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

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Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol Samuel Bru , Lydie Michaillat Mayer , Geun-Don Kim , Danye Qiu , View ORCID Profile Henning Jacob Jessen , View ORCID Profile Andreas Mayer doi: https://doi.org/10.1101/2025.05.30.656997 Samuel Bru 1 Département d’immunobiologie, Université de Lausanne , Chemin des Boveresses 155, 1066 Epalinges, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Lydie Michaillat Mayer 1 Département d’immunobiologie, Université de Lausanne , Chemin des Boveresses 155, 1066 Epalinges, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Geun-Don Kim 1 Département d’immunobiologie, Université de Lausanne , Chemin des Boveresses 155, 1066 Epalinges, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Danye Qiu 2 Institute of Organic Chemistry University of Freiburg , Albertstrasse 21, 79104 Freiburg, Germany & CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Henning Jacob Jessen 2 Institute of Organic Chemistry University of Freiburg , Albertstrasse 21, 79104 Freiburg, Germany & CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg , Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Henning Jacob Jessen Andreas Mayer 1 Département d’immunobiologie, Université de Lausanne , Chemin des Boveresses 155, 1066 Epalinges, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Andreas Mayer For correspondence: andreas.mayer{at}unil.ch Abstract Full Text Info/History Metrics Data/Code Preview PDF Abstract Cells experience strong variations in the consumption and availability of inorganic phosphate (P i ). Since P i is an essential macronutrient but excess P i has negative impacts on nucleotide hydrolysis and metabolism, its concentration must be maintained in a suitable range. Conserved storage organelles, acidocalcisomes, provide this buffering function. We used acidocalcisome-like yeast vacuoles to study how such organelles are set up to for this task. Our combined in vitro and in vivo analyses revealed that their ATP-driven polyphosphate polymerase VTC converts cytosolic P i into inorganic polyphosphates (polyP), which it transfers into the vacuole lumen. Luminal polyphosphatases immediately hydrolyse this polyP to establish a growing reservoir of vacuolar P i . Product inhibition by this P i pool silences the polyphosphatases, caps P i accumulation, and favours vacuolar polyP storage. Upon cytosolic P i scarcity, the declining inositol pyrophosphate levels activate the vacuolar P i exporter Pho91 to replenish cytosolic P i . In this way, acidocalcisome-like vacuoles constitute a feedback-regulated buffering system for cytosolic P i , which the cells can switch between P i accumulation, P i release, and high-capacity phosphate storage through polyP. Introduction Cells actively manage the concentration of P i in their cytosol because they must strike a balance between conflicting goals ( Austin & Mayer, 2020 ). On the one hand, P i is a product of nucleotide-hydrolysing reactions. Its concentration has a significant impact on the free energy that these reactions can provide to drive metabolism. Since, for his reason, excessive P i concentrations might stall metabolism, we can expect that cytosolic P i must remain limited. But P i is also an essential macronutrient. As a major constituent of nucleic acids and phospholipids, and as an important modifier of proteins, carbohydrates and many metabolites, it is consumed in large quantities for anabolic reactions. This can lead to situations where progression of the S-phase of the cell cycle is limited by the P i uptake capacity of the cells ( Bru et al , 2016 , 2017 ; Gillies et al , 1981 ; Neef & Kladde, 2003 ). Rapidly dividing cells, such as yeast cells, remedy this problem by maintaining phosphate stores in the form of inorganic polyP. PolyPs are chains of P i linked through phosphoric anhydride bonds, which can be stored in acidocalcisome-like organelles ( Urech et al , 1978 ; Okorokov et al , 1980 ). Acidocalcisomes are lysosome-related organelles that have been characterised by pioneering work mainly in Trypanosomes, but they have been isolated from a multitude of other organisms ( Docampo et al , 2005 ). Their defining chemical features are their acidity, high concentrations of polyP, Ca, Mg, Zn, Fe, K, Na, and basic amino acids and they are implicated in osmoregulation and the homeostasis of phosphate and metals ( Docampo et al , 2005 ; Girard-Dias et al , 2023 ; Docampo, 2024 ). Yeast vacuoles share all these properties and are hence good and easily accessible models for studying aspects of acidocalcisomal mechanisms, such as the synthesis and turnover of polyP ( Dürr et al , 1979 ; Huber-Wälchli & Wiemken, 1979 ; Urech et al , 1978 ; Austin & Mayer, 2020 ). But they have an additional, lysosome-like face, enabling them to assume also hydrolytic degradative functions for the cells ( Li & Kane, 2009 ). Therefore, we refer to them as acidocalcisome-like organelles. The polyP stores of vacuoles are accessed by cells when P i becomes limiting in the environment, or when they face a sudden enhanced P i consumption, e.g. during metabolic transitions from respiratory growth to fermentation, which requires much more phosphorylated metabolites ( Shirahama et al , 1996 ; Thomas & O’Shea, 2005 ; Gillies et al , 1981 ; Nicolay et al , 1982 , 1983 ). In such cases, cellular polyphosphate content decreases. It has therefore been proposed that polyPs are re-converted into P i to buffer shortages in cytosolic P i ( Nicolay et al , 1983 , 1982 ). While this hypothesis is straightforward, we are lacking a coherent concept of how a P i buffer based on an acidocalcisome-like organelle might work. Pioneering work on these organelles has revealed several characteristic and conserved features, such as their acidity, and their high content of basic amino acids and divalent cations ( Docampo & Huang, 2016 ; Docampo, 2024 ). Of direct relevance to P i homeostasis is the high capacity of acidocalcisome-like organelles for storing polyP. PolyP can greatly vary in length, from two to hundreds of phosphate units. The membrane of acidocalcisome-like organelles can carry polyP polymerases, such as VTC, and P i transporters, such as Pho91 ( Jimenez & Docampo, 2015 ; Hürlimann et al , 2007 ; Wang et al , 2015 ; Huang & Docampo, 2015 ; Gerasimaite & Mayer, 2016 ; Müller et al , 2003 , 2002 ). VTC is a coupled polyP polymerase and translocase ( Gerasimaite et al , 2014 ), which synthesizes polyP by transferring the ψ-phosphate of cytosolic ATP onto an elongating polyP chain ( Hothorn et al , 2009 ). PolyP is generated by a catalytic domain in the centre of the Vtc4 subunit of the complex ( Hothorn et al , 2009 ). The activity of this subunit is controlled through the SPX domains of VTC ( Wild et al , 2016 ), which may associate into a dimerized inactive state ( Pipercevic et al , 2023 ). They can be released from this state through the action of inositol pyrophosphates, signalling molecules that accumulate when P i is abundant in the cytosol and activate P i storage in the form of polyP. The P i state of the cytosol is communicated to VTC through a specific inositol pyrophosphate, 1,5-IP 8 ( Gerasimaite et al , 2017 ; Chabert et al , 2023 ). The postulated direct channelling of polyP from the site of its synthesis through the vacuolar membrane ( Gerasimaite et al , 2014 ) is facilitated by the