We needed to establish methods to measure key parameters of the INPHORS pathway in order to identify its components and characterise its dynamics. Concentration of cytosolic Pi and of inositol pyrophosphates (InsPPs) had to become measurable. InsPPs are crucial signaling molecules in INPHORS, which may communicate information about cytosolic Pi concentration to SPX domains. Published assays for InsPPs proved unusable for our purposes and published biosensors to report cytosolic Pi concentrations did not pass the quality controls in our experimental system. They could hence not be used as foreseen. New solutions could be found. A novel technique for InsPP separation and quantification became available that allows to separate the entire spectrum of different InsPPs. We applied this technique in a comprehensive analysis of InsPP dynamics under a variety of starvation and stress conditions, and correlated them with gene regulation under these conditions. These experiments refuted the widely accepted view of Pi signalling in the cytosol and established a new concept, in which the abundance of Pi is signalled through a corresponding accumulation of a specific InsPP isomer, 1,5-IP8 and SPX-dependent INPHORS signalling. This is a conserved mechanism, which applies not only to our yeast model, but is also valid in humans, mammals, and plants.
The complexity and redundancy of proteins that stabilize cytosolic Pi pose a challenge. Yeast cells dispose of 7 protein systems to bring Pi into the cytosol or withdraw it from there. They are all carry SPX domains and are regulated through INPHORS. The resulting redundancy and resilience of the system explains why mutations in any one of these systems show only weak phenotypes. Therefore, we generated yeast cells lacking all INPHORS-responsive protein systems. This was lethal for the cells, confirming the essential nature of INPHORS signalling. By generating simplified "minimal" yeast, which expresses only a single SPX protein at a time, we could circumventing redundancy and reveal that cells require at least one of their systems to eliminate Pi from the cytosol in order to survive. This establishes that Pi is not only an essential macronutrient, but also surprisingly toxic when cells cannot extrude or inactivate an excess of it. This is of practical interest in several respects. These results can explain why a large number of cancer cell types can only grow when they have a high activity of the INPHORS-controlled Pi exporter XPR1. Cancer cells have an increased need for this Pi homeostasis pathway because their metabolism differs from that of non-dividing cells and employs much greater quantities of Pi-containing metabolites. Pharmacological targeting of the INPHORS pathway is thus of potential therapeutic interest. Another implication concerns food safety. Polyphosphates are very common - supposedly harmless - additives in processed food. Upon their degradation and conversion into Pi in the intestinal tract, they lead to exaggerated Pi uptake and toxic side effects.
This new concept of Pi toxicity led us to explore how cells avoid such toxicity. We elucidated how acidocalcisomes, a poorly studied organelle, which is nevertheless present in most eukaryotic organisms, can operate as Pi buffers for the cytosol. They can remove exaggerated Pi concentrations by polymerising Pi into inorganic polyphosphate (polyP), which they then sequester in their lumen. Upon Pi scarcity, they remobilise this polyP and release it back into the cytosol. We discovered a novel feedback loop, which allows acidocalcisome to switch between polyP accumulation and Pi remobilisation, a transition that is at the heart of their function as Pi buffer. Remobilised Pi can then be released from these organelles through a Pi transporter, which is activated by its cytosolic SPX domain when Pi in the cytosol is low. This reveals an INPHORS-controlled buffer system stabilising cytosolic Pi. Key components of this buffer system are conserved in other eukaryotes, which suggests that the identified mechanism applies also to other eukaryotic cells.
Metabolomic analyses led us to discover a new pathway of metabolic signalling to control transcription as a function of Pi availability. This novel pathway is distinct from INPHORS and operates through the Pi-dependent accumulation of primary metabolites, which directly alter chromatin structure, nucleosome positioning and transcription.
