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• W4386896889 abstract "Full text Figures and data Side by side Abstract eLife assessment Introduction Results Discussion Materials and methods Data availability References Peer review Author response Article and author information Metrics Abstract Eukaryotic cells control inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impact on reactions liberating phosphate. Phosphate homeostasis depends on the conserved INPHORS signaling pathway that utilizes inositol pyrophosphates and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates of yeast, 1-IP7, 5-IP7, and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8 triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8 inactivates the cyclin-dependent kinase inhibitor Pho81 through its SPX domain. This stimulates the cyclin-dependent kinase Pho85-Pho80 to phosphorylate the transcription factor Pho4 and repress the PHO pathway. Combining our results with observations from other systems, we propose a unified model where 1,5-IP8 signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses. eLife assessment This fundamental study describes the mechanisms for regulation of the phosphate starvation response in baker's yeast, clarifies the interpretations of prior data, and suggests a unifying mechanism across eukaryotes. The study provides compelling data, based on biochemical analyses, protein localization by fluorescence, and genetic approaches that 1,5-InsP8 is the phosphate nutrient messenger in yeast. https://doi.org/10.7554/eLife.87956.3.sa0 About eLife assessments Introduction Inorganic phosphate (Pi) is an essential nutrient for all living systems. While required in large amounts for synthesis of nucleic acids, phospholipids, and phosphorylated carbohydrates and proteins, an overaccumulation of Pi decreases the free energy provided by Pi-liberating reactions, such as nucleotide hydrolysis, which might stall metabolism (Austin and Mayer, 2020). In fungi, plants, and animals, control of Pi homeostasis involves myo-inositol pyrophosphates and a family of evolutionarily conserved SPX domains, constituting the core of a postulated signaling pathway that we termed INPHORS (Austin and Mayer, 2020; Azevedo and Saiardi, 2017). SPX domains interact with or form part of a large variety of proteins that affect Pi homeostasis by transporting Pi across membranes, converting it into polyphosphates (polyP) or other metabolites, or regulating Pi-dependent transcription (Secco et al., 2012). Direct regulation of an SPX-containing protein by synthetic inositol pyrophosphates was shown for the polyP polymerase VTC, the Pi transporters Pho91 and the PHR transcription factors in plants (Gerasimaite et al., 2017; Potapenko et al., 2018; Wild et al., 2016; Ried et al., 2021; Wang et al., 2015; Liu et al., 2016; Dong et al., 2019; Liu et al., 2023; Pipercevic et al., 2023; Guan et al., 2023). A firm link suggesting that the SPX domain as the receptor for inositol pyrophosphate regulation was provided by point mutants in the SPX domain that rendered VTC either independent of activation by inositol pyrophosphates or non-responsive to them (Wild et al., 2016). Structural analysis revealed that many of these mutations localized to a highly charged region that can bind inositol poly- and pyrophosphates with high affinity. However, this binding site discriminates poorly between different inositol pyrophosphates at the level of binding (Wild et al., 2016). By contrast, strong differences are observed in the agonist properties, leading to the suggestion that binding affinity is a poor predictor of inositol pyrophosphate specificity and activity (Austin and Mayer, 2020; Gerasimaite et al., 2017; Pipercevic et al., 2023). Inositol pyrophosphates that can be found in a wide variety of organisms carry seven (IP7) or eight (IP8) phosphates occupying every position around myo-inositol ring. The IP7 isomers 1PP-InsP5 and 5PP-InsP5 carry diphosphate groups at the 1- or 5-position, respectively, and the 1,5(PP)2-InsP4 carries two diphosphate groups at the 1- and 5-position. For convenience, we refer to these inositol pyrophosphates as 5-IP7, 1-IP7, and 1,5-IP8 from hereon. All three inositol pyrophosphates are linked to phosphate homeostasis by the fact that genetic ablation of the enzymes making them, such as IP6Ks (inositol hexakisphosphate kinases), PPIP5Ks (diphosphoinositol pentakisphosphate kinases), and ITPKs (inositol tris/tetrakisphosphate kinases), alters the phosphate starvation response. Considerable discrepancies exist in the assignment of these inositol pyrophosphates to different aspects of Pi homeostasis. In mammalian cells, 1,5-IP8 activates the only SPX protein expressed in this system, the Pi exporter XPR1 (Li et al., 2020; Wilson et al., 2019). 