Inorganic phosphate (P_i) 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 P_i decreases the free energy provided by P_i-liberating reactions, such as nucleotide hydrolysis, which might stall metabolism (Austin and Mayer, 2020). In fungi, plants, and animals, control of P_i 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 P_i homeostasis by transporting P_i across membranes, converting it into polyphosphates (polyP) or other metabolites, or regulating P_i-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 P_i 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 P_i homeostasis. In mammalian cells, 1,5-IP₈ activates the only SPX protein expressed in this system, the P_i 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 P_i-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 P_i-responsive transcription factor PHR2 revealed only a minor, twofold difference in the K_d values for 5-IP₇ and 1,5-IP₈. 1-IP₇, 5-IP₇, and 1,5-IP₈ all decrease upon P_i 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 P_i 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 P_i 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 P_i content (Riemer et al., 2021). Although 1,5-IP₈ is the inositol pyrophosphate that is the most responsive to P_i starvation and P_i re-feeding (Riemer et al., 2021) this has rendered it difficult to clearly resolve whether IP₇, IP₈, or both signal cellular P_i status to the P_i 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 P_i 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 P_i-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 P_i 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 P_i-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 P_i 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 P_i, 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 P_i starvation, Pho81 inhibits the Pho85-Pho80 kinase, leading to dephosphorylation and activation of Pho4 and the ensuing expression of P_i-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 P_i 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 P_i-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 P_i 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.

So far, analysis of the role of inositol pyrophosphates in P_i homeostasis and P_i 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 P_i depletion or replenishment, and ablation of enzymes adding phosphate groups at the 1- or 5-positions of the inositol ring induces similar P_i 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 P_i starvation.

Figure 1

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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 P_i-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).

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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 P_i-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 P_i withdrawal. 5-IP₇ was the predominant inositol pyrophosphate species in wildtype cells growing on P_i-replete media (Figure 2B). Within 30 min of P_i 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 P_i starvation medium (Figure 2C). After 2 hr of starvation, it was still twofold above the concentration measured in P_i-replete wildtype cells. Even after 3.5–4 hr, P_i-starved vip1Δ cells had just reached the 5-IP₇ concentration of P_i-replete wildtype cells. A comparable decline of all inositol pyrophosphate species upon P_i starvation could be observed in other fungi, such as S. pombe and C. neoformans (Figure 3), suggesting that this response is conserved.

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Taken together, P_i 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 P_i 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 Pho4^yEGFP 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 Pho4^yEGFP 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 P_i 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 P_i-replete conditions, Pho4^yEGFP 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 P_i-free medium, Pho4^yEGFP relocated into the nucleus and PHO84 and PHO5 were strongly induced. By contrast, kcs1Δ cells showed Pho4^yEGFP constitutively in the nucleus already under P_i-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 P_i starvation program, but the genetic ablation of these compounds is sufficient for a forced triggering of this response in P_i-replete conditions. Therefore, we explored the hypothesis that inositol pyrophosphates repress the PHO pathway, and that their loss upon P_i starvation creates the signal that activates the starvation response.

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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 P_i-replete medium. Upon withdrawal of P_i, 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 pyrophosphate species strongly decline upon P_i starvation instead of showing the increase postulated by Lee et al. Second, vip1Δ cells show a more than 15-fold overaccumulation of 5-IP₇. Genetic ablation of this pool by deleting KCS1 is epistatic to the vip1Δ mutation. A kcs1Δ vip1Δ double mutant constitutively activates the PHO pathway already in presence of P_i, despite its strong reduction of 1-IP₇ and the complete absence of 1,5-IP₈. Third, ddp1Δ cells, which show a 10-fold quite selective increase in 1-IP₇ under P_i-replete conditions, did not induce the PHO84 and PHO5 promotors, nor did they show Pho4 accumulation in the nucleus in P_i-replete medium. Thus, even a strong increase in 1-IP₇ is not sufficient to activate the PHO pathway, while reductions of inositol pyrophosphates do this.

