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

Cells actively manage the concentration of P_i in their cytosol because they must strike a balance between conflicting goals (Austin and 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 this 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., 2017; Bru et al., 2016; Gillies et al., 1981; Neef and 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 (Okorokov et al., 1980; Urech et al., 1978). 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, 2024; Docampo et al., 2005; Girard-Dias et al., 2023). 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 (Austin and Mayer, 2020; Dürr et al., 1979; Huber-Wälchli and Wiemken, 1979; Urech et al., 1978) or metal uptake (Klompmaker et al., 2017). But they have an additional, lysosome-like face, enabling them to assume also hydrolytic degradative functions for the cells (Li and 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 increase in P_i consumption, e.g., during metabolic transitions from respiratory growth to fermentation, which requires many more phosphorylated metabolites (Gillies et al., 1981; Nicolay et al., 1983; Nicolay et al., 1982; Shirahama et al., 1996; Thomas and O’Shea, 2005). 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; Nicolay et al., 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, 2024; Docampo and Huang, 2016). 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 (Gerasimaitė and Mayer, 2016; Huang and Docampo, 2015; Hürlimann et al., 2007; Jimenez and Docampo, 2015; Müller et al., 2002; Müller et al., 2003; Wang et al., 2015). VTC is a coupled polyP polymerase and translocase (Gerasimaitė et al., 2014), which synthesises polyP by transferring the γ-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 dimerised 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-IP₈ (Chabert et al., 2023; Gerasimaitė et al., 2017; Kim et al., 2025; Kim et al., 2023).

The postulated direct channelling of polyP from the site of its synthesis through the vacuolar membrane (Gerasimaitė 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 (Gerasimaitė et al., 2014; Guan et al., 2023; Liu et al., 2023; Müller et al., 2002; Müller et al., 2003). 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 (Freimoser et al., 2006; Gerasimaitė et al., 2014; Wild et al., 2016; 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 homologues from other organisms are consistent with this view. Its ablation increases vacuolar P_i and polyP content, in yeast, as well as in trypanosomes (Farofonova et al., 2023; Hürlimann et al., 2007; Jimenez and Docampo, 2015), and it weakly induces the phosphate starvation programme 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., 2019; Potapenko et al., 2018; Wang et al., 2015). In the presence of a pH gradient, the Pho91 homologue 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 (Gerasimaitė and Mayer, 2016; Kulakovskaya et al., 2021; Lander et al., 2016; McCarthy and Downey, 2023), which can convert polyP back into P_i. In baker’s yeast, two vacuolar polyphosphatases are known: Ppn1 and Ppn2 (Andreeva et al., 2019; Gerasimaitė and Mayer, 2017; Sethuraman et al., 2001). This means that a chain of polyP, when being synthesised 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-synthesising 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 downregulation 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 and O’Shea, 2005). We hence explored the capacity of isolated yeast vacuoles to interconvert polyP and P_i, and we characterised 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.

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 (D’Agostino and Mayer, 2019; Kunz et al., 2004; Michaillat et al., 2012; Sattler and Mayer, 2000). They also contain active polyP polymerase (VTC) and active polyphosphatases (Ppn1 and Ppn2) and they can synthesise and import polyP (Gerasimaitė et al., 2014; Gerasimaitė 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 synthesise polyP in vitro (Gerasimaitė et al., 2014). After different periods of incubation, the organelles were sedimented and solubilised 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-InsP₈ (Gerasimaitė et al., 2017; Gerasimaitė et al., 2014). Since 1,5-InsP₈ is not commercially available, we used saturating concentrations of 5-InsP₇ for our experiments (Pavlovic et al., 2015). This compound stimulates VTC with a higher EC₅₀ but to the same maximal activity as 1,5-InsP₈, and it was more readily available to us for routine experiments. Under the conditions used here, the organelles rapidly and efficiently produced polyP (Figure 1), in line with previous results (Gerasimaitė et al., 2017; Gerasimaitė et al., 2014). This production was stimulated by 5-InsP₇, reaching 0.7 nmol of phosphate units per µg of vacuolar protein per 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 synthesise polyP. This signal was entirely dependent on VTC, because it was not observed in a mutant lacking the catalytic subunit of VTC (vtc4Δ).

Figure 1

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Isolated wildtype vacuoles also accumulated P_i with significant efficiency, at an initial rate of at least 0.3 nmol/µg/min (Figure 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. Thus, vacuolar P_i accumulation depended on polyP synthesis through VTC. But 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 (Figure 2A and 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 (Figure 2A and B) or strains carrying hyperactivating substitutions in the SPX domains of VTC (vtc3^K126A vtc4^K129A; Figure 2C and 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 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 (Figure 2—figure supplement 1) and 5*10⁷ vacuoles/µg of protein, we can estimate that P_i accumulation in vacuoles incubated with 5-InsP₇ 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. 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 ³¹P-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.