structure of the VTC complex. The catalytic domain of VTC has its exit for polyP right at the entry of a polyP-conducting channel, which is formed through the transmembrane domains of the VTC complex itself ( Liu et al , 2023 ; Guan et al , 2023 ; Müller et al , 2002 , 2003 ; Gerasimaite et al , 2014 ). This channel was proposed to exist in an open and closed conformation and to be gated through polyP ( Liu et al , 2023 ). PolyP translocation may be driven by the electrochemical potential across the membrane, which could move the highly negatively charged polyP chain through electrophoresis. This can explain why efficient polyP synthesis depends on proton pumps such as the V-ATPase or H + -pumping pyrophosphatases ( Gerasimaite & Mayer, 2016 ; Gerasimaite et al , 2014 ; Freimoser et al , 2006 ; Lemercier et al , 2004 ). The transporter Pho91 was proposed to export vacuolar P i ( Hürlimann et al , 2007 ). Several properties of yeast Pho91 and its homologs from other organisms are consistent with this view. Its ablation increases vacuolar P i and polyP content, in yeast as well as in trypanosomes ( Hürlimann et al , 2007 ; Jimenez & Docampo, 2015 ; Farofonova et al , 2023 ), and it weakly induces the phosphate starvation program in yeast ( Pinson et al , 2004 ), suggesting that it may induce cytosolic P i scarcity. Furthermore, patch-clamp analyses of Pho91-like channels from yeast, trypanosomes and plants have shown their P i permeability and the dependence of their directionality on a pH gradient across the membrane ( Potapenko et al , 2018 , 2019 ; Wang et al , 2015 ). In the presence of a pH gradient, the Pho91 homolog from rice, OsSpx-MFS3, mediates P i flux along the proton gradient, consistent with a function in exporting P i from the acidic lumen of vacuoles ( Wang et al , 2015 ) towards the cytosol. Acidocalcisome-like organelles also contain polyphosphatases in their lumen ( Lander et al , 2016 ; Gerasimaite & Mayer, 2016 ; Kulakovskaya et al , 2021 ; McCarthy & Downey, 2023 ), which can convert polyP back into P i . In baker’s yeast, two vacuolar polyphosphatases are known, Ppn1 and Ppn2 ( Gerasimaite & Mayer, 2017 ; Sethuraman et al , 2001 ; Andreeva et al , 2019 ). This means that a chain of polyP, when being synthesized by VTC and arriving in the vacuolar lumen, is immediately exposed to hydrolytic enzymes that will degrade it. While this seems at first sight paradoxical, we explored the hypothesis that the co-existence of polyP-synthesizing and polyP-hydrolysing activities might be a key feature conveying to acidocalcisome-like organelles the capacity to buffer cytosolic P i . That these organelles have a critical role to play in this process is illustrated by observations in yeast, where artificial up- or down-regulation of vacuolar polyP synthesis suffices to drive the cytosol into a state of P i starvation or P i excess, respectively ( Desfougères et al , 2016a ). Furthermore, the presence of polyP reserves delays the activation of the transcriptional phosphate starvation response, the PHO pathway ( Thomas & O’Shea, 2005 ). We hence explored the capacity of isolated yeast vacuoles to interconvert polyP and P i , and we characterized the roles played by the vacuolar polyphosphatases Ppn1 and Ppn2 and the vacuolar P i transporter Pho91. Our observations can be combined with previous findings to yield a coherent model of how an acidocalcisome-like organelle can operate as a P i buffer for the cytosol. Results We explored the interplay of VTC, polyphosphatases and Pho91 in the accumulation of polyP and P i inside vacuoles using an in vitro system with purified organelles. Vacuoles can be isolated in intact form when the cells are gently opened by enzymatic digestion of the cell wall and disruption of the cell membrane by low concentrations of DEAE-dextran ( Dürr et al , 1975 ). Organelles isolated in this way can perform many of their normal cellular functions, such as membrane fusion, membrane fission and autophagy ( Michaillat et al , 2012 ; Sattler & Mayer, 2000 ; D’Agostino & Mayer, 2019 ; Kunz et al , 2004 ). They also contain active polyP polymerase (VTC) and active polyphosphatases (Ppn1 and Ppn2) and they can synthesize and import polyP ( Gerasimaite et al , 2014 ; Gerasimaite & Mayer, 2017 ; Gerasimaite et al , 2017 ). Vacuolar P i accumulation depends on polyP synthesis Purified vacuoles were incubated with an ATP-regenerating system under conditions that allow these organelles to synthesize polyP in vitro ( Gerasimaite et al , 2014 ). After different periods of incubation, the organelles were sedimented and solubilized in detergent. Vacuolar accumulation of polyP was assayed through DAPI fluorescence and P i was measured through malachite green. VTC is stimulated by a variety of inositol pyrophosphates, most efficiently by 1,5-InsP 8 ( Gerasimaite et al , 2014 , 2017 ). Since 1,5-InsP 8 is not commercially available, we used saturating concentrations of 5-InsP 7 for our experiments ( Pavlovic et al , 2015 ). This compound stimulates VTC with a higher EC 50 but to the same maximal activity as 1,5-InsP 8 , and it was more readily available to us for routine experiments. Under the conditions used here, the organelles rapidly and efficiently produced polyP ( Fig. 1 ), in line with previous results ( Gerasimaite et al , 2014 , 2017 ). This production was stimulated by 5-InsP 7 , reaching 0.7 nmol of phosphate units per µg of vacuolar protein and min. This means that already within 10 min, wildtype vacuoles produced a mass of polyP equivalent to their total protein content, indicating how efficiently these organelles synthesize polyP. This signal was entirely dependent on VTC, because it was not observed in a mutant lacking the catalytic subunit of VTC ( vtc4Δ ). Download figure Open in new tab Fig. 1: VTC- and 5-IP 7 -dependent polyP synthesis by isolated vacuoles Vacuoles were isolated from logarithmic cultures of BY4742 wildtype cells (WT) or from isogenic vtc4Δ cells strains. They were incubated in polyphosphate synthesis assays without ( A ) or (B) in presence of 50 µM 5-InsP 7 . At indicated times, aliquots were withdrawn, solubilized in Triton X-100 and polyphosphate was quantified through the polyP-dependent fluorescence of added DAPI. Means ± SD (standard deviation) of three independent experiments are shown. * p<0.05, ** p<0.005 for a paired t-test comparing WT with and without 5-InsP 7 at each time point. Isolated wildtype vacuoles also accumulated P i with significant efficiency, at an initial rate of at least 0.3 nmol µg -1 min -1 ( Fig. 2A ). Vacuoles from vtc4Δ cells showed only 8% of this P i signal. Most of it was not time-dependent and hence may represent a background signal from the organelle preparation. In sum, vacuolar P i accumulation depended on polyP synthesis through VTC. Download figure Open in new tab Figure 2. VTC-dependent accumulation of P i in isolated vacuoles Vacuoles were isolated from the indicated, logarithmically growing BY4742 (WT) cells. PolyP overproduction was achieved either through (A, B) overexpressing VTC5 from the strong GPD promotor, or (C,D) by expressing the hyperactivating vtc3 k126A and vtc4 K129A alleles form their native promotors as the sole source of these two proteins. The vacuoles were incubated with an ATP-regenerating system and 50 µM 5-InsP 7 under conditions allowing polyP synthesis. At indicated times, the vacuoles were solubilized with Triton X-100 and P i was assayed through