1,5-IP8 was also proposed as a regulator of the phosphate starvation response in Arabidopsis because 1,5-IP8, but not 5-IP7, promotes the interaction of SPX1 with the Pi-responsive transcription factor PHR1 in vitro (Dong et al., 2019). On the other hand, quantitative measurements of the interaction of inositol pyrophosphates with rice SPX4 and its cognate Pi-responsive transcription factor PHR2 revealed only a minor, twofold difference in the Kd values for 5-IP7 and 1,5-IP8. 1-IP7, 5-IP7, and 1,5-IP8 all decrease upon Pi starvation and mutants lacking either of the enzymes necessary for their synthesis in plants, the ITPKs and the PPIP5Ks, induce the phosphate starvation response (Dong et al., 2019; Riemer et al., 2021; Laha et al., 2015; Zhu et al., 2019). Analysis of respective Arabidopsis mutants revealed that Pi concentration in shoots correlates poorly with their content of IP7 and IP8 (Riemer et al., 2021). While itpk1 mutants, lacking one of the enzymes that generates 5-IP7, have similar 1,5-IP8 content as wildtype, their Pi content is almost twofold increased and their 5-IP7 content is reduced by a factor of four. Inversely, mutants lacking the PPIP5K VIH2 show a fivefold reduction of 1,5-IP8 but normal Pi content (Riemer et al., 2021). Although 1,5-IP8 is the inositol pyrophosphate that is the most responsive to Pi starvation and Pi re-feeding (Riemer et al., 2021) this has rendered it difficult to clearly resolve whether IP7, IP8, or both signal cellular Pi status to the Pi starvation program in vivo. That plants are composed of multiple source and sink tissues for phosphate may complicate the analysis, because systemic knockouts of inositol pyrophosphate synthesizing enzymes may exert their main effect in a tissue that is different from the one where the starvation response is scored. Pioneering studies on the regulation of the Pi starvation program of Saccharomyces cerevisiae, the PHO pathway, concluded that this transcriptional response is triggered by an increase in 1-IP7 (Lee et al., 2007; Lee et al., 2008), whereas subsequent studies in the pathogenic yeast Cryptococcus neoformans proposed 5-IP7 as the necessary signaling compound (Desmarini et al., 2020). Studies using the S. cerevisiae polyphosphate polymerase VTC as a model suggested that this enzyme, which is necessary for polyP accumulation under Pi-replete conditions, is stimulated by 5-IP7 in vivo (Gerasimaite et al., 2017; Liu et al., 2023; Lonetti et al., 2011; Auesukaree et al., 2005), whereas studies in Schizosaccharomyces pombe proposed IP8 as the stimulator (Pascual-Ortiz et al., 2021). S. pombe has similar enzymes for IP8 synthesis and hydrolysis as S. cerevisiae (Zhai et al., 2015; Randall et al., 2020; Pascual-Ortiz et al., 2018; Topolski et al., 2016; Pöhlmann et al., 2014). In contrast to S. cerevisiae, genetic ablation of 1-IP7 and IP8 production in S. pombe leads to hyper-repression of the transcriptional phosphate starvation response (Pascual-Ortiz et al., 2018; Sanchez et al., 2019; Benjamin et al., 2022) and interferes with the induction of the transcriptional phosphate starvation response. However, the transcriptional phosphate starvation response in S. pombe occurs through a different set of protein mediators. For example, it lacks homologs of Pho81 and uses Csk1 instead of Pho85-Pho80 for phosphate-dependent transcriptional regulation (Estill et al., 2015; Henry et al., 2011; Carter-O’Connell et al., 2012; Schwer et al., 2021). Furthermore, PHO pathway promotors in S. pombe overlap with and are strongly regulated by lncRNA transcription units (Sanchez et al., 2018; Schwer et al., 2015; Schwer et al., 2014). It is