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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 (Figure 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 of Mg²⁺, which favours Ppn1 activity, or in the presence of Zn²⁺, which supports activity of Ppn1 and Ppn2 (Gerasimaitė et al., 2017). In the presence of Zn²⁺, the substrate was consumed in less than 3 min (Figure 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 Mg²⁺ instead of Zn²⁺, which stimulates the activity of Ppn1 much more than that of Ppn2, polyP degradation was slower and genetic ablation of Ppn1 sufficed to stabilise polyP. The addition of 30 mM potassium phosphate, which is equivalent to the maximal P_i accumulation in vacuoles that we observed in Figure 2, attenuated degradation of polyP 5- to 10-fold, both through Ppn1 (assayed in ppn2Δ and in samples without Zn²⁺) 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.

Figure 3

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The transporter 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 (Hürlimann et al., 2007; Potapenko et al., 2019; Potapenko et al., 2018; Wang et al., 2015). However, its vacuolar localisation is challenged by reports that yeast can show significant growth mediated by Pho91 as the sole Pi transporter, and that, upon overexpression, Pho91 rescues growth to a similar degree as any of the four yeast plasma membrane P_i transporters, Pho84, Pho87, Pho89, and Pho90 (Wykoff et al., 2007; Wykoff and O’Shea, 2001). Since this capacity to feed the cells is difficult to reconcile with a vacuolar localisation – unless the cells would absorb P_i through fluid phase endocytosis and subsequent export from the vacuole (Klompmaker et al., 2017) – we reinvestigated this aspect. Instead of relying on a downregulatable PHO84 background, which results in a low but constitutive background expression of the potent P_i importer Pho84 in addition to Pho91 (Wykoff et al., 2007; Wykoff and O’Shea, 2001), we took 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 and O’Shea, 2001), we could not confirm that Pho91 supports slower growth. In our plasmid shuffling approach, Pho91 did not support growth at all (Figure 4).

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An overexpressed GFP-Pho91 fusion had been reported to localise to vacuoles (Elbaz-Alon et al., 2014; Hürlimann et al., 2007), 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 localisation in the absence of a strong, overexpressing promoter. When we expressed C- or N-terminal GFP fusions of Pho91 from the authentic PHO91 promoter, we observed considerably weaker signals and different and complex localisation patterns (see Figure 4—figure supplement 1 for examples). These localisation 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 N- and C-terminal fluorescent protein tags interfere with intracellular trafficking of Pho91.

We hence used mass spectrometry of purified vacuoles to test whether the non-tagged, endogenous Pho91 is a vacuolar protein. To this end, we determined the enrichment of proteins in the vacuolar fraction relative to a whole cell extract (Supplementary file 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 file 1). Its co-enrichment with vacuolar proteins 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). Thus, Pho91 is indeed a vacuolar P_i transporter, but C- or N-terminal protein fusions easily interfere with correct sorting and cannot serve as reliable reporters for localisation of this protein.

We tested the impact of Pho91 on P_i accumulation by vacuoles in vitro, using the same approach as above (Figure 5). 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). The difference between pho91Δ and wildtype vacuoles vanished when the vacuoles were incubated in the presence of the inositol pyrophosphate 5-InsP₇. 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-InsP₇ could be caused by downregulation of Pho91, which would make the wildtype vacuoles behave similarly to pho91Δ vacuoles. Alternatively, 5-InsP₇ 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 (Figure 6) revealed that 5-InsP₇ stimulated polyP synthesis in wildtype and pho91Δ vacuoles to similar degrees. Furthermore, as shown above (Figure 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-InsP₇ to Pho91. Upon P_i scarcity, the declining inositol pyrophosphate levels should then activate Pho91 to replenish cytosolic P_i.

Figure 5

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Figure 6

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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 (Auesukaree et al., 2004; Chabert et al., 2023; Desfougères et al., 2016a; O’Neill et al., 1996). 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 utilisation for biosynthesis may exceed the uptake capacity of the cell and consume its polyP stores (Bru et al., 2016; Neef and Kladde, 2003; Pondugula et al., 2009). Therefore, even on P_i-rich media, some cells can show nuclear accumulation of Pho4-GFP (Vardi et al., 2014; Vardi et al., 2013; Wykoff et al., 2007). Since S-phase occupies only around 20% of the yeast cycle (Donaldson et al., 1998; Koren et al., 2010), 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 (Figure 7). 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 (Gerasimaitė et al., 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.

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In line with this, the fraction of ppn1Δ ppn2Δ double mutants that showed nuclear Pho4-GFP was threefold 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 (Chabert et al., 2023; Lonetti et al., 2011), we tested the impact of Pho91, Ppn1, and Ppn2 on the cellular levels of these metabolites using capillary electrophoresis coupled to mass spectrometry (CE-MS) (Qiu et al., 2023; Qiu et al., 2021; Qiu et al., 2020). The cells were harvested from the same P_i-replete growth conditions as for the microscopic assays above (Figure 8). The ppn1Δ ppn2Δ double mutants showed a decrease of 30–50% in the inositol pyrophosphates 1,5-InsP₈, 5-InsP₇, and 1-InsP₇, whereas InsP₆ 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., 2025; Kim et al., 2023). This is consistent with the threefold increase of cells with nuclear Pho4-GFP (Figure 7). pho91Δ cells did not show significant changes for all four metabolites although they showed a partial shift of Pho4-GFP into the nucleus (Figures 7 and 8). We attribute this discrepancy to the different nature of the assays. The microscopic assay for Pho4-GFP localisation 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.