malachite green. Graphs represent the mean ± SEM of three independent experiments for each strain. * p<0.05, ** p<0.005, *** p<0.001 from a paired t-test comparing each time WT vs VTC5 overexpression or vtc3 k126A vtc4 K129A . The kinetics of polyP and P i accumulation were remarkably different. Whereas polyP continued to accumulate throughout the entire incubation period, P i accumulated rapidly in the initial phase but reached a plateau within 30 min ( Fig. 2A, B ). Thus, although polyP production was essential for P i accumulation, it could not become the limiting factor for it in the second phase. This is further illustrated by P i accumulation in strains showing enhanced polyP synthesis, such as overexpressors of VTC5 ( Fig. 2A, B ) or strains carrying hyperactivating substitutions in the SPX domains of VTC ( vtc3 K126A vtc4 K129A ; Fig. 2 C, D ) ( Desfougères et al , 2016b ; Wild et al , 2016 ). Vacuoles from these strains accumulated polyP at a two to seven times higher initial rate than the wildtype and to higher concentrations. Nevertheless, their accumulation of P i arrested at the same level as that of wildtype vacuoles, at 5 nmol µg -1 vacuolar protein. To estimate the corresponding luminal concentration of P i , we measured the diameters of 100 isolated wildtype vacuoles and counted the number of vacuoles per µg of vacuolar protein. With an average diameter of 0.8 µm ( Suppl. Fig. 1 ) and 5*10 7 vacuoles/µg of protein we can estimate that P i accumulation in vacuoles incubated with 5-InsP 7 reached a plateau at a luminal concentration of around 30 mM. vtc4Δ vacuoles showed only a very small increase of less than 3 mM in luminal P i content. Download figure Open in new tab Supplementary Figure 1: Isolated vacuoles during polyP synthesis BJ 4742 (WT) vacuoles were incubated in vitro as in Fig. 1B . After 10 min, 0.1μM of the fluorophore FM4-64 was added to stain the membranes. The suspension was immediately imaged on a Yokogawa spinning disc microscope with a Gataca LiveSR unit and a 100x 1.45 NA objective. Stacks were taken at 0.2 µM z-distance. The image shows an optical section out of such a stack. To determine vacuole diameters, the stacks were subjected to maximum intensity projection in ImageJ. The largest visible diameter for 100 organelles was measured manually using ImageJ, yielding an average diameter of 0.8 µm with an SD of 0.2 µm Scale bar: 1 µm The 30 mM of P i accumulating in wildtype vacuoles is a substantial concentration, which is in the range of the in vivo P i concentration of 25 mM that was measured by 31 P-NMR spectroscopy of yeast cells under conditions where vacuolar P i dominates the signal ( Okorokov et al , 1980 ). Since, upon P i scarcity, cytosolic P i drops to 1 mM ( Okorokov et al , 1980 ), a substantial P i concentration gradient across the vacuolar membrane could drive rapid replenishment of the cytosol from a readily accessible vacuolar pool of P i . Vacuolar P i accumulation is limited by feedback inhibition of Ppn1 and Ppn2 We asked why vacuolar P i accumulation quickly forms a plateau whereas vacuolar polyP continues to accumulate. To this end, we explored the hypothesis that vacuolar polyphosphatases become inhibited when their product, P i , has accumulated. To test polyphosphatase activity we liberated the polyphosphatases from isolated vacuoles by detergent lysis, followed by incubation with synthetic polyP as a substrate and DAPI as an indicator for polyP. PolyP was efficiently degraded by this extract. The apparent polyPase activity was significantly reduced by 3 mM P i and efficiently silenced by 30 mM P i ( Fig. 3A ). To differentiate the contributions of Ppn1 and Ppn2, we also analysed the respective deletion mutants and performed the assay at higher polyP concentration in the presence Mg 2+ , which favours Ppn1 activity, or in the presence of Zn 2+ , which supports activity of Ppn1 and Ppn2 ( Gerasimaite & Mayer, 2017 ). In the presence of Zn 2+ , the substrate was consumed in less than 3 min ( Fig. 3 ). Degradation was delayed in vacuoles from ppn1Δ or ppn2Δ mutants, and it was suppressed in vacuoles from a ppn1Δ ppn2Δ mutant, in which both polyphosphatases were ablated. In incubations with only Mg 2+ instead of Zn 2+ , which stimulates the activity of Ppn1 much more than that of Ppn2, polyP degradation was slower and genetic ablation of Ppn1 sufficed to stabilize polyP. The addition of 30 mM potassium phosphate, which is equivalent to the maximal P i accumulation in vacuoles that we observed in Fig. 2 , attenuated degradation of polyP 5-to 10-fold, both through Ppn1 (assayed in ppn2Δ and in samples without Zn 2+ ) and through Ppn2 (assayed in ppn1Δ ). That both polyphosphatases are inhibited by P i at this concentration is consistent with the notion that product inhibition of Ppn1 and Ppn2 might limit the conversion of polyP into P i in the vacuolar lumen and thus define the maximal concentration of the vacuolar P i reservoir. Download figure Open in new tab Figure 3. Effect of P i on polyphosphatase activity. A. P i titration. Vacuoles were isolated from logarithmically growing wildtype cells (BY4742). The organelles were diluted in polyphosphatase assay reaction buffer, which contained 0.1% Triton X-100 and hence liberated the luminal polyphosphatases. This lysate was incubated with 30 μM polyP 300 as a substrate and supplemented with 1 mM ZnCl 2 and the indicated concentrations of K-P i pH 6.8. After the indicated times of incubation, the remaining polyP was quantified through DAPI. The DAPI signal at the beginning of the incubation served as 100% reference. Graphs represent the means ± SEM of three independent experiments. B, C. Differentiation of P i effects on Ppn1 and Ppn2. Vacuoles were isolated from the indicated, logarithmically growing strains, lysed and used in polyP degradation assays with 300 µM polyP 300 as in (A). These assays were performed in the presence or absence of 30 mM K-P i pH 6.8 and 1 mM MgCl 2 (B) or, instead of this, with 1 mM ZnCl 2 (C) as cation supporting catalytic activity. Pho91 limits vacuolar P i accumulation in an inositol pyrophosphate-dependent manner To allow vacuoles to function as a P i buffer for the cytosol, the P i in the vacuolar lumen should become accessible in a regulated manner. The P i transporter Pho91 is a prime candidate for mediating regulatable efflux because it is regulated by InsPPs through its SPX domain ( Potapenko et al , 2018 , 2019 ; Wang et al , 2015 ; Hürlimann et al , 2007 ). Furthermore, an overexpressed GFP fusion was reported to localize to vacuoles ( Hürlimann et al , 2007 ; Elbaz-Alon et al , 2014 ), a phenotype that we confirmed (not shown). Since overexpression of membrane proteins in yeast easily leads to their erroneous accumulation in vacuoles, we re-investigated Pho91 localization in the absence of a strong, overexpressing promotor. When we expressed C- or N-terminal Pho91-GFP fusions from the authentic PHO91 promotor, we observed different and complex localization patterns (see Supplementary Figure 2 for examples). Download figure Open in new tab Supplementary Figure 2: Effects of N- and C-terminal fluorescent protein tags on the localization of Pho91. PHO91 was fused with a variety of N- or C-terminal protein tags and peptide spacers as indicated. They were expressed from the endogenous PHO91 promotor or, where indicated, from the overexpressing ADH1 or GPD1 promotors. Cells were logarithmically grown overnight in SC