hence difficult to compare the downstream events in this system to the PHO pathway of S. cerevisiae. The discrepancies mentioned above could either reflect a true divergence in the signaling properties of different inositol pyrophosphates in different organisms, in which case a common and evolutionarily conserved signaling mechanism may not exist. Alternatively, the diverging interpretations could reflect limitations in the analytics of inositol pyrophosphates and in the in vivo assays for the fundamental processes of Pi homeostasis. The analysis of inositol pyrophosphates is indeed very challenging in numerous ways. Their cellular concentrations are very low, the molecules are highly charged, and they exist in multiple isomers that differ only in the positioning of the pyrophosphate groups. Inositol pyrophosphate analysis has traditionally been performed by ion exchange HPLC of extracts from cells radiolabeled through 3H-inositol (Wilson and Saiardi, 2017). This approach requires constraining and slow labeling schemes, is costly and time-consuming. Furthermore, HPLC-based approaches in most cases did not resolve isomers of IP7 or IP8. These factors severely limited the number of samples and conditions that could be processed and the resolution of the experiments. The recent use of capillary electrophoresis coupled to mass spectrometry (CE-MS) has dramatically improved the situation, permitting resolution of many regio-isomers of IP7 and IP8 without radiolabeling, and at superior sensitivity and throughput. We harnessed the potential of this method to dissect the role of the three known inositol pyrophosphates that accumulate in S. cerevisiae. We analyzed their impact on the PHO pathway, which is a paradigm for a Pi-controlled transcriptional response and the regulation of phosphate homeostasis (Austin and Mayer, 2020; Eskes et al., 2018; Conrad et al., 2014). Beyond this physiological function, however, the PHO pathway also gained widespread recognition as a model for promotor activation, transcription initiation, chromatin remodeling, and nucleosome positioning (Korber and Barbaric, 2014). In the PHO pathway, the cyclin-dependent kinase inhibitor (CKI) Pho81 translates intracellular Pi availability into an activation of the cyclin-dependent kinase (CDK) complex Pho85-Pho80 (Lee et al., 2007; Schneider et al., 1994; Ogawa et al., 1995; Yoshida et al., 1989). At high Pi, Pho85-Pho80 phosphorylates and inactivates the key transcription factor of the PHO pathway, Pho4, which then accumulates in the cytosol (Kaffman et al., 1998b; Kaffman et al., 1998a; Komeili and O’Shea, 1999). During Pi starvation, Pho81 inhibits the Pho85-Pho80 kinase, leading to dephosphorylation and activation of Pho4 and the ensuing expression of Pi-responsive genes (PHO genes). The activation of Pho85-Pho80 through Pho81 has been explored in detail, leading to a series of highly influential studies that have gained wide acceptance in the field. A critical function was ascribed to 1-IP7 in activating the CDK inhibitor PHO81, allowing it to inhibit Pho85-Pho80 (Lee et al., 2007). IP7 concentration was reported to increase upon Pi starvation, and this increase was considered as necessary and sufficient to inactivate Pho85-Pho80 kinase through Pho81 and trigger the PHO pathway. The 1-IP7 binding site on Pho81 was mapped to a short stretch of 80 amino acids, the ‘minimum domain’ (Lee et al., 2007; Lee et al., 2008; Ogawa et al., 1995; Huang et al., 2001). This domain is in the central region and distinct from the N-terminal SPX domain of Pho81. Since overexpression of the minimum domain rescued Pi-dependent regulation of the PHO pathway to some degree, the key regulatory function was ascribed to this minimum domain and the SPX domain was considered as of minor importance. By contrast, earlier studies based on unbiased mutagenesis, which subsequently received much less attention, had identified mutations in other regions of PHO81 with significant impact on PHO pathway activation (Ogawa et al., 1995; Spain et al., 1995; Toh-E and Oshima, 1974; Creasy et al., 1993; Creasy et al., 1996). The situation is complicated by further studies, which found global IP7 levels to decrease rather than increase upon Pi