polyP mobilization to sustain cytosolic phosphate levels. This regulatory circuit ensures metabolic flexibility, particularly during critical processes such as glycolysis, nucleotide synthesis, and cell cycle progression, where phosphate demand fluctuates dramatically.

From my viewpoint, one of the most important findings is the demonstration that vacuoles act as a rapidly accessible Pi reservoir, capable of switching between storage (as polyP) and release (as free Pi) in response to metabolic cues. The energetic cost of polyP synthesis-driven by ATP and the vacuolar proton gradient-highlights the evolutionary importance of this buffering system. The study also draws parallels between yeast vacuoles and acidocalcisomes in other eukaryotes, such as Trypanosoma and Chlamydomonas, suggesting a conserved role for these organelles in phosphate homeostasis.

Weaknesses:

While the manuscript is highly insightful, referring to yeast vacuoles as "acidocalcisome-like" may warrant further discussion. Canonical acidocalcisomes are structurally and chemically distinct (e.g., electron-dense, in most cases spherical, and not routinely subjected to morphological changes, and enriched with specific ions), whereas yeast vacuoles have well-established roles beyond phosphate storage. A comment on this terminology could strengthen the comparative analysis and avoid potential confusion in the field.

Reviewer #3 (Public review):

Bru et al. investigated how inorganic phosphate (Pi) is buffered in cells using S. cerevisiae as a model. Pi is stored in cells in the form of polyphosphates in acidocalcisomes. In S. cerevisiae, the vacuole, which is the yeast lysosome, also fulfills the function of Pi storage organelle. Therefore, yeast is an ideal system to study Pi storage and mobilization.

They can recapitulate in their previously established system, using isolated yeast vacuoles, findings from their own and other groups. They integrate the available data and propose a working model of feedback loops to control the level of Pi on the cellular level.

This is a solid study, in which the biological significance of their findings is not entirely clear. The data analysis and statistical significance need to be improved and included, respectively. The manuscript would have benefited from rigorously testing the model, which would also have increased the impact of the study.

Author response:

Reviewer #1 (Public review):

The manuscript by Bru et al. focuses on the role of vacuoles as a phosphate buffering system for yeast cells. The authors describe here the crosstalk between the vacuole and the cytosol using a combination of in vitro analyses of vacuoles and in vivo assays. They show that the luminal polyphosphatases of the vacuole can hydrolyse polyphosphates to generate inorganic phosphate, yet they are inhibited by high concentrations. This balances the synthesis of polyphosphates against the inorganic phosphate pool. Their data further show that the Pho91 transporter provides a valve for the cytosol as it gets activated by a decline in inositol pyrophosphate levels. The authors thus demonstrate how the vacuole functions as a phosphate buffering system to maintain a constant cytosolic inorganic phosphate pool.

This is a very consistent and well-written manuscript with a number of convincing experiments, where the authors use isolated vacuoles and cellular read-out systems to demonstrate the interplay of polyphosphate synthesis, hydrolysis, and release. The beauty of this system the authors present is the clear correlation between product inhibition and the role of Pho91 as a valve to release Pi to the cytosol to replenish the cytosolic pool. I find the paper overall an excellent fit and only have a few issues, including:

(1) Figure 3: The authors use in their assays 1 mM ZnCl2 or 1mM MgCl2. Is this concentration in the range of the vacuolar luminal ion concentration? Did they also test the effect of Ca2+, as this ion is also highly concentrated in the lumen?

The concentrations inside vacuoles can reach those values. However, given that polyP is a potent chelator of divalent metal ions, what would matter are the concentrations of free Zn²⁺ or Mg²⁺ inside the organelle. These are not known. This is not critical since we use those two conditions only as a convenient tool to differentiate Ppn1 and Ppn2 activity in vitro. In our initial characterisation of Ppn2 (10.1242/jcs.201061), we had also tested Mn, Co, Ca, Ni, Cu. Only Zn and Co supported activity. Ca did not. Andreeva et al. (10.1016/j.biochi.2019.06.001) reached similar conclusions and extended our results.

(2) Regarding the concentration of 30 mM K-PI, did the authors also use higher and lower concentrations? I agree that there is inhibition by 30 mM, but they cannot derive conclusions on the luminal concentration if they use just one in their assay. A titration is necessary here.

The concentration of 30 mM was not arbitrarily chosen. It is the luminal P_i concentration that the vacuoles could reach through when they entered a plateau of luminal Pi. We consider this as an upper limit because polyP kept increasing which luminal P_i did not. Thus, there is in principle no physiological motivation for trying higher values. But we will probably add a titration to the revised version.

(3) What are the consequences on vacuole morphology if the cells lack Pho91?