The data described above argues against a diminution of 1-IP₇ as a critical factor for induction of the PHO pathway because vip1Δ cells, which have virtually no 1-IP₇, do not constitutively activate the PHO pathway. By contrast, vip1Δ kcs1Δ double mutants, which not only have low levels of 1-IP₇ and 1,5-IP₈ but also strongly reduced 5-IP₇, show constitutive PHO pathway activity (Figure 4). We hence tested the hypothesis that a decline of 5-IP₇ might trigger the PHO pathway. To define the critical concentration at which this happens in vip1Δ cells, we assayed the induction of the PHO pathway over time (Figure 5). While Pho4^yEGFP was cytosolic in wildtype cells under high P_i supply, a large fraction of it rapidly relocated into the nucleus within the first 15 min of P_i starvation (Figure 5A and B). In vip1Δ, Pho4^yEGFP relocation followed a sigmoidal curve. The nuclear/cytosolic ratio strongly increased after 1 hr, reaching a plateau after 3 hr. Thus, relocation was stimulated when the 20-fold exaggerated 5-IP₇ levels of the vip1Δ cells had declined below 5 µM (Figure 2C), which is six to seven times above the maximal concentration observed in P_i-replete wildtype cells. Since 5-IP₇ is the only inositol pyrophosphate that exists in vip1Δ cells in significant amounts, this observation places the critical concentration of 5-IP₇ that can repress the PHO pathway at 5 µM. In P_i-replete wildtype cells, 5-IP₇ never reaches 5 µM (Figure 2) and, therefore, 5-IP₇ is unlikely to be a physiological repressor of the PHO pathway in wildtype cells. The 5-IP₇-dependent PHO pathway repression in vip1Δ cells must then be considered as a non-physiological reaction resulting from the strong overaccumulation of 5-IP₇ in these cells.

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This interpretation is also consistent with the phenotype of siw14Δ cells. Although these cells accumulate 5-IP₇, they accumulate it fourfold less than vip1Δ, remaining below the 5 µM threshold where we should expect non-physiological repression of the PHO pathway. However, siw14Δ cells contain the same concentration of 1,5-IP₈ as the wildtype (Figure 2A), degrade it upon P_i starvation as the wildtype (Figure 2—figure supplement 3), and relocalize Pho4^yEGFP with similar kinetics as the wildtype (Figure 5B). Taken together with the evidence presented above, 1,5-IP₈ thus ends up being the only inositol pyrophosphate that correlates with the activation of the PHO pathway in all mutants and conditions. This points to 1,5-IP₈ as the controller of the PHO pathway.

The PHO pathway is controlled by the SPX domain of Pho81

Our results strongly argue against an increase of 1-IP₇ as the key factor for inactivating the CDK Pho85-Pho80 through the CDK inhibitor Pho81. This prompted us to also re-evaluate the regulation of Pho81 because previous studies had proposed a small peptide from the central region of Pho81, called the minimum domain (amino acids 645–724), as a receptor for 1-IP₇ that triggers the PHO pathway (Lee et al., 2007; Lee et al., 2008). This model rests mainly on results with the isolated minimum domain used in vitro or, in highly overexpressed form, in vivo. This expression of the minimum domain outside its normal molecular context may be problematic because earlier work had suggested that the N- and C-terminal portions of Pho81, that is regions outside the minimum domain, could provide competing inhibitory and stimulatory functions for Pho81 (Ogawa et al., 1995).

We targeted the N-terminal SPX domain of Pho81 through various mutations, leaving the rest of Pho81 and its minimum domain intact (Figure 6). First, we asked whether Pho81 lacking the N-terminal 200 amino acids, corresponding to a deletion of its SPX domain, could still activate the PHO pathway. This Pho81^ΔSPX is likely to be folded because a yEGFP-tagged version of Pho81^ΔSPX localized to the nucleus (Figure 6C and D), which requires Pho81 to interact with nuclear import factors and with Pho85-Pho80 (Huang et al., 2001). We tested the effect of Pho81^ΔSPX on relocation of Pho4^yEGFP (Figure 6A and B). Whereas wildtype cells efficiently relocated Pho4^yEGFP to the nucleus upon P_i starvation, pho81^ΔSPX cells partially maintained Pho4^yEGFP in the cytosol. Thus, while the SPX domain is not essential for nuclear targeting of Pho81, it is required for efficient nuclear relocation of Pho4 and the induction of the PHO pathway under P_i starvation.