starvation (Wild et al., 2016; Li et al., 2020; Lonetti et al., 2011; Kim et al., 2023). The analytics used in most studies could not distinguish 1-IP7 and 5-IP7, however, leaving open the possibility that an increase of 1-IP7 might be masked by a decrease of a much larger pool of 5-IP7. To resolve these discrepancies and revisit the regulation of the PHO pathway by inositol pyrophosphates and Pho81, we capitalized on recent advances in the non-radioactive analysis of inositol pyrophosphates through CE-MS, offering superior resolution, sensitivity, and throughput (Qiu et al., 2020; Qiu et al., 2023). We combined comprehensive analyses of PHO pathway activation and inositol pyrophosphate profiles in mutants of key enzymes involved in inositol pyrophosphate metabolism of yeast to determine the inositol pyrophosphate species relevant to PHO pathway control and their impact on Pho85-Pho80 kinase. This led us to a revised model of PHO pathway regulation. Results So far, analysis of the role of inositol pyrophosphates in Pi homeostasis and Pi starvation responses relied mainly on the use of mutants which ablate one of the pathways of inositol pyrophosphate synthesis (Figure 1). Yet, as shown in plants, several inositol pyrophosphates can change in a similar manner upon Pi depletion or replenishment, and ablation of enzymes adding phosphate groups at the 1- or 5-positions of the inositol ring induces similar Pi starvation responses (Dong et al., 2019; Riemer et al., 2021). This renders it difficult to distinguish a role for an individual inositol pyrophosphate from the alternative hypothesis that all inositol pyrophosphates collectively contribute to signaling. To dissect this issue in yeast, we performed time course analyses of mutants in inositol pyrophosphate phosphatases and kinases, in which we correlate the levels of all inositol pyrophosphates with the induction of the phosphate starvation response in yeast. The rationale was to seek for upper or lower thresholds of inositol pyrophosphate concentrations during PHO pathway induction and using those to sieve out the inositol pyrophosphate responsible for signaling Pi starvation. Figure 1 Download asset Open asset Pathways of inositol pyrophosphate metabolism in S. cerevisiae. Dynamics of the cytosolic concentrations of 5-IP7, 1-IP7, and 1,5-IP8 In yeast, the myo-inositol hexakisphosphate kinases Vip1 and Kcs1 generate 1-IP7 and 5-IP7, respectively, and they are both required for synthesis of 1,5-IP8 (Figure 1; Mulugu et al., 2007; Saiardi et al., 1999; Zong et al., 2022; Wang et al., 2012; Zong et al., 2021). Inositol pyrophosphatases, such as Ddp1 and Siw14, dephosphorylate these compounds at the 1- and 5-position, respectively (Figure 1A; Lonetti et al., 2011; Wang et al., 2018; Steidle et al., 2020). We analyzed the kinetics and the role of these inositol pyrophosphates in PHO pathway activation. To this end, yeasts were cultured in liquid synthetic complete (SC) media to early logarithmic phase and then transferred to Pi-free SC medium. The cells were extracted with perchloric acid and inositol phosphates were analyzed by capillary electrophoresis coupled to electrospray ionization (ESI) mass spectrometry (Qiu et al., 2020). Three inositol pyrophosphates were detectable: 1-IP7, 5-IP7, and 1,5-IP8. We note that the CE-MS approach does not differentiate pyrophosphorylation of the inositol ring at the 1- and 3-positions. Thus, our assignments of the relevant species as 1-IP7 and 1,5-IP8 are based on previous characterization of the reaction products and specificities of IP6Ks and PPIPKs (Mulugu et al., 2007; Saiardi et al., 1999; Zong et al., 2022; Wang et al., 2012; Zong et al., 2021; Dollins et al., 2020). Quantitation of inositol pyrophosphates by CE-MS was facilitated by spiking the samples with synthetic, 13C-labeled inositol pyrophosphate standards (Harmel, 2019; Puschmann et al., 2019). The recovery rate of inositol pyrophosphates during the extraction was determined by adding known quantities of synthetic standards to the cells already before the extractions. This demonstrated that 89% of 1-IP7, 90% of 5-IP7, and 75% of 1,5-IP8 were recovered in the extract (Figure 2—figure supplement 1). To estimate the cellular concentrations of these compounds, the volume of the cells was determined by fluorescence microscopy after staining of the cell wall with trypan blue (Figure 2—figure supplement 2). This yielded an average cell volume of 42 fL. Detailed morphometric studies of yeast showed that the nucleus occupies around 8% of this volume and that all other organelles collectively account for approx. 