medium, harvested at OD 600nm of 1-2, and analysed by fluorescence microscopy. These localization patterns depended on the nature of the fluorescent tag and the linker peptides used to attach it to Pho91, and they often showed significant accumulation in the ER. This suggests that the fluorescent protein tags interfere with intracellular trafficking of Pho91. We hence analysed purified vacuoles to test whether the non-tagged, endogenous Pho91 is a vacuolar protein. Mass spectrometry was used to determine the enrichment of proteins in the vacuolar fraction relative to a whole cell extract (Suppl. Table 1). Peptides from Pho91 were enriched in the purified vacuoles to a similar degree (39-fold) as peptides from vacuolar marker proteins, such as the vacuolar polyP polymerase subunit Vtc3 (35-fold), the vacuolar amino acid transporter Avt3 (48-fold), the v-ATPase subunit Vma9 (46-fold), or the alkaline phosphatase Pho8 (34-fold). By contrast, typical plasma membrane proteins were barely enriched, such as the iron permease Ftr1 (2.5-fold), the P i importer Pho87 (2.4-fold) or the polyamine importer Tpo5 (2.2-fold) (Supplementary data file 1). This co-enrichment suggests that the major fraction of non-tagged Pho91 indeed resides in the vacuole. In agreement with this, Pho91 engages the AP3-dependent vesicular trafficking pathway, which leads from the Golgi to the vacuole ( Eising et al , 2022 ). In contrast to the other four yeast Pi transporters, Pho84, Pho87, Pho89, and Pho90, which individually suffice to support vigorous growth of yeast, Pho91 was reported to do support slower but significant growth and to fully rescue upon overexpression ( Wykoff & O’Shea, 2001 ; Wykoff et al , 2007 ). Since this capacity to feed the cells is difficult to reconcile with a vacuolar localisation, unless the cells would feed through fluid phase endocytosis and subsequent export from the vacuole ( Klompmaker et al , 2017 ), we reinvestigated this aspect. Instead of relying a down-regulatable PHO84 background, which results in a low but constitutive background expression of the potent Pi importer Pho84 in addition to Pho91, we relied on a more stringent approach, using plasmid shuffling to completely remove PHO89 when PHO91 is brought in. To this end, we generated a quintuple knockout strain lacking these five P i transporters (Pho84, Pho87, Pho89, Pho90, Pho91), which was kept alive through expression of Pho89 from a URA-based plasmid. Plasmid shuffling allowed to exchange this plasmid against others expressing an individual P i transporter. Whereas an individual plasma-membrane P i transporter such as Pho89 supported normal colony formation, confirming previous observations ( Wykoff et al , 2007 ; Wykoff & O’Shea, 2001 ), we could not confirm that Pho91 supports slower growth. In our plasmid shuffling approach, Pho91 did not support growth, at all ( Suppl. Fig. 3 ). This supports that Pho91 is indeed a vacuolar P i transporter, but that C- or N-terminal protein fusions interfere with correct sorting and cannot serve as reliable reporters for localization of this protein. Download figure Open in new tab Supplementary Figure 3: Pho91 cannot replace other P i transporters to support growth of yeast. We generated a BY4741 strain with a quintuple deletion of the genes for the known P i transporters, PHO84, PHO87, PHO89, PHO90 and PHO91 (D5m). These cells were kept alive by expressing the gene for the plasma membrane P i transporter PHO89 from a URA3-based centromeric (single copy) plasmid (pRS416). PHO91 was expressed from a HIS3-based centromeric plasmid (pRS315). Cells were plated in a dilution series on SC lacking histidine (SC-HIS) to verify that the cells had the HIS3-based PHO91 plasmid, or on SC with 5-fluoro-orotic acid (5-FOA), a drug that forces cells to lose the URA3-based pRS416 and thus to live without PHO89. PHO91 as the sole P i transporter gene (on SC + 5-FOA) does not allow cells to grow. We tested the impact of Pho91 on P i accumulation by vacuoles in vitro, using the same approach as above ( Fig. 4 ). Vacuoles lacking Pho91 ( pho91Δ ) accumulated P i two times faster than the wildtype and the maximal accumulated concentration was two times higher. This is consistent with a function of Pho91 as vacuolar P i exporter ( Hürlimann et al , 2007 ; Potapenko et al , 2018 ; Wang et al , 2015 ). Download figure Open in new tab Fig. 4: Accumulation of P i in isolated vacuoles VTC- and Pho91-dependence. Vacuoles were isolated from the indicated, logarithmically growing strains. The purified organelles were incubated as in Fig. 1 , i.e. in a buffer with an ATP-regenerating system that allows the synthesis of polyP, and either ( A) without ( w/o ) or ( B) in the presence of 50 µM 5-IP 7 . After the indicated periods of incubation at 27°C, an 80 µl aliquot was withdrawn, the vacuoles were sedimented by centrifugation, washed and lysed. Released vacuolar P i was determined by malachite green assay. Graphs represent the mean ± SEM of three independent experiments. p<0.05 from a paired t-test comparing each time WT versus pho91Δ . The difference between pho91Δ and wildtype vacuoles vanished when the vacuoles were incubated in the presence of the inositol pyrophosphate 5-InsP 7 . Since, as we showed above, vacuolar P i accumulation depends on polyP synthesis through VTC, the relative enhancement of P i accumulation in wildtype vacuoles through 5-InsP 7 could be caused by downregulation of Pho91, which would make the wildtype vacuoles behave similarly as pho91Δ vacuoles. Alternatively, 5-InsP 7 might stimulate polyP synthesis in wildtype more than in pho91Δ vacuoles. We could rule out the latter explanation based on two observations: An assay of polyP synthesis during the incubation ( Fig. 5 ) revealed that 5-InsP 7 stimulated polyP synthesis in wildtype and pho91Δ vacuoles to similar degrees. Furthermore, as shown above ( Fig. 2 ), polyP synthesis activity in wildtype cells is not rate-limiting for vacuolar P i accumulation. Therefore, we attribute the enhancement of P i accumulation through 5-InsP 7 to Pho91. Upon P i scarcity, the declining inositol pyrophosphate levels should then activate Pho91 to replenish cytosolic P i . Download figure Open in new tab Fig. 5: PolyP accumulation in pho91 mutant vacuoles. Vacuoles from BY4742 cells (WT) and isogenic pho91Δ cells were isolated and incubated under conditions supporting polyP synthesis and P i accumulation as in Fig. 4 , in the absence (w/o) or presence of 50 µM 5-InsP 7 . At the indicated timepoints, aliquots were withdrawn, the vacuoles were lysed in detergent, and polyP was assayed through DAPI fluorescence. Graphs show the means and SEM from three independent experiments. p>0.05 from a paired t-test for all differences between WT and pho91Δ . Interference with vacuolar polyP turnover provokes cytosolic P i scarcity We tested the effects of this postulated cycle of polyP synthesis, polyphosphatase activity and Pho91-mediated P i export on P i homeostasis in vivo. Pho4-GFP was used as a reporter, because this transcription factor shuttles between nucleus and cytosol. Under cellular P i scarcity and correspondingly low inositol pyrophosphate levels it is predominantly nuclear, but it shifts to the cytosol under P i -replete conditions ( O’Neill et al , 1996 ; Chabert et al , 2023 ; Desfougères et al , 2016a ; Auesukaree et al , 2004 ). Pho4-GFP localisation can hence serve as a readout for cytosolic P i signalling. In wildtype