Inositol Pyrophosphate Dynamics Reveals Control of the Yeast Phosphate Starvation Program Through 1,5-IP8and the SPX Domain of Pho81

AbstractEukaryotic 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 (IPPs) and SPX receptor domains. Since cells synthesize various IPPs and SPX domains bind them promiscuously, it is unclear whether a specific IPP regulates SPX domains in vivo, or whether multiple IPPs act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the IPP 1-IP7, we now show that the levels of all detectable IPPs of yeast, 1-IP7, 5-IP7and 1,5-IP8, strongly decline upon phosphate starvation. Among these, specifically the decline of 1,5-IP8triggers the transcriptional phosphate starvation response, the PHO pathway. 1,5-IP8inactivates 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-IP8signals cytosolic phosphate abundance to SPX proteins in fungi, plants, and mammals. Its absence triggers starvation responses.Significance statementCytosolic Piis of prime importance for cellular bioenergetics because Piinfluences free energy of nucleotide hydrolysis and the metabolite fluxes through glycolysis and oxidative phosphorylation. Eukaryotic cells use the INPHORS pathway to signal Pivia SPX domains and their ligands, inositol pyrophosphates (IP7, IP8), which control Pihomeostasis 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 in yeast and dissected the isomer that is relevant to intracellular Pisignaling. Our results can be combined with existing observations from plants, mammals, and other fungi to support a unified model of Pisignaling across all eukaryotic kingdoms, which is in accord with the fundamental importance of Pimanagement for metabolism.