18% (Uchida et al., 2011). Taking this into account we can estimate the concentrations in the cytosolic space (including the nucleus, which is permeable to small molecules) of cells growing logarithmically on SC medium as 0.5 µM for 1-IP7, 0.7 µM for 5-IP7, and 0.3 µM for 1,5-IP8 (Figure 2A). Figure 2 with 3 supplements see all Download asset Open asset Cytosolic concentrations of 5-IP7, 1-IP7, and 1,5-IP8. (A) Inositol pyrophosphate concentrations in the cytosol. The indicated strains were grown logarithmically in synthetic complete (SC) medium containing 7.5 mM of inorganic phosphate (Pi) (30°C, 150 rpm, overnight). When OD600nm reached 1 (1 × 107 cells/mL), 1 mL of culture was extracted with perchloric acid and analyzed for inositol pyrophosphates by CE-ESI-MS. The y-axis provides the estimated cytosolic concentrations based on an average cell volume of 42 fL. Means (n=4) and standard deviations are indicated. ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; n.s. not significant, tested with Student’s t-test. (B) Evolution of inositol pyrophosphate species during Pi starvation. Cells were grown as in A, washed twice with Pi starvation medium, and further incubated in Pi starvation medium. The inoculum for the samples bound to be extracted after different times of further incubation in starvation medium was adjusted such that all samples had similar OD600nm at the time of harvesting (OD600nm=0.5 for 30 min and 60 min samples; OD600nm=0.4 for 120 min and 240 min samples; OD600nm=0.25 for 360 min samples). At the indicated times in starvation media, 1 mL aliquots were extracted and analyzed for inositol pyrophosphates as in A. The data was normalized by the number of cells harvested before calculating cytosolic concentrations. Means and standard deviations are given (n=3). (C) Depletion of 5-IP7 in starving vip1Δ cells. The indicated cells were grown in Pi-replete medium and then transferred to Pi starvation medium as in B. At the indicated times, samples were extracted and analyzed for 5-IP7 as in A. Means and standard deviations (n=4) are shown as solid lines and shaded areas, respectively. Next, we determined the impact of Kcs1, Vip1, Siw14, and Ddp1 on the inositol pyrophosphate levels in the cells (Figure 2A). 5-IP7 was not detected in the kcs1Δ mutant and 1-IP7 was strongly reduced in the vip1Δ strain. 1,5-IP8 was undetectable in kcs1Δ and decreased by 75% in vip1Δ. The nature of the residual 1,5-IP8 and 1-IP7 signals is currently unclear. They may represent inositol pyrophosphates synthesized by enzymes other than Kcs1 and Vip1, such as the inositol polyphosphate multi-kinases, which can also produce inositol pyrophosphates (Riemer et al., 2021; Zong et al., 2022; Laha et al., 2019; Adepoju et al., 2019; Whitfield et al., 2020). Importantly, residual 1,5-IP8 and 1-IP7 were not observed in Pi-starved wildtype cells (Figure 2B). This may be due to presence of the Vip1 phosphatase activity, which is missing in vip1Δ cells, but which may quench weak production of inositol pyrophosphates such as 1-IP7 or 1,5-IP8 by other enzymes in wildtype cells. Since this aspect is not central to the question of our study, it was not pursued further. kcs1Δ mutants showed a two- to threefold decrease in 1-IP7, suggesting that the accumulation of 1-IP7 depends on 5-IP7. This might be explained by assuming that, in the wildtype, most 1-IP7 stems from the conversion of 5-IP7 to 1,5-IP8, followed by dephosphorylation of 1,5-IP8 to 1-IP7. A systematic analysis of this interdependency will require rapid pulse-labeling approaches for following the turnover of the phosphate groups, which are not yet established for inositol pyrophosphates (Wilson and Saiardi, 2017; Harmel, 2019; Nguyen Trung et al., 2022; Azevedo and Saiardi, 2006). An unexpected finding was the up to 20-fold overaccumulation of 5-IP7 in the vip1Δ mutant. By contrast, ddp1Δ cells showed normal levels of 5-IP7 and 1,5-IP8 but a 10-fold increase in 1-IP7. siw14Δ cells showed a fivefold increase in 5-IP7, but similar levels of 1,5-IP8 and 1-IP7 as wildtype. We performed time course experiments to analyze how inositol pyrophosphate levels change under Pi withdrawal. 