Inositol pyrophosphate dynamics reveals control of the yeast phosphate starvation program through 1,5-IP8 and the SPX domain of Pho81

Abstract

Eukaryotic cells control cytosolic inorganic phosphate to balance its role as essential macronutrient with its negative bioenergetic impacts. Phosphate homeostasis depends on a conserved signaling pathway including inositol pyrophosphates (PP-IPs) and SPX receptor domains. Since cells synthesize various PP-IPs and SPX domains bind them promiscuously, it is unclear whether a specific PP-IP regulates SPX domains in vivo, or whether multiple PP-IPs act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased 1-IP7 production, we now show that the levels of all detectable PP-IPs 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. Cytosolic Pi is of prime importance for cellular bioenergetics because Pi influences free energy of nucleotide hydrolysis and the metabolite fluxes through glycolysis and oxidative phosphorylation. Eukaryotic cells signal Pi via SPX domains binding critical ligands, inositol pyrophosphates (IP7, IP8), which control Pi homeostasis through a network of target proteins that import, export, store or detoxify Pi. Studies with different systems failed to yield a coherent model on this regulation. We performed the first time-resolved profiling of the full isomer spectrum of inositol pyrophosphates and dissected the isomer that is relevant to intracellular Pi signaling. Our results support a unified model of Pi signaling across all eukaryotic kingdoms, which is in accord with the fundamental importance of Pi management for metabolism.

Inositol pyrophosphate catabolism by three families of phosphatases controls plant growth and development

“…We speculate that M. polymorpha may contain a second, sequence-divergent PP-InsP kinase able to synthesize 1,5-InsP 8 . In line with this, vip1Δ (the single PPIP5K in baker's yeast) mutants still contain detectable levels of 1,5-InsP 8 (Chabert et al, 2023). Linking Mppfa-dsp1 ge , Mpnudt1 ge or Mpvip1 ge mutant phenotypes to isomer-specific PP-InsP level changes is complicated by compensatory changes in gene expression for other PP-InsP metabolizing enzymes, as indicated by our RNA-seq experiments (Supplementary Figure 8B).…”

“…PP-InsPs are ubiquitous in eukaryotes where they perform diverse signaling functions. Their central role in cellular inorganic phosphate (Pi) / polyphosphate (polyP) homeostasis is conserved among fungi (Azevedo and Saiardi, 2017; Chabert et al, 2023; Guan et al, 2023; Wild et al, 2016), protozoa (Cordeiro et al, 2017), algae (Couso et al, 2016), plants (Stevenson-Paulik et al, 2005; Zhu et al, 2019; Dong et al, 2019; Ried et al, 2021; Guan et al, 2022) and animals (Gu et al, 2017; Haykir et al, 2024; Li et al, 2020; Wang et al, 2020).…”

“…In Arabidopsis, complementation of the seedling lethal phenotype of vih1-2 vih2-4 mutant plants with the full-length PPIP5K VIH2 containing a catalytically inactive phosphatase domain restored growth back to wild-type levels with only minor Pi accumulation defects (Zhu et al, 2019). Baker’s yeast PFA-DSP siw14 Δ strains showed enhanced environmental stress responses (Steidle et al, 2020) and increased 5-InsP 7 levels (Chabert et al, 2023). T-DNA insertion lines in the pfa-dsp1 locus had no apparent phenotypes and wild-type-like cellular PP-InsP levels (Gaugler et al, 2022).…”

Conservation of heat stress acclimation by the inositol polyphosphate multikinase, IPMK responsible for 4/6-InsP7production in land plants

“…Inositol phosphates (InsP) are phosphoesters of myo -inositol (Ins) that are synthesized by the combinatorial phosphorylation of hydroxyl group (-OH) of myo -inositol ring forming InsP 1 -InsP 6 . Diphosphate-containing inositol phosphates, also known as inositol pyrophosphates (PP-InsPs) are sub-family of InsP and are versatile metabolites characterized by the presence of ‘‘high energy’’ phosphoanhydride bonds, with InsP 7 and InsP 8 being the most characterized species (Barker et al, 2009; Chabert et al, 2023; Monserrate and York, 2010; Nguyen Trung et al, 2022; Shears, 2015; Thota and Bhandari, 2015; Wilson et al, 2013). In yeast and metazoans, PP-InsPs serve as the critical cellular messengers controlling a large array of physiological processes including phosphate homeostasis (Chabert et al, 2023; Lee et al, 2007; Li et al, 2020; Wilson et al, 2019), cellular energetics (Szijgyarto et al, 2011) and metabolism (Gu et al, 2021; Qin et al, 2023).…”

“…Diphosphate-containing inositol phosphates, also known as inositol pyrophosphates (PP-InsPs) are sub-family of InsP and are versatile metabolites characterized by the presence of ‘‘high energy’’ phosphoanhydride bonds, with InsP 7 and InsP 8 being the most characterized species (Barker et al, 2009; Chabert et al, 2023; Monserrate and York, 2010; Nguyen Trung et al, 2022; Shears, 2015; Thota and Bhandari, 2015; Wilson et al, 2013). In yeast and metazoans, PP-InsPs serve as the critical cellular messengers controlling a large array of physiological processes including phosphate homeostasis (Chabert et al, 2023; Lee et al, 2007; Li et al, 2020; Wilson et al, 2019), cellular energetics (Szijgyarto et al, 2011) and metabolism (Gu et al, 2021; Qin et al, 2023). To date, the metabolic pathways leading to the production of PP-InsPs are well established in yeast, amoeba and metazoans (Desfougeres et al, 2022; Mulugu et al, 2007; Saiardi et al, 1999; Wang et al, 2011).…”