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

Abstract

Eukaryotic cells control cytosolic 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.

Metabolic Consequences of Polyphosphate Synthesis and Imminent Phosphate Limitation

“…Early studies postulated that P i starvation triggers an increase in 1-IP 7 , which should inactivate Pho85/Pho80 kinase by direct interaction with a central part of its regulatory subunit Pho81 ( 12 , 13 ). However, several subsequent studies failed to confirm the starvation-induced increase in IP 7 and instead showed a strong decline in the inositol phosphate pools after Pi starvation ( 5 , 9 , 14 ). These qualitatively different results on the inositol pyrophosphate pools and additional characterization of Pho81 led to a revised model of PHO pathway regulation in which 1,5-IP 8 controls Pho81 through its SPX domain.…”

“…In Saccharomyces cerevisiae , INPHORS triggers the PHO pathway, the transcriptional phosphate starvation response that controls expression of genes for P i scavenging, uptake, and recycling ( 9 ). The PHO pathway in yeast is controlled through the transcription factor Pho4, which accumulates in the nucleus after P i starvation and interacts with another transcription factor, Pho2, to activate many P i -dependent genes.…”

“…The decline of 1,5-IP 8 , which is triggered by P i starvation, allows Pho81 to bind Pho85/Pho80 more tightly and inhibit its kinase activity, favoring dephosphorylation and activation of Pho4. Pho4-dependent transcription depends on the interaction of Pho4 with Pho2, which is stimulated by Pho4 dephosphorylation ( 9 – 11 ). This interaction is also stimulated by an intermediate of purine synthesis, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR), but it is unknown whether AICAR levels respond to P i availability ( 15 – 17 ).…”

“…Synthesis of poly(P) by VTC is stimulated by inositol pyrophosphates ( 5 , 49 , 50 ). The levels of all these compounds (in yeast: 1-IP 7 , 5-IP 7 , and 1,5-IP 8 ) increase in abundance with increasing P i availability ( 9 , 14 ) and will hence favor accumulation and storage of P i in the form of poly(P) only if P i is abundant. How the turnover of poly(P) inside the organelle is regulated and coordinated with the release of P i into the cytosol is unknown.…”

Pools of independently cycling inositol phosphates revealed by pulse labeling with18O-water

“…18 O-water can be obtained in 99.5% pure form, enabling labeling to very high specific isotope content. This facilitates detection of inositol phosphates by mass spectrometry, some of which exist is sub-micromolar concentrations in cells (Chabert et al , 2023; Harmel et al , 2019; Barker et al , 2004; Albert et al , 1997). 18 O-water labeling can be performed in any medium and under any growth condition.…”