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Several lysines located on α-helix 4 of SPX domains form part of an inositol pyrophosphate-binding patch (Ried et al., 2021; Pipercevic et al., 2023). Substituting them by alanine severely reduces the affinity of the domains for inositol pyrophosphates and can thus mimic the inositol pyrophosphate-free state. We determined the corresponding lysines in the SPX domain of Pho81 (Figure 6—figure supplement 1), created a point mutation in the genomic PHO81 locus that substitutes one of them, K154, by alanine, and investigated the impact on the PHO pathway. Pho81^K154A-yEGFP should be correctly folded because it concentrates in the nuclei of the cells (Huang et al., 2001). pho81^K154A cells constitutively activated the PHO pathway because Pho4^yEGFP accumulated in their nucleus already under P_i-replete conditions (Figure 6A and B). This constitutive activation was also observed in a pho81^K154A vip1Δ double mutant, confirming that 1-IP₇ synthesis by Vip1 is not necessary for PHO pathway induction. Induction of the PHO pathway through SPX substitutions is further illustrated by PHO81 expression, which itself is under the control of the PHO pathway (Figure 6E and F). Western blots of protein extracts from cells grown under P_i-replete conditions showed increased levels of Pho81 in pho81^K154A cells, like those observed in strains that constitutively activate the PHO pathway due to the absence of inositol pyrophosphates, such as kcs1Δ and vip1Δ/kcs1Δ. They were significantly higher than in cells that do not constitutively activate the PHO pathway, such as wildtype or vip1Δ. In sum, these results suggest that the PHO pathway is regulated through binding of inositol pyrophosphates to the SPX domain of Pho81.

This critical role of the SPX domain for controlling Pho81 is also consistent with the results from two random mutagenesis approaches (Ogawa et al., 1995; Creasy et al., 1993), which had received little attention in the previous model of PHO pathway regulation (Lee et al., 2007; Lee et al., 2008). These random mutagenesis approaches generated multiple pho81^c alleles, which constitutively activate the PHO pathway and carry the substitutions G4D, G147R, 158-SGSG-159, or E79K. All these alleles affect the SPX domain at residues in the immediate vicinity of the putative inositol pyrophosphate-binding site (Figure 7). Three of the alleles (G4D, G147R, 158-SGSG-159) replace small glycine residues by bulky residues or insert additional amino acids, respectively, at sites where they should sterically interfere with binding of inositol pyrophosphates. We hence expect them to mimic the inositol pyrophosphate-free state and constitutively activate the PHO pathway for this reason.

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SPX and minimum domains contribute to the interaction of Pho81 with Pho85-Pho80 in vivo

The traditional model of PHO pathway activation has tied the effect of 1-IP₇ to the minimum domain of Pho81, proposing that a complex of the two competitively inhibits Pho85 kinase (Lee et al., 2008). Since the results presented above argued against a role of 1-IP₇ in inhibiting Pho85-Pho80 and triggering the PHO pathway, we explored potential alternative roles of the minimum domain by modeling the Pho80-Pho81 interaction with a Google Colab notebook for Alphafold multimer v3 (Mirdita et al., 2022). The non-templated structure prediction of the complex (Figure 8) yields a Pho80 structure that agrees with an available crystal structure (Huang et al., 2007), showing R121 and E154 of Pho80 forming a salt bridge in a groove, which is critical for controlling Pho85 kinase (Huang et al., 2001). The prediction shows the groove binding a long unstructured loop of Pho81, which corresponds to the minimum domain. Substitutions in the minimum domain, and specifically of the residues binding the Pho80 groove in the prediction (residues 690–701), constitutively repress the PHO pathway when introduced into full-length Pho81, and they destabilize the interaction of Pho81 with Pho85-Pho80 (Ogawa et al., 1995; Huang et al., 2001). The same effects are also shown by the pho80^R121K and pho80^E154V alleles, which ablate the critical salt bridge in the Pho80 groove (Huang et al., 2001; Huang et al., 2007). The similarity of the effects of these substitutions validates the predicted interaction of the minimum domain with the R121/E154-containing groove. It invites a re-interpretation of the role of the minimum domain and suggests that this domain may serve as an anchor for Pho80 on Pho81 rather than acting directly on the Pho85 kinase.

