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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.

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.