cultures growing logarithmically, cells can experience a transient shortage of P i during S-phase, when P i utilization for biosynthesis may exceed the uptake capacity of the cell and consume its polyP stores ( Bru et al , 2016 ; Neef & Kladde, 2003 ; Pondugula et al , 2009 ). Therefore, even on P i -rich media, some cells can show nuclear accumulation of Pho4-GFP ( Wykoff et al , 2007 ; Vardi et al , 2013 , 2014 ). Since S-phase occupies only around 20% of the yeast cycle ( Koren et al , 2010 ; Donaldson et al , 1998 ), this fraction is expected to be correspondingly small. However, it is significant, and it can be clearly quantified by microscopy, which scores individual cells and not the population as an ensemble. We exploited this natural fluctuation of intracellular P i availability as a sensitising situation to assay how the vacuolar systems influence P i balance in the cytosol. We scored the fraction of logarithmically growing cells that show nuclear Pho4-GFP even in P i -replete medium. This fraction was 7% for wildtype cells ( Fig. 6 ). It increased fourfold upon hyperactivation of polyP synthesis, which we achieved by overexpression of the regulatory VTC subunit Vtc5 ( Desfougères et al , 2016a ). Ablation of polyP synthesis by deletion of the catalytic subunit Vtc4 had the opposite effect and reduced the frequency of cells with nuclear Pho4-GFP by half. Deletion of the vacuolar polyphosphatases Ppn1 and Ppn2 prevents polyP turnover and leads to the accumulation of extremely long polyP chains ( Gerasimaite & Mayer, 2017 ; Sethuraman et al , 2001 ). We may thus expect phosphate to remain fixed in the form of polyP instead of being made available for P i reflux into the cytosol. Download figure Open in new tab Fig. 6: Impact of the vacuolar polyP/P i cycle on cytosolic P i signalling A) Illustration of nucleo-cytoplasmic relocation of Pho4-GFP in response to P i availability. Wildtype yeast cells were grown in SC medium under P i replete conditions. During exponential phase (OD 600nm =1), cells were transferred for 30 min to synthetic complete media with 200 µM phosphate (-P i ) or 7.5 mM P i (+P i ) and imaged by fluorescence microscopy. B) The indicated yeast strains were logarithmically grown over night in SC medium with 7.5 mM P i, , harvested at OD 600nm =1 and immediately imaged by fluorescence microscopy as in A. The graph shows the means and SEM of the percentage of cells showing Pho4 predominantly in the nucleus. n=3 independent experiments with 200 cells quantified per sample. ** p<0.005, *** p<0.001, ****p<0.0001 from a paired t-test comparing WT with the mutants. In line with this, the fraction of ppn1Δ ppn2Δ double mutants that showed nuclear Pho4-GFP was 3-fold higher than in wildtype. pho91Δ cells, in which we expect P i export from the vacuoles to be impaired, showed two times higher frequency of nuclear Pho4-GFP than wildtype. pho91Δ vtc4Δ double mutants showed an even lower frequency of nuclear Pho4-GFP than wildtype, suggesting that the state of P i starvation that pho91Δ favours is dependent on vacuolar polyP accumulation. Cells expressing pho91 K237A as the sole source of Pho91 also showed a 50% lower frequency of nuclear Pho4-GFP. The pho91 K237A allele generates an amino acid substitution in the InsPP-binding patch of the Pho91 SPX domain. It mimics the InsPP-free state ( Wild et al , 2016 ) and hence low-P i conditions ( Chabert et al , 2023 ). Collectively, our results are consistent with the notion that loss of InsPP binding activates Pho91 to export P i from vacuoles to the cytosol, enhancing repression of the PHO pathway. Since InsPP levels in yeast decline in response to cellular P i availability ( Lonetti et al , 2011 ; Chabert et al , 2023 ), we tested the impact of Pho91, Ppn1 and Ppn2 on the cellular levels of these metabolites using CE-MS, capillary electrophoresis coupled to mass spectrometry (CE-MS) ( Qiu et al , 2020 , 2023 , 2021 ). The cells were harvested from the same P i -replete growth conditions as for the microscopic assays above ( Fig. 7 ). Download figure Open in new tab Fig. 7: Impact of Pho91 and vacuolar polyphosphatases on InsPP levels. pho91Δ and ppn1Δppn2Δ cells, as well as their isogenic wildtypes, were logarithmically grown in P i -replete SC medium as in Fig. 6 . At OD 600nm =1 the cells were extracted with perchloric acid as previously described ( Wilson et al , 2015 ) and analysed for the indicated InsPs through capillary electrophoresis coupled to mass spectrometry (CE-MS) as described ( Qiu et al , 2023 ) . n=3. * p<0.05 from a paired t-test comparing WT with the mutants. The ppn1Δ ppn2Δ double mutants showed a decrease by 30 to 50% in the inositol pyrophosphates 1,5-InsP 8 , 5-InsP 7 and 1-InsP 7 , whereas InsP 6 remained at a similar level as in wildtype. InsPP changes at this scale are functionally significant, because they suffice to trigger the initial phase of the P i starvation response ( Chabert et al , 2023 ; Kim et al , 2023 , 2025 ). This is consistent with the 3-fold increase of cells with nuclear Pho4-GFP ( Fig. 6 ). pho91Δ cells did not show significant changes for all four metabolites although they showed a partial shift of Pho4-GFP into the nucleus ( Fig. 6 ). We attribute this discrepancy to the different nature of the assays. The microscopic assay for Pho4-GFP localization can pick up effects in a fraction of the cells because it offers single cell resolution. Inositol pyrophosphate analysis is an ensemble assay, in which changes in a smaller fraction of the population become diluted through the major pool that does not show the effect. For this reason, a change in inositol pyrophosphates affecting only 10% of the pho91Δ cells that show nuclear Pho4-GFP remains undetectable. The microscopic assay suggests that cytosolic P i scarcity in ppn1Δ ppn2Δ affects more cells, perhaps because it is more profound, and it hence becomes detectable even in a whole-population-analysis. Discussion Our observations can be integrated with existing data on the properties of VTC ( Wild et al , 2016 ; Hothorn et al , 2009 ; Müller et al , 2002 ; Gerasimaite et al , 2014 , 2017 ; Liu et al , 2023 ; Guan et al , 2023 ; Pipercevic et al , 2023 ) and Pho91 ( Hürlimann et al , 2007 ; Wang et al , 2015 ; Potapenko et al , 2018 ) to generate a working model explaining how an acidocalcisome-like organelle such as the yeast vacuole is set up to function as a P i buffer for the cytosol. Under P i -replete conditions, high InsPP levels activate VTC to polymerize P i into polyP and translocate it into the vacuolar lumen. Here, the vacuolar polyphosphatases degrade polyP into P i , filling the lumen with P i ( Sethuraman et al , 2001 ; Shi & Kornberg, 2005 ; Lichko et al , 2010 ; Gerasimaite & Mayer, 2017 ). Since the P i exporter Pho91 is downregulated through InsPP binding to its SPX domain ( Potapenko et al , 2018 ; Hürlimann et al , 2007 ; Wang et al , 2015 ), the P i liberated through polyP hydrolysis accumulates in the vacuoles. Product inhibition of the polyphosphatases attenuates polyP hydrolysis once the vacuolar lumen has reached a P i concentration above 30 mM. When the cells experience P i scarcity, InsPP levels decline ( Chabert et al , 2023 ). This activates Pho91 to release P i from the vacuolar pool into the cytosol and stabilizes cytosolic P i . Download figure Open in new tab Fig. 8: Working model of acidocalcisome-like vacuoles as P i buffering systems. (A) Under Pi-replete conditions, ATP