We had not observed significant abnormalities during a screen of the genome-wide deletion collection of yeast (10.1371/journal.pone.0054160)

(4) Discussion: The authors do not refer to the effect of calcium, even though I would expect that the levels of the counterion should affect the phosphate metabolism. I would appreciate it if they would extend their discussion accordingly.

We will pick this up in the discussion. However, the situation is much more complex because major pools of counterions (up to hundreds of mM) are constituted by vacuolar lysine, arginine, polyamines, Mg, Zn etc. Their interplay with polyP is probably complex and worth to be treated in a dedicated project.

(5) I would appreciate a brief discussion on how phosphate sensing and control are done in human cells. Do they use a similar lysosomal buffer system?

Mammalian cells have their Pi exporter XPR1 mainly on a lysosome-like compartment (10.1016/j.celrep.2024.114316). Whether and how it functions there for Pi export from the cytosol is not entirely clear. We will address this situation in the revision.

Reviewer #2 (Public review):

Summary:

This manuscript presents a well-conceived and concise study that significantly advances our understanding of polyphosphate (polyP) metabolism and its role in cytosolic phosphate (Pi) homeostasis in a model unicellular eukaryote. The authors provide evidence that yeast vacuoles function as dynamic regulatory buffers for Pi homeostasis, integrating polyP synthesis, storage, and hydrolysis in response to cellular metabolic demands. The work is methodologically sound and offers valuable insights into the conserved mechanisms of phosphate regulation across eukaryotes.

Strengths:

The results demonstrate that the vacuolar transporter chaperone (VTC) complex, in conjunction with luminal polyphosphatases (Ppn1/Ppn2) and the Pi exporter Pho91, establishes a finely tuned feedback system that balances cytosolic Pi levels. Under Pi-replete conditions, inositol pyrophosphates (InsPPs) promote polyP synthesis and storage while inhibiting polyP hydrolysis, leading to vacuolar Pi accumulation.

Conversely, Pi scarcity triggers InsPP depletion, activating Pho91-mediated Pi export and polyP mobilization to sustain cytosolic phosphate levels. This regulatory circuit ensures metabolic flexibility, particularly during critical processes such as glycolysis, nucleotide synthesis, and cell cycle progression, where phosphate demand fluctuates dramatically.

From my viewpoint, one of the most important findings is the demonstration that vacuoles act as a rapidly accessible Pi reservoir, capable of switching between storage (as polyP) and release (as free Pi) in response to metabolic cues. The energetic cost of polyP synthesis-driven by ATP and the vacuolar proton gradient-highlights the evolutionary importance of this buffering system. The study also draws parallels between yeast vacuoles and acidocalcisomes in other eukaryotes, such as Trypanosoma and Chlamydomonas, suggesting a conserved role for these organelles in phosphate homeostasis.

Weaknesses:

While the manuscript is highly insightful, referring to yeast vacuoles as "acidocalcisome-like" may warrant further discussion. Canonical acidocalcisomes are structurally and chemically distinct (e.g., electron-dense, in most cases spherical, and not routinely subjected to morphological changes, and enriched with specific ions), whereas yeast vacuoles have well-established roles beyond phosphate storage. A comment on this terminology could strengthen the comparative analysis and avoid potential confusion in the field.

Yeast vacuoles show all major chemical features of acidocalcisomes. They are acidified, contain high concentrations of Ca, polyP (which make them electron-dense, too), other divalent ions, such as Mg, Zn, Mn etc, and high concentrations of basic amino acids. Thus, they clearly have an acidocalcisome-like character. In addition, they have hydrolytic, lysosome-like functions and, depending on the strain background, they can be larger than acidocalcisomes described e.g. in protists. We will elaborate this point, which is obvious to us but probably not to most readers, in the revised version.

Reviewer #3 (Public review):

Bru et al. investigated how inorganic phosphate (Pi) is buffered in cells using S. cerevisiae as a model. Pi is stored in cells in the form of polyphosphates in acidocalcisomes. In S. cerevisiae, the vacuole, which is the yeast lysosome, also fulfills the function of Pi storage organelle. Therefore, yeast is an ideal system to study Pi storage and mobilization.

They can recapitulate in their previously established system, using isolated yeast vacuoles, findings from their own and other groups. They integrate the available data and propose a working model of feedback loops to control the level of Pi on the cellular level.

This is a solid study, in which the biological significance of their findings is not entirely clear. The data analysis and statistical significance need to be improved and included, respectively. The manuscript would have benefited from rigorously testing the model, which would also have increased the impact of the study.

It is not clear to us what the reviewer would see as a more rigorous test of the model.