5-IP7 was the predominant inositol pyrophosphate species in wildtype cells growing on Pi-replete media (Figure 2B). Within 30 min of Pi starvation, the concentration of all three inositol pyrophosphate species rapidly decreased by 75% for 1,5-IP8, by 47% for 1-IP7, and by 40% for 5-IP7. This decline continued, so that 1-IP7 and 1,5-IP8 became undetectable and only 3% of 5-IP7 remained after 4 hr, corresponding to a concentration below 50 nM. The high excess of 5-IP7 in vip1Δ cells also declined as soon as the cells were transferred to Pi starvation medium (Figure 2C). After 2 hr of starvation, it was still twofold above the concentration measured in Pi-replete wildtype cells. Even after 3.5–4 hr, Pi-starved vip1Δ cells had just reached the 5-IP7 concentration of Pi-replete wildtype cells. A comparable decline of all inositol pyrophosphate species upon Pi starvation could be observed in other fungi, such as S. pombe and C. neoformans (Figure 3), suggesting that this response is conserved. Figure 3 Download asset Open asset Inositol pyrophosphate analysis in C. neoformans and S. pombe. Inositol pyrophosphates were measured in C. neoformans (A) and S. pombe (B). Both fungi were logarithmically grown in synthetic complete (SC) medium for 17 h up to an OD600nm of 1. Cells were sedimented by centrifugation, resuspended in SC without Pi, and incubated further. At the indicated times, aliquots were extracted with perchloric acid. Inositol pyrophosphates were enriched on TiO2 beads and analyzed by CE-MS. Concentrations of 1,5-IP8, 5-IP7 and 1-IP7 in the extracts were determined by comparison with added synthetic 13C-labeled inositol pyrophosphate standards. The graphs provide the concentrations in the extracts. n=4 for C. neoformans, and n=3 for S. pombe; means and standard deviations are indicated. The inositol pyrophosphate values were normalized to the OD600 of the culture for every given time point. Taken together, Pi starvation leads to a virtually complete depletion of all three inositol pyrophosphate species, with 1,5-IP8 declining faster than 1-IP7 and 5-IP7. Furthermore, inositol pyrophosphatase mutants provide the possibility to generate relatively selective increases in 5-IP7 and 1-IP7. We used this information to dissect the impact of 5-IP7, 1-IP7, and 1,5-IP8 on control of the PHO pathway. 1,5-IP8 signals cytosolic Pi levels to the PHO pathway To this end, we correlated the measured inositol pyrophosphate concentrations to the induction of the PHO pathway. We assayed a key event of PHO pathway activation, partitioning of the fluorescently tagged transcription factor Pho4yEGFP between the cytosol and the nucleus. Pho4 shuttles between nucleus and cytosol and its phosphorylation through Pho85-Pho80 favors Pho4 accumulation in the cytosol. The relocation of Pho4 can hence serve as an in vivo indicator of PHO pathway activation (Thomas and O’Shea, 2005; Wykoff et al., 2007; O’Neill et al., 1996). It provides a readout for Pho85-Pho80 activity. PHO4 was tagged at its genomic locus, making Pho4yEGFP the sole source of this transcription factor. Pho4 relocation was assayed through automated image segmentation and analysis. The artificial intelligence-based segmentation algorithm recognized more than 90% of the cells in a bright-field image and delimited their nuclei based on a red-fluorescent nuclear mCherry marker (Figure 4—figure supplement 1). This segmentation allows quantitative measurements of Pho4 distribution between the cytosol and nucleus in large numbers of cells. In addition, we assayed PHO pathway activation through fluorescent yEGFP reporters expressed from the PHO5 (prPHO5-yEGFP) and PHO84 (prPHO84-yEGFP) promotors. These are classical assays of PHO pathway activation, but their output is further downstream and hence comprises additional regulation, for example at the level of chromatin or RNA, or the direct activation of Pho4 through