drives the conversion of P i into polyP and its translocation into the organelle. Here, polyP is degraded by the vacuolar polyphosphatases Ppn1 and Ppn2 to establish a vacuolar pool of free P i . Feedback inhibition of P i gradually reduces polyP degradation, enabling the buildup of a vacuolar polyP stock. Red lines and SPX colouring indicate inhibitory action, green colouring stimulation. (B) Cytosolic P i scarcity decreases InsPP levels, which triggers two compensatory, SPX-controlled effects: The transfer of P i from the cytosol into vacuoles through VTC ceases; and Pho91-dependent export of P i from vacuoles is activated. Both measures synergize to stabilize cytosolic P i . The export of P i from the vacuole lifts product inhibition on the polyphosphatases Ppn1 and Ppn2 and stimulates a compensatory degradation of polyP. Yeast cells do not only accumulate P i as a rapidly accessible buffer for the cytosol. Under P i -replete conditions they accumulate hundreds of millimolar of phosphate in the form of polyP ( Urech et al , 1978 ). In contrast to the vacuolar reserve of P i , which is presumed to be accessible immediately, mobilizing the polyP store takes minutes to hours ( Nicolay et al , 1982 ; Pondugula et al , 2009 ; Bru et al , 2016 ). But the polyP store offers advantages in the form of high capacity - hundreds of millimolar of phosphate units can be stored in the form of polyP - and low osmotic activity of polyP ( Dürr et al , 1979 ). Keeping such a large stock of a critical resource, which is often growth-limiting in nature, is relevant for the cells. In case of phosphate shortage, the vacuolar polyP store can be mobilized to enable the cells to complete the cell cycle and transition into G 0 phase (Spain et al , 2015; Westenberg et al , 1989 ; Müller et al , 1992 ). This can consume substantial amounts of phosphate, because we can estimate that replicating the entire DNA (1.2*10 7 base pairs) immobilizes roughly 1 mM P i in the cells; and cellular RNA is even 50 times more abundant than DNA ( Warner, 1999 ), accounting for 50 mM phosphate. Phospholipids, which must also be synthesized to complete a cell cycle, fix phosphate in similar amounts ( Lange & Heijnen, 2001 ). Thus, a large polyP store is necessary to guarantee that the cells can finish S-phase upon a shortage of phosphate sources. In accord with this notion, absence of the polyP store impairs cell cycle progression, nucleotide synthesis and induces genome instability ( Bru et al , 2016 , 2017 ). Also, a shift from non-fermentable carbon sources to fermentation of glucose leads to a strong requirement for P i because the activation of glucose uptake and glycolysis depends on large amounts of phosphate-containing sugars and glycolytic intermediates ( Nicolay et al , 1982 , 1983 ; Gillies et al , 1981 ). Shortage of P i restrains the abundance of these metabolites ( Kim et al , 2023 ). The properties of the regulatory circuit described above imply an inbuilt switch from vacuolar P i accumulation to large-scale stocking of vacuolar polyP. P i -replete conditions generate high cellular InsPP levels. These will not only reduce P i efflux from the vacuoles through Pho91 and inactivate the vacuolar polyphosphatases, but at the same time stimulate continued polyP synthesis by VTC. Coincidence of these effects will favour storage and high accumulation of phosphate in the form of polyP. Conversely, depletion of the vacuolar P i reservoir upon P i scarcity in the medium will activate the vacuolar polyphosphatases. In combination with the downregulation of the polyP polymerase VTC through the decline of InsPPs this will mobilize the large vacuolar polyP reserve once the immediately available vacuolar P i pool is gradually depleted. The concentration of P i inside vacuoles as a rapidly accessible P i reserve, and the synthesis of a large polyP stock, come at an energetic cost because the transformation of P i into polyP requires the formation of phosphoric anhydride bonds ( Hothorn et al , 2009 ; Gerasimaite et al , 2014 ) and vacuolar P i reaches 30 mM. This exceeds the cytosolic P i concentration, which was measured through 31 P-NMR in a variety of yeasts, yielding values of 5-17 mM ( Nicolay et al , 1982 , 1983 ). Cytosolic P i can also be estimated based on data from several other studies ( Auesukaree et al , 2004 ; Hürlimann et al , 2009 ; Theobald et al , 1996 ; Zhang et al , 2015 ; Pinson et al , 2004 ). Assuming that the cytosolic volume of a BY4741 yeast cell is 40 fL ( Chabert et al , 2023 ), and 1 g of dry weight contains 40 * 10 9 yeast cells, these studies point to cytosolic values of 10-15 mM in P i -replete media. Upon P i starvation, this value rapidly drops up to fivefold, resulting in a strong P i gradient across the vacuolar membrane ( Okorokov et al , 1980 ; Shirahama et al , 1996 ). To replenish the cytosolic pool under P i scarcity, Pho91 can exploit not only this P i concentration gradient, but also the vacuolar electrochemical potential, which was shown to stimulate P i export through the Pho91 homolog OsSPX-MFS3 from plant vacuoles ( Wang et al , 2015 ). Vacuolar P i accumulation is driven indirectly through ATP in two ways. VTC uses ATP as a substrate and transfers the phosphoric anhydride bond of the ψ-phosphate onto a polyP chain ( Hothorn et al , 2009 ). The growing polyP chain exits from the catalytic site directly towards the transmembrane part of VTC ( Liu et al , 2023 ; Guan et al , 2023 ). This transmembrane part likely forms a controlled channel that can guide polyP through the membrane ( Liu et al , 2023 ). Coupled synthesis and translocation require the V-ATPase ( Gerasimaite et al , 2014 ), probably because polyP is highly negatively charged and therefore follows the electrochemical potential across the vacuolar membrane of 180 mV (inside positive) and 1.7 pH units ( Kakinuma et al , 1981 ), which is generated through the proton pumping V-ATPase. Thus, the combination of the VTC complex and vacuolar polyphosphatases can be considered as a P i pump that is driven by ATP through polyP synthesis and through the electrochemical potential for polyP translocation and P i export. It is likely that acidocalcisome- and lysosome-like organelles of other organisms act as buffers for cytosolic P i similarly as described in our model for yeasts. This notion is supported by the conserved molecular setup of acidocalcisome-like organelles as well as by phenotypic similarities. The acidocalcisomes of Trypanosomes contain VTC, a Pho91 homolog and proton pumps in their membranes, and polyphosphatases in their lumen ( Huang & Docampo, 2015 ; Billington et al , 2023 ; Fang et al , 2007 ; Lander et al , 2013 ; Ulrich et al , 2013 ; Scott et al , 1997 ). Also the acidocalcisome-like organelles of the alga Chlamydomonas contain such proteins and they accumulate polyP through VTC as a function of the availability of P i , a proton gradient and metal ions ( Zúñiga-Burgos et al , 2024 ; Blaby-Haas & Merchant, 2014 ; Long et al , 2023 ; Hong-Hermesdorf et al , 2014 ; Aksoy et al , 2014 ; Ruiz et al , 2001 ; Goodenough et al , 2019 ). Like in yeast, the polyP stores are mobilized upon P i limitation ( Sanz-Luque et al , 2020 ; Plouviez et al , 2021 ). Drosophila has a potentially lysosome-related compartment, which is acidic, carries V-ATPase and a homolog of the Pi exporter XPR1, impacts cytosolic P i and diminishes upon P i starvation ( Xu et al , 2023 ). Mammalian lysosome-like