Figure 8

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Our observations can be integrated with existing data on the properties of VTC (Gerasimaitė et al., 2017; Gerasimaitė et al., 2014; Guan et al., 2023; Hothorn et al., 2009; Liu et al., 2023; Müller et al., 2002; Pipercevic et al., 2023; Wild et al., 2016) and Pho91 (Hürlimann et al., 2007; Potapenko et al., 2018; Wang et al., 2015) 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 (Figure 9). Under P_i-replete conditions, high InsPP levels activate VTC to polymerise 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 (Gerasimaitė et al., 2017; Lichko et al., 2010; Sethuraman et al., 2001; Shi and Kornberg, 2005). Since the P_i exporter Pho91 is downregulated through InsPP binding to its SPX domain (Hürlimann et al., 2007; Potapenko et al., 2018; 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 stabilises cytosolic P_i.

Figure 9

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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, mobilising the polyP store takes minutes to hours (Bru et al., 2016; Nicolay et al., 1982; Pondugula et al., 2009). 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 mobilised to enable the cells to complete the cell cycle and transition into G₀ phase (Müller et al., 1992; Jiménez et al., 2015; Westenberg et al., 1989). This can consume substantial amounts of phosphate, because we can estimate that replicating the entire DNA (1.2*10⁷ base pairs) immobilises 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 synthesised to complete a cell cycle, fix phosphate in similar amounts (Lange and 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 accordance with this notion, the absence of the polyP store impairs cell cycle progression and nucleotide synthesis, and induces genome instability (Bru et al., 2017; Bru et al., 2016). 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 (Gillies et al., 1981; Nicolay et al., 1983; Nicolay et al., 1982). A 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 mobilise 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, comes at an energetic cost because the transformation of P_i into polyP requires the formation of phosphoric anhydride bonds (Gerasimaitė et al., 2014; Hothorn et al., 2009) and vacuolar P_i reaches 30 mM. This exceeds the cytosolic P_i concentration, which was measured through ³¹P-NMR in a variety of yeasts, yielding values of 5–17 mM (Nicolay et al., 1983; Nicolay et al., 1982). Cytosolic P_i can also be estimated based on data from several other studies (Auesukaree et al., 2004; Hürlimann et al., 2009; Pinson et al., 2004; Theobald et al., 1996; Zhang et al., 2015). 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*10⁹ 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 homologue 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 γ-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 (Guan et al., 2023; Liu 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 (Gerasimaitė 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 homologue and proton pumps in their membranes, and polyphosphatases in their lumen (Billington et al., 2023; Fang et al., 2007; Huang and Docampo, 2015; Lander et al., 2013; Scott et al., 1997; Ulrich et al., 2013). 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 (Aksoy et al., 2014; Blaby-Haas and Merchant, 2014; Goodenough et al., 2019; Hong-Hermesdorf et al., 2014; Long et al., 2023; Ruiz et al., 2001; Zúñiga-Burgos et al., 2024). Like in yeast, the polyP stores are mobilised upon P_i limitation (Plouviez et al., 2021; Sanz-Luque et al., 2020). Drosophila has a potentially lysosome-related compartment, which is acidic, carries V-ATPase and a homologue 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 and 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.

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Author details

1. Samuel Bru

   Département d'immunobiologie, Université de Lausanne, Epalinges, Switzerland

   Present address

   Department of Biomedical Sciences, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Barcelona, Spain

   Contribution

   Conceptualization, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-7005-8609

2. Lydie Michaillat Mayer

   Département d'immunobiologie, Université de Lausanne, Epalinges, Switzerland

   Contribution

   Data curation, Formal analysis, Validation, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0008-3292-5899

3. Geun-Don Kim

   Département d'immunobiologie, Université de Lausanne, Epalinges, Switzerland

   Contribution

   Formal analysis, Investigation, Methodology, Writing – original draft

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-3580-2471

4. Danye Qiu

   Institute of Organic Chemistry University of Freiburg and CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

   Contribution

   Data curation, Formal analysis, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

5. Henning J Jessen

   Institute of Organic Chemistry University of Freiburg and CIBSS – Centre for Integrative Biological Signalling Studies, University of Freiburg, Freiburg, Germany

   Contribution

   Resources, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-1025-9484

6. Andreas Mayer

   Département d'immunobiologie, Université de Lausanne, Epalinges, Switzerland

   Contribution

   Resources, Data curation, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Writing – original draft, Project administration, Writing – review and editing

   For correspondence

   andreas.mayer@unil.ch

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-6131-313X

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