Figure 8

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The recruitment of Pho81 to the nucleus and its interaction with Pho85-Pho80 in wildtype cells is independent of the P_i regime, suggesting that it is dominated by a constitutive interaction (Figure 6C; Huang et al., 2001). This constitutive recruitment vanishes when the minimum domain-binding site is compromised in pho80^R121K and pho80^E154V cells. Then, recruitment of Pho81 to the nucleus and to Pho85-Pho80 becomes P_i-sensitive and is stimulated by P_i starvation (Figure 9). We hence used pho80^R121K and pho80^E154V as tools to ask whether Pho81 might be tied to Pho85-Pho80 through a second layer of interaction that provides P_i dependence. As a bona fide P_i-responsive element, the SPX domain of Pho81 was a prime candidate. We combined pho80^R121K and pho80^E154V with substitutions in the putative inositol pyrophosphate-binding site of a fluorescent Pho81^yEGFP fusion protein, and with deletions of VIP1 or KCS1. Under P_i-replete conditions, pho80^R121K or pho80^E154V cells showed Pho81^yEGFP in the cytosol, but they accumulated the protein in the nucleus upon P_i starvation (Figure 9). This rescue by starvation was more pronounced in pho80^R121K than in pho80^E154V. Pho81^K154A-yEGFP, with a substitution in its putative inositol pyrophosphate binding site designed to mimic the low-P_i state, accumulated in the nucleus of pho80^R121K and pho80^E154V cells already under P_i-replete conditions, that is, it behaved constitutively as under P_i starvation. vip1Δ mutants behaved like the wildtype, accumulating Pho81^yEGFP in the nucleus of pho80^R121K and pho80^E154V cells upon P_i starvation, whereas kcs1Δ cells showed nuclear Pho81^yEGFP already under P_i-replete conditions. Thus, interfering with inositol pyrophosphate signaling, be it through P_i starvation, ablation of inositol pyrophosphate synthesis, or substitution of the putative inositol pyrophosphate binding site of Pho81, partially compensated for the destabilization of the minimum domain binding site in Pho80.

Figure 9

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Two in vivo observations are consistent with the notion that this compensation may reflect an interaction of the SPX domain with Pho85-Pho80: First, Pho81^ΔSPX-yEGFP, which lacks this SPX domain, did not show starvation-induced nuclear accumulation in pho80^R121K and pho80^E154V cells (Figure 9). Second, a yEGFP fusion of only the SPX domain of Pho81 (Pho81SPX^yEGFP), which lacks the rest of the protein and thereby excludes a constitutive interaction through the minimum domain, localized mainly to the cytosol of PHO80 wildtype cells under P_i-replete conditions, but it concentrated in the nucleus upon P_i starvation (Figure 10). This concentration was not observed in pho80Δ cells, which lack nuclear Pho85-Pho80. By contrast, pho80^R121K or pho80E^154V cells, which only target the minimum domain-Pho80 interaction but leave Pho85-Pho80 kinase active and in the nucleus (Huang et al., 2007), retained the capacity to accumulate Pho81SPX^yEGFP in their nuclei upon P_i starvation. These results suggest that the SPX domain contributes to the interaction of Pho81 and Pho85-Pho80. Our observations are consistent with a model in which this SPX-Pho85-Pho80 interaction is independent of the minimum domain but controlled by P_i through inositol pyrophosphates, which can destabilize it and relieve the inhibition of Pho85-Pho80.

Figure 10

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The transcriptional response of S. cerevisiae to varying P_i supply is an important model system for studying gene regulation, promotor structure, and the impact of chromatin structure (Korber and Barbaric, 2014; Wolff et al., 2021). However, identification of the inositol pyrophosphates relaying phosphate availability to this PHO pathway has been challenging. This is mainly due to limitations in analytics. The recent development of a CE-MS method has provided a potent tool, allowing inositol pyrophosphate analysis without radiolabeling and under any physiological condition. The improved separation, higher throughput, and lower cost of this approach led us to re-investigate the role of inositol pyrophosphates in PHO pathway regulation. The results inspire a revised model that differs from the traditional hypothesis in three aspects: We propose that P_i scarcity is signaled by a decrease in inositol pyrophosphates rather than by an increase; a critical function is ascribed to the loss of 1,5-IP₈ instead of 1-IP₇; and we postulate that the N-terminal SPX domain of Pho81 rather than the central minimum domain is the primary receptor mediating inositol pyrophosphate control over the PHO pathway.