organelles also participate in P i homeostasis. They can accumulate polyP and take up P i ( Pisoni, 1991 ; Pisoni & Lindley, 1992 ). They carry the P i exporter XPR1, which interacts with the plasma membrane P i importer PiT1 to regulate its degradation ( Li et al , 2024 ). We hence propose that acidocalcisome-like vacuoles may have a general role as feedback-controlled, rapidly accessible P i buffers for the cytosol, addressing a critical parameter for metabolism. However, given that acidocalcisome-like organelles accumulate not only phosphate but also multiple other metabolites and ions ( Docampo, 2024 ), they are probably interlinked with cellular metabolism in multiple ways and might form an important hub for its homeostasis. Materials and Methods Yeast strains and growth conditions Saccharomyces cerevisiae cells were grown on yeast extract-peptone-dextrose (YPD: 1% yeast extract, 2% peptone and 2% dextrose) or in synthetic complete (SC) medium from Formedium, supplemented with sodium phosphate as needed). Yeast backgrounds used in this study were BY4741 and BY4742. Genetic manipulations of yeast were performed by homologous recombination according to published procedures and/or transformation with the indicated plasmids ( Gietz & Schiestl, 2007 ; Güldener et al , 1996 ). Strains used are listed in Table 1 . View this table: View inline View popup Table 1: PHO4 localization Cells transformed with pRS415-P PHO4 -PHO4-GFP, a plasmid expressing a PHO4-GFP fusion from the PHO4 promotor ( Chabert et al , 2023 ), were grown exponentially overnight in SC medium without leucine (SC-Leu). Care was taken that the culture did not grow beyond a density of OD 600 =0.7. At this point, the cells were collected by brief centrifugation (15 sec, 3000 x g) in a tabletop centrifuge and resuspended in 1/10th to 1/20th of their own supernatant. Pho4-GFP localization was immediately checked by microscopy. An aliquot of the cells was washed twice with SC-Leu without P i and then diluted in SC-Leu with 200 μM phosphate to OD 600 =0.7. After 30 minutes of incubation in this medium, Pho4-GFP localization was analysed by fluorescence microscopy on a LEICA DMI6000B inverted microscope equipped with a Hamamatsu ORCA-R2 (C10600-10B) camera, an XCite ® series 120Q UV lamp and a Leica 100x 1.4 NA lens. Vacuole preparation Vacuoles were purified from yeast cells essentially as described ( D’Agostino & Mayer, 2019 ). Briefly, yeast cells were grown in 1L of YPD to an OD 600 of 1.5. 330 ml of cells were collected by centrifugation and resuspended in 50 ml of 30 mM Tris-HCl pH 8.9, 10 mM DTT buffer. Suspensions were incubated in a 30° C water bath for 5 min and collected by centrifugation. The pellet was resuspended in 15 ml of spheroplasting solution (50 mM K-phosphate pH 7.5, 600 mM sorbitol in YPD with 0.2% D-glucose and 3600 U/ml lyticase) and incubated for 25 min at 30°C. Spheroplasts were collected by centrifugation (2500 x g, 3 min) and resuspended in 2.5 ml of 15% Ficoll 400 in PS buffer (10 mM PIPES-KOH pH 6.8, 200 mM sorbitol). 80 μg of DEAE-dextran were added under gentle mixing. After incubation on ice for 2 min and then at 30 °C for 80 sec, spheroplasts were transferred into Beckman SW41.1 tubes, overlaid with cushions of 8%, 4% and 0% Ficoll 400 in PS buffer, and centrifuged (150’000 x g, 90 min, 2 °C). Vacuoles were collected from the 0-4 % Ficoll interface. Their protein concentration was determined through Bradford assay using BSA as a standard. Polyphosphatase activity of vacuolar lysates Polyphosphatase activity was assayed as described previously ( Gerasimaite & Mayer, 2017 ), with the following modifications. Isolated vacuoles were diluted to a final protein concentration of 0.002 mg/ml in 1 ml of reaction buffer (20 mM PIPES/KOH pH 6.8, 150 mM KCl, 1 mM ZnCl 2 or MgCwwl 2 , 0.1% Triton X-100, 1xPIC, 1 mM PMSF and 30 or 300 µM polyP 300 ) in the presence or absence of various concentrations of KH 2 PO 4 and incubated at 27°C. At the indicated times, 80 µl aliquots were collected and the reaction was stopped by dilution with 160 µl of stop solution (10 mM PIPES/KOH pH 6.8, 150 mM KCl, 12 mM EDTA pH 8.0, 0.1% Triton X-100, 15 µM DAPI). Remaining polyP was quantified by measuring polyP-DAPI fluorescence (λ exc. 415 nm, λ em. 550 nm) in a black 96 well plate in a Spectramax Gemini microplate fluorometer (Molecular Devices). A reaction containing boiled vacuoles was used as negative control. Polyphosphate synthesis by isolated vacuoles Polyphosphate synthesis was assayed as described ( Gerasimaite et al , 2014 ). Isolated vacuoles were diluted to final protein concentration of 0.02 mg/ml on 1 ml of reaction buffer (10 mM PIPES/KOH pH 6.8, 150 mM KCl, 0.5 mM MnCl2, 200 mM sorbitol) and the reaction was started by adding an ATP regenerating system (1 mM ATP-MgCl 2 , 40 mM creatine phosphate and 0.25 mg/ml creatine kinase). The mix was incubated at 27 °C. At different timepoints, 80 µl aliquots were mixed with 160 µl of stop solution (10 mM PIPES/KOH pH 6.8, 150 mM KCl, 200 mM sorbitol, 12 mM EDTA, 0.15% Triton X-100 and 15 μM DAPI). PolyP synthesis was quantified through polyP-DAPI fluorescence (λ exc. 415 nm, λ em. 550 nm) in a black 96 well plate. A calibration curve was prepared using commercial polyP 60 as a standard. Phosphate quantification in isolated vacuoles Isolated vacuoles were incubated as described for the polyP synthesis assay above. At different time points, 80 µl aliquots were centrifuged (3 min, 2000 x g, 2°C), the pellets were washed with 500 µl of washing solution (10 mM PIPES/KOH pH 6.8, 200 mM Sorbitol, 150 mM KCl) and centrifuged as before. The final pellet was resuspended with 100 µl of lysis buffer (10 mM PIPES/KOH pH 6.8, 200 mM sorbitol, 150 mM KCl, 12 mM EDTA, 0.1 % Triton). Free phosphate was quantified by adding 150 µl of molybdate-malachite green solution (1 mM malachite green, 10 mM ammonium molybdate,1M HCl) and reading the absorbance at 595 nm in a microplate photometer. Inositol pyrophosphate synthesis, extraction and quantification 5-InsP7 was synthesized as described ( Capolicchio et al , 2013 ; Wang et al , 2014 ). For quantification from cells, InsPPs extraction was performed as described previously ( Kim et al , 2023 ), with the following modifications. Briefly, 3 ml of yeast cell culture at OD 600nm =1 was collected using rapid vacuum-filtration on a polytetrafluoroethylene membrane filter (1.2 μm; Piper Filter GmbH, Germany). After snap freezing in liquid nitrogen, yeast cells on the membrane were resuspended in 400 μL of 1 M perchloric acid and lysed by bead beating (glass beads; 0.25-0.5 mm) for 10 min at 4 °C. After centrifugation at 13,000 rpm for 3 min at 4 °C, the supernatant was transferred into a new tube containing 3 mg of titanium dioxide (TiO 2 ) beads (GL Sciences, Japan) which had been pre-washed twice with H 2 O and 1 M perchloric acid. The sample was gently rotated for 15 min at 4 °C. The TiO 2 beads were collected by centrifugation at 13,000 rpm for 1 min at 4 °C and washed twice using 1 M perchloric acid. After the second washing step, the TiO 2 beads were resuspended in 300 μL of 3 % (v/v) NH 4 OH and rotated gently at room temperature. After centrifugation at 13,000 rpm for 1 min, the eluants were transferred into a new tube and dried in SpeedVac (Labogene, Denmark) at 42 °C. InsPPs were measured through capillary electrophoresis coupled to mass spectrometry (CE-MS) as described ( Qiu et al , 2021 ). Quantification of the vacuolar proteome Vacuoles were prepared as described above. During the purification procedure, a sample of the sedimented spheroplasts was withdrawn before DEAE dextran was added. These withdrawn spheroplasts constitute the “whole cell” extract. After the flotation step, the vacuoles withdrawn from the 4%-0% Ficoll interface were used as the vacuole fraction. For each fraction samples with 100 µg protein were precipitated by adding a final concentration 12.5% TCA for 10 min on ice. The proteins were sedimented (12’000 x g, 5 min, room temperature), the supernatant discarded, and the pellets were washed with ice-cold acetone and centrifuged as above twice. The final pellet was dried and dissolved in reducing SDS sample buffer (10% glycerol, 50 mM Tris-HCl pH 6.8, 2 mM DTT, 2% SDS, 0.002% bromophenol blue). Samples dissolved in Laemmli buffer (50 mM Tris pH 6.8, 10 mM DTT, 2 % SDS, 0.1 % bromophenol blue, 10 % glycerol) were equalized in concentration by dilution in SP3 buffer (2% SDS, 10 mM DTT, 50 mM Tris, pH 7.5). 