metabolites such as AICAR (Nishizawa et al., 2008; Almer et al., 1986; Barbaric et al., 2007; Lam et al., 2008; Pinson et al., 2009). Upon Pi withdrawal, both promotors are induced by the PHO pathway but the PHO84 promotor reacts in a more sensitive manner and is induced more rapidly than the PHO5 promotor (Thomas and O’Shea, 2005). In wildtype cells grown under Pi-replete conditions, Pho4yEGFP was cytosolic and the PHO5 and PHO84 promotors were inactive, indicating that the PHO pathway was repressed (Figure 4). Within 30 min of starvation in Pi-free medium, Pho4yEGFP relocated into the nucleus and PHO84 and PHO5 were strongly induced. By contrast, kcs1Δ cells showed Pho4yEGFP constitutively in the nucleus already under Pi-replete conditions, and PHO5 and PHO84 promoters were activated. These cells have strongly reduced 1,5-IP8 and 5-IP7, and 50% less 1-IP7 than the wildtype. Thus, a decline of inositol pyrophosphates not only coincides with the induction of the Pi starvation program, but the genetic ablation of these compounds is sufficient for a forced triggering of this response in Pi-replete conditions. Therefore, we explored the hypothesis that inositol pyrophosphates repress the PHO pathway, and that their loss upon Pi starvation creates the signal that activates the starvation response. Figure 4 with 1 supplement see all Download asset Open asset Inhibition of the PHO pathway by excessive 5-IP7. The indicated cells producing Pho4yEGFP and the histone Hta2mCherry as a nuclear marker were logarithmically grown in inorganic phosphate (Pi)-replete synthetic complete (SC) medium, washed, and transferred to Pi starvation medium as in Figure 2A. (A) Subcellular localization of Pho4yEGFP was analyzed on a spinning disc microscope. Cells are shown in the presence of 7.5 mM of Pi (+Pi) or 30 min after the shift to Pi starvation (- Pi) medium. Yellow lines surrounding the cells illustrate the segmentation performed by the algorithm that was used to quantify Pho4yEGFP distribution in B. Scale bar: 5 μM. λex: 470 nm; λem: 495–560 nm. (B) Average intensity of Pho4yEGFP fluorescence was determined by automated image segmentation and analysis. Pho4yEGFP localization is quantified by the ratio of the average fluorescence intensities in the nucleus over the average fluorescence intensity in the cytosol (IN/IC). 100–200 cells were analyzed per condition and experiment. n=3. Means and standard deviation are indicated. (C) Activation of the PHO5 promotor. Cells expressing the prPHO5-yEGFP reporter construct from a centromeric plasmid were grown in Pi-replete medium (7.5 mM Pi) as in Figure 2A, and then shifted to Pi starvation medium or kept in Pi-replete medium. After 4 hr of further incubation, fluorescence intensity of the same number of cells was measured in a Spectramax EM microplate reader. λex: 480 nm; λem: 510 nm. n=3. Means and standard deviations are indicated. (D) Activation of the PHO84 promotor. Cells expressing the prPho84-yEGFP reporter construct from a centromeric plasmid were treated and analyzed as in C. For B, C, and D: ****p<0.0001; ***p<0.001; **p<0.01; *p<0.05; n.s. not significant, tested with Turkey’s test. In this case, we must explain the behavior of the vip1Δ mutation, which strongly reduces 1-IP7 and 1,5-IP8 and maintains the PHO pathway repressed in Pi-replete medium. Upon withdrawal of Pi, vip1Δ cells did not show nuclear relocation of Pho4 after 30 min and, even after 4 hr of starvation, PHO5 was not expressed. The PHO84 promoter remained partially repressed in comparison with the wildtype. These results are at first sight consistent with the proposal that 1-IP7 activates the PHO pathway (Lee et al., 2007; Lee et al., 2008). Several further observations draw this hypothesis into question, however. First, all three inositol pyrop" @default.
• W4386896889 created "2023-09-21" @default.
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• W4386896889 date "2023-09-20" @default.
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• W4386896889 title "eLife assessment: Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81" @default.
• W4386896889 doi "https://doi.org/10.7554/elife.87956.3.sa0" @default.
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