Our observations reveal an effect that misled the interpretation of several preceding studies. This includes our own work on the activation of the polyphosphate polymerase VTC (Hothorn et al., 2009), which is controlled by SPX domains (Gerasimaite et al., 2017; Wild et al., 2016; Pipercevic et al., 2023; Gerasimaitė and Mayer, 2016). Our in vitro activity assays had shown a clear order of potency of inositol pyrophosphates in stimulating VTC, with 1,5-IP₈ showing an apparent EC₅₀ 30-fold below that of 5-IP₇ and 50-fold below that of 1-IP₇ (Gerasimaite et al., 2017). In this work, we nevertheless dismissed 1,5-IP₈ as a physiological stimulator of the VTC SPX domains, based on the argument that a vip1Δ mutant, which lacks the enzyme for 1-IP₇ and 1,5-IP₈ synthesis, shows quite normal polyP accumulation in vivo. We proposed 5-IP₇ for this role because a kcs1Δ mutant, which lacks the IP6K making this compound, also lacks polyP. The inositol pyrophosphate analyses that we present now call for a re-interpretation of this data. The presence of polyP in a vip1Δ mutant can well be explained by the 20-fold overaccumulation of 5-IP₇ in this mutant, which can compensate for the lower EC₅₀ and permit stimulation of VTC through 5-IP₇. In wildtype cells, however, where 5-IP₇ is only twice as abundant as 1,5-IP₈ (Figure 2), 1,5-IP₈ should be the relevant activator of VTC for polyP synthesis under P_i-replete conditions.

Then, the situation is also compatible with results from S. pombe, where ablation of the Vip1 homolog Asp1 impairs polyP synthesis (Pascual-Ortiz et al., 2021; Schwer et al., 2022). The transcriptional phosphate starvation response in S. pombe also depends on inositol pyrophosphates. However, the downstream executers differ from those present in S. cerevisiae. Fission yeast contains no homologs of Pho81, and the responsible transcription factor is not controlled through a Pho85-Pho80-like kinase (Estill et al., 2015; Henry et al., 2011; Carter-O’Connell et al., 2012). P_i-dependent transcription strongly depends on lncRNA-mediated interference, which may be linked to inositol pyrophosphates through an SPX domain containing ubiquitin E3 ligase (Sanchez et al., 2019; Schwer et al., 2021; Sanchez et al., 2018; Schwer et al., 2022). Nevertheless, important basic properties of the IP6K and PPIP5K system are conserved. Like in baker’s yeast, deletion of the Vip1 homolog Asp1 leads to a strong increase in IP₇ (Pascual-Ortiz et al., 2018). Characterization of the purified enzyme also showed that P_i stimulates net production of IP₈ by inhibiting the phosphatase domain of Asp1 (Benjamin et al., 2022). In line with this, we observed that IP₈ rapidly decreased upon P_i starvation also in S. pombe (Figure 3). As for Vip1 in baker’s yeast, genetic ablation of Asp1 does not interfere with the induction of the phosphate-regulated gene PHO1 by P_i starvation, suggesting that 1-IP₇ and 1,5-IP₈ are not essential for this transcriptional response (Estill et al., 2015). In P_i-replete media, however, ablation of Asp1 hyper-represses PHO1, and putative overproduction of 1-IP₇ and/or 1,5-IP₈ by selective inactivation of only the Asp1 phosphatase domain induces this gene (Estill et al., 2015; Garg et al., 2020). It remains to be seen whether these discrepancies originate from a similar disequlibration of IP₇ species as in S. cerevisiae (ablation of Asp1 increases the IP₇ pool four- to fivefold Pascual-Ortiz et al., 2018), or whether they are due to other processes such as lncRNA-mediated interference, which plays an important role in P_i homeostasis in S. pombe (Sanchez et al., 2018; Schwer et al., 2015; Schwer et al., 2014).

The PHO pathway in C. neoformans, a pathogenic yeast, shares many protein components with that of S. cerevisiae and might hence be expected to function in a similar manner (Toh-e et al., 2015). Yet a recent study on this organism proposed 5-IP₇ as the critical inducer of the PHO pathway (Desmarini et al., 2020). This model relies on the observation that kcs1Δ mutants, which lack 5-IP₇, cannot induce the PHO pathway in this yeast. This is the opposite behavior compared to S. cerevisiae. It was hence concluded that, despite an experimentally observed moderate decline in 5-IP₇ upon short P_i starvation, the remaining pool of 5-IP₇ was required for PHO pathway activity. Substitution of basic residues in the inositol pyrophosphate-binding pocket of C. neoformans Pho81 interfered with PHO pathway activation, lending further support to the conclusion. However, the same study also showed that Pho81 was lacking completely from kcs1Δ mutants. When the Pho81 inositol pyrophosphate-binding site was compromised by amino acid substitutions, the Pho81 protein level was significantly reduced, and the protein almost vanished upon P_i starvation. This offers an alternative explanation of the effects because absence of the critical inhibitor Pho81 activates Pho85/80 kinase and represses the PHO pathway independently of P_i availability (Schneider et al., 1994; Ogawa et al., 1995). Destabilization of Pho81 will then mask effects of inositol pyrophosphates. Thus, it remains possible that the PHO pathway in C. neoformans is controlled as in S. cerevisiae, that is that P_i starvation is signaled by a decline of 1,5-IP₈. In line with this, we observed that C. neoformans and S. pombe cells lose all inositol pyrophosphates within 2 hr of phosphate starvation (Figure 3). As in S. cerevisiae, 1,5-IP₈ showed a faster and more profound response. Therefore, we consider it as likely that the loss of 1,5-IP₈ signals P_i starvation also in C. neoformans and S. pombe.