150 µg of protein per sample were heated at 95 °C for 5 min and cooled down. Reduced cysteine residues were alkylated by adding iodoacetamide (30 mM final) and incubating for 45 min at room temperature in the dark. Digestion was done by the SP3 method ( Hughes et al , 2019 ) using magnetic Sera-Mag Speedbeads (Cytiva 45152105050250, 50 mg/ml). Beads were added at a ratio 10:1 (w:w) to samples, and proteins were precipitated on beads with ethanol (final concentration: 60 %). After 3 washes with 80% ethanol, beads were digested in 50 μl of 100 mM ammonium bicarbonate with 3.0 μg of trypsin (Promega #V5073). After 1 h of incubation at 37 °C, the same amount of trypsin was added to the samples for an additional 1 h of incubation. Supernatants were then recovered, transferred to new tubes, acidified with formic acid (0.5% final concentration), and dried by centrifugal evaporation. To remove traces of SDS, two sample volumes of isopropanol containing 1% TFA were added to the digests, and the samples were desalted on a strong cation exchange (SCX) plate (Oasis MCX; Waters Corp., Milford, MA) by centrifugation. After washing with isopropanol/1%TFA and 2% acetonitrile/0.1% FA, peptides were eluted in 200µl of 80% MeCN, 19% water, 1% (v/v) ammonia, and dried by centrifugal evaporation. Data-dependent LC-MS/MS analyses of samples were carried out on a Fusion Tribrid Orbitrap mass spectrometer (Thermo Fisher Scientific) interfaced through a nano-electrospray ion source to an Ultimate 3000 RSLCnano HPLC system (Dionex). Peptides were separated on a reversed-phase custom packed 45 cm C18 column (75 μm ID, 100Å, Reprosil Pur 1.9 μm particles, Dr. Maisch, Germany) with a 4-90% acetonitrile gradient in 0.1% formic acid at a flow rate of 250 nl/min (total time 140 min). Full MS survey scans were performed at 120’000 resolution. A data-dependent acquisition method controlled by Xcalibur software (Thermo Fisher Scientific) was used that optimized the number of precursors selected (“top speed”) of charge 2+ to 5+ while maintaining a fixed scan cycle of 0.6 s. Peptides were fragmented by higher energy collision dissociation (HCD) with a normalized energy of 32%. The precursor isolation window used was 1.6 Th, and the MS2 scans were done in the ion trap. The m/z of fragmented precursors was then dynamically excluded from selection during 60 s. Data files were analysed with MaxQuant 1.6.14.0 ( Cox & Mann, 2008 ) incorporating the Andromeda search engine ( Cox et al , 2011 ). Cysteine carbamidomethylation was selected as fixed modification while methionine oxidation and protein N-terminal acetylation were specified as variable modifications. The sequence databases used for searching were the S. cerevisiae reference proteome based on the UniProt database ( https://www.uniprot.org , version of June 6th, 2021, containing 6050 sequences), and a “contaminant” database containing the most usual environmental contaminants and enzymes used for digestion (keratins, trypsin, etc). Mass tolerance was 4.5 ppm on precursors (after recalibration) and 20 ppm on MS/MS fragments. Both peptide and protein identifications were filtered at 1% FDR relative to hits against a decoy database built by reversing protein sequences. The match between runs feature was enabled. Data availability Source data from the proteomic analysis have been deposited at the PRIDE database under the identifier PXD060102. Author contributions AM conceived the study, analysed data and wrote the paper. SB performed in vitro and in vivo experiments with yeast. GDK, LM, GL, and HJJ analysed inositol pyrophosphates. All authors assembled and corrected the manuscript. Competing interests None Acknowledgements Mass spectrometry-based proteomics work was performed by the Protein Analysis Facility of the Faculty of Biology and Medicine, University of Lausanne. This study was supported by grants from the SNSF (320030-228119, 31003A_179306 and 310030_204713) and ERC (788442) to AM, by the HFSP (LT000588/2019) to GDK, by the Deutsche Forschungsgemeinschaft (CIBSS, EXC-2189, Project ID 390939984, to HJJ) and the Volkswagen Foundation (VW Momentum Grant 98604 to HJJ). Funder Information Declared Swiss National Science Foundation, https://ror.org/00yjd3n13 , 320030-228119 , 31003A_179306 , 310030_204713 European Research Council , 788442 Deutsche Forschungsgemeinschaft , EXC-2189 Project 390939984 Volkswagen Foundation, https://ror.org/03bsmfz84 , 98604 International Human Frontier Science Program Organization , LT000588/2019 Footnotes Titration of Pi sensitivity of polyPases Sample picture of isolated vacuoles https://www.ebi.ac.uk/pride/archive/projects/PXD060102 References ↵ Aksoy M , Pootakham W & Grossman AR ( 2014 ) Critical Function of a Chlamydomonas reinhardtii Putative Polyphosphate Polymerase Subunit during Nutrient Deprivation . Plant Cell 26 : 4214 – 4229 OpenUrl Abstract / FREE Full Text ↵ Andreeva N , Ledova L , Ryazanova L , Tomashevsky A , Kulakovskaya T & Eldarov M ( 2019 ) Ppn2 endopolyphosphatase overexpressed in Saccharomyces cerevisiae: Comparison with Ppn1, Ppx1, and Ddp1 polyphosphatases . 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Share Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol Samuel Bru , Lydie Michaillat Mayer , Geun-Don Kim , Danye Qiu , Henning Jacob Jessen , Andreas Mayer bioRxiv 2025.05.30.656997; doi: https://doi.org/10.1101/2025.05.30.656997 Share This Article: Copy Citation Tools Acidocalcisome-like vacuoles constitute a feedback-controlled phosphate buffering system for the cytosol Samuel Bru , Lydie Michaillat Mayer , Geun-Don Kim , Danye Qiu , Henning Jacob Jessen , Andreas Mayer bioRxiv 2025.05.30.656997; doi: https://doi.org/10.1101/2025.05.30.656997 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Cell Biology Subject Areas All Articles Animal Behavior and Cognition (7617) Biochemistry (17633) Bioengineering (13856) Bioinformatics (41840) Biophysics (21398) Cancer Biology (18529) Cell Biology (25422) Clinical Trials (138) Developmental Biology (13352) Ecology (19860) Epidemiology (2067) Evolutionary Biology (24281) Genetics (15582) Genomics (22461) Immunology (17700) Microbiology (40289) Molecular Biology (17138) Neuroscience (88413) Paleontology (666) Pathology (2823) Pharmacology and Toxicology (4813) Physiology (7632) Plant Biology (15107) Scientific Communication and Education (2042) Synthetic Biology (4283) Systems Biology (9807) Zoology (2267)