Our results revise the widely accepted model of increased 1-IP₇ as the trigger of the PHO pathway and of the minimum domain of Pho81 as its receptor (Lee et al., 2007; Lee et al., 2008). This model rests upon an increase of 1-IP₇ upon P_i starvation, a reported constitutive activation of the PHO pathway in a ddp1Δ mutant, and upon the lack of rapid PHO pathway induction in a vip1Δ mutant. A later study from O’Shea and colleagues revealed that vip1Δ cells finally induced the PHO pathway after prolonged P_i starvation (Choi et al., 2017), but this did not lead to a revision of the model. Our results rationalize the strong delay of vip1Δ in PHO pathway induction by the overaccumulation of 5-IP₇ in this mutant, which leads to aberrant PHO pathway silencing through this compound. Under P_i starvation, it takes several hours longer to degrade this exaggerated pool, which provides a satisfying explanation for the delayed PHO pathway induction in vip1Δ cells. Even though earlier studies, due to the limited resolution of the radio-HPLC approach, could not differentiate between 1- and 5-IP₇, these studies reported an increase in the IP₇ pool upon P_i starvation (Lee et al., 2007). Other studies using a similar radio-HPLC approach suggested a decline of the IP₇ pool upon long P_i withdrawal, but they analyzed only a single timepoint and could not exclude an earlier increase in 1-IP₇ as a trigger for the PHO pathway (Wild et al., 2016; Desmarini et al., 2020; Lonetti et al., 2011). In our CE-MS analyses, we consistently observed strong, continuous declines of 1,5-IP₈, 5-IP₇, and 1-IP₇ and, upon several hours of P_i starvation, their almost complete disappearance. Furthermore, we could not confirm the reported constitutive activation of the PHO pathway in a ddp1Δ mutant (Lee et al., 2007), although this mutant did show the expected increased 1-IP7 concentration. These observations argue against a role of 1-IP₇, 5-IP₇, or 1,5-IP₈ as activators for Pho81 and the PHO pathway.

The link between 1-IP₇ and Pho81 activation relied mainly on the use of the 80 amino acid minimum domain from the central region (Lee et al., 2007; Lee et al., 2008; Huang et al., 2001). This domain was used in in vitro experiments, where 1-IP₇ stimulated binding of the minimum domain to Pho85-Pho80 and inhibition of the enzyme. Given that 1-IP₇ disappears under P_i starvation, it appears unlikely that this in vitro effect is physiologically relevant. Furthermore, the apparent dissociation constant of the complex of 1-IP₇ with the minimum domain and Pho80-Pho85 was determined to be 20 µM (Lee et al., 2008). The minimum domain is thus unlikely to respond to 1-IP₇ in the cells because its K_d is almost 50-fold above the cellular concentrations of 1-IP₇ in P_i-replete medium (0.5 µM; Figure 2), and at least three orders of magnitude above the concentration remaining on P_i starvation media (<20 nM). Another discrepancy is that the in vitro inhibition of Pho85-Pho80 by the minimum domain was reported to be specific for 1-IP₇ whereas 5-IP₇ had no effect (Lee et al., 2008). This contrasts with the in vivo situation, where we find that even massive overaccumulation of 1-IP₇ by ablation of DDP1 cannot trigger the PHO pathway, but where suppression of 5-IP₇ and 1,5-IP₈ production by ablation of KCS1 does activate it constitutively (Figure 4; Auesukaree et al., 2005).

An argument supporting a role of the minimum domain in mediating P_i responsiveness was provided by in vivo experiments in a pho81Δ mutant, where P_i-dependent activation of the PHO pathway could be partially rescued through overexpression of the minimum domain (Ogawa et al., 1995; Huang et al., 2001). PHO pathway activation was assayed through PHO5 expression. This is relevant because the PHO5 promotor is not only controlled through Pho85-Pho80-mediated phosphorylation of Pho4, but also through changes in nucleosome occupancy, which substantially influence activity and activation threshold of the PHO5 promotor (Almer et al., 1986; Lam et al., 2008). It is thus conceivable that constitutive overexpression of the minimum domain reduces Pho85-Pho80 activity moderately, but sufficiently to allow regulation of PHO5 expression through nucleosome positioning, which changes in a P_i-dependent manner (Almer et al., 1986).

Even though the minimum domain is unlikely to function as a receptor for inositol pyrophosphates, this does not call into question that the minimum domain is critical for Pho81 function. A role of this domain is supported by substitutions and deletions in this domain, introduced into full-length Pho81, which constitutively repress the PHO pathway (Ogawa et al., 1995; Huang et al., 2001). Our modeling approach provides a satisfying link of these residues to Pho85-Pho80 regulation. It suggests the minimum domain to be an extended unstructured loop that binds Pho80 at a groove that encompasses R121 and E154, residues that emerged from a genetic screen as essential for recruiting Pho81 to Pho85-Pho80 and for inhibiting the kinase (Huang et al., 2001). Therefore, we propose that the minimum domain does provide a critical connection between Pho81 and Pho80, which is per se inositol pyrophosphate-independent, whereas the SPX domain of Pho81 may create additional contacts to Pho80-Pho85 and impart regulation through inositol pyrophosphates.

Taking all these aspects together, we propose the following working model (Figure 11): P_i sufficiency in the cytosol is signaled through the accumulation of 1,5-IP₈. 1,5-IP₈ binding to the SPX domain of Pho81 labilizes the interaction of this domain with Pho85-Pho80, allowing the kinase to become active. The now un-inhibited kinase phosphorylates Pho4, triggers its relocation into the cytosol and represses the PHO pathway. P_i limitation is signaled by a decline in 1,5-IP₈, resulting in the ligand-free form of the SPX domain and the activation of Pho81. Inhibition of Pho85-Pho80 requires the Pho81 minimum domain to bind a groove in the cyclin Pho80, stabilizing the complex and inhibiting Pho85 kinase. An obvious question arising from this model is whether the Pho81 SPX domain itself could activate Pho85-Pho80 by directly binding these proteins. Alternatively, or in addition, Pho85-Pho80 might be controlled through the availability of the minimum domain for Pho80 binding, which might be restricted through an inositol pyrophosphate-dependent SPX-minimum domain interaction. These aspects will be explored in future work.

Figure 11

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This revision of PHO pathway regulation permits to develop a coherent picture on intracellular phosphate signaling across the eukaryotic kingdoms. Phosphate abundance being indicated by the accumulation of 1,5-IP₈ is compatible with the situation in mammalian cells, where 1,5-IP₈ triggers P_i export through the putative phosphate transporter XPR1 (Li et al., 2020; Giovannini et al., 2013), and with the situation in plants, where IP₈ is particularly abundant upon shift to high-P_i substrate and inhibits transcription factors for the phosphate starvation response (Ried et al., 2021; Dong et al., 2019; Riemer et al., 2021; Zhu et al., 2019). Thus, loss of 1,5-IP₈ may generally signal phosphate scarcity in eukaryotic cells.

The structure predictions shown in the manuscript and the script used for image analysis have been deposited at Figshare under the https://doi.org/10.6084/m9.figshare.c.6700281.

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Author details

1. Valentin Chabert

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

   Contribution

   Software, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Contributed equally with

   Geun-Don Kim

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-0797-2481

2. Geun-Don Kim

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

   Contribution

   Investigation, Methodology, Writing – review and editing

   Contributed equally with

   Valentin Chabert

   Competing interests

   No competing interests declared

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

3. Danye Qiu

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

   Contribution

   Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

4. Guizhen Liu

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

   Contribution

   Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1748-6687

5. Lydie Michaillat Mayer

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

   Contribution

   Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

6. Muhammed Jamsheer K

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

   Contribution

   Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-2135-8760

7. Henning J Jessen

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

   Contribution

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

   Competing interests

   No competing interests declared

8. Andreas Mayer

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

   Contribution

   Conceptualization, Formal analysis, Supervision, 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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