Periodic Reporting for period 3 - INPHORS (Intracellular phosphate reception and signaling: A novel homeostatic system with roles for an orphan organelle?)
Reporting period: 2021-09-01 to 2023-02-28
Cells face major changes in demand for and supply of inorganic phosphate (Pi). Pi is a major constituent of DNA, RNA, proteins, many metabolites, and in carbohydrates that form the extracellular matrix or cell wall. Pi is often limiting in the environment, particularly for plants and micro-organisms. Cells experience strong peaks of Pi demand, e.g. when they duplicate and all nucleic acids (DNA, RNA), yet we can also expect that they must safeguard themselves against an excess of Pi because Pi is a product of all nucleotide hydrolyzing reactions. Such reactions drive most metabolic pathways. If they are perturbed, metabolism can be arrested, with potentially lethal consequences. An accumulation of Pi shifts the equilibria of these reactions and reduces the free energy that they can provide to drive metabolism. Thus, while Pi starvation may retard growth, elevated cytosolic Pi might kill a cell by stalling metabolism. In accord with this perturbed Pi homeostasis can be lethal in micro-organisms, severely retard growth in plants, and cause embryonic lethality, neurodegeneration, or renal Fanconi syndrome in mammals. Intracellular Pi homeostasis is thus not only a fundamental problem of cell biology but also of interest for medicine and agriculture.
The arguments outlined above raise the expectation that cells should control and coordinate their systems for uptake, export and storage of Pi in order to strike the delicate balance between the biosynthetic requirements for Pi and the risks of elevated cytoplasmic Pi. We postulates a dedicated signaling network that senses Pi and exerts this coordinating function. We also postulate a buffering system for cytosolic Pi in the form of acidocalcisomes. These highly conserved but poorly understood organelles, found from micro-organisms to men, accumulate hundreds of mM of Pi in the form of polyphosphate (polyP), polymers of up to a thousand Pi units linked through phosphoric anhydride bonds. PolyP is stored inside acidocalcisome. Acidocalcisomes might thereby become buffering devices for cytosolic Pi because they also carry polyphosphatases that might hydrolyze polyP to mobilize Pi and re-export it into the cytosol.
The goal of this proposal is to elucidate the intracellular signaling and buffering network that stabilizes cytosolic Pi. We term this network the INPHORS pathway.
We provided first hints for the existence of this pathway, which may coordinate multiple systems for import, export and acidocalcisomal storage of Pi. Yeast cells serve us as a powerful model system for exploring this pathway. Yeast Pi transport and storage proteins are known. Furthermore, we can establish cell-free in vitro systems that reconstitute Pi-regulated transport and storage processes, providing an excellent basis for identifying signaling complexes and studying their dynamics.
In this project, we (A) generate novel tools to uncouple, individually manipulate and measure key parameters for the INPHORS pathway; (B) identify its components, study their interactions and regulation; (C) elucidate how acidocalcisomes are targeted by INPHORS and how they contribute to Pi homeostasis; (D) study the crosstalk between INPHORS and Pi-regulated transcriptional responses; (E) test the relevance of INPHORS for Pi homeostasis in mammalian cells.
The arguments outlined above raise the expectation that cells should control and coordinate their systems for uptake, export and storage of Pi in order to strike the delicate balance between the biosynthetic requirements for Pi and the risks of elevated cytoplasmic Pi. We postulates a dedicated signaling network that senses Pi and exerts this coordinating function. We also postulate a buffering system for cytosolic Pi in the form of acidocalcisomes. These highly conserved but poorly understood organelles, found from micro-organisms to men, accumulate hundreds of mM of Pi in the form of polyphosphate (polyP), polymers of up to a thousand Pi units linked through phosphoric anhydride bonds. PolyP is stored inside acidocalcisome. Acidocalcisomes might thereby become buffering devices for cytosolic Pi because they also carry polyphosphatases that might hydrolyze polyP to mobilize Pi and re-export it into the cytosol.
The goal of this proposal is to elucidate the intracellular signaling and buffering network that stabilizes cytosolic Pi. We term this network the INPHORS pathway.
We provided first hints for the existence of this pathway, which may coordinate multiple systems for import, export and acidocalcisomal storage of Pi. Yeast cells serve us as a powerful model system for exploring this pathway. Yeast Pi transport and storage proteins are known. Furthermore, we can establish cell-free in vitro systems that reconstitute Pi-regulated transport and storage processes, providing an excellent basis for identifying signaling complexes and studying their dynamics.
In this project, we (A) generate novel tools to uncouple, individually manipulate and measure key parameters for the INPHORS pathway; (B) identify its components, study their interactions and regulation; (C) elucidate how acidocalcisomes are targeted by INPHORS and how they contribute to Pi homeostasis; (D) study the crosstalk between INPHORS and Pi-regulated transcriptional responses; (E) test the relevance of INPHORS for Pi homeostasis in mammalian cells.
We need to be able to measure key parameters of this pathway in order to identify its components. Thus, the first half of this project focused on tool building and the description of basic system properties.
The complexity and redundancy of mechanisms that stabilize cytosolic Pi pose a challenge. Yeast cells dispose of 7 protein systems that are regulated through INPHORS and that either bring Pi into the cytosol or withdraw it from there. All 7 systems carry SPX domains, which are key constituents of INPHORS. The resulting high degrees of redundancy and resilience of the system can explanain why mutations in any one of these systems show disappointingly weak phenotypes, which are not at all in accord with the essential role of Pi homeostasis that we postulate. Therefore, we generated yeast cells lacking all INPHORS-responsive (SPX-carrying) protein systems. To our relief, this is lethal for the cells, confirming the essential nature of INPHORS signaling. We have thus generated simplified "minimal" yeast, which express only a single SPX protein. This leads to a viable cell that allows us to study the properties of a single INPHORS-responsive system at a time, circumventing the problems of redundancy and resilience.
Concentration of cytosolic Pi and of inositol pyrophosphates (InsPPs)n must also 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. At the end of 2020, a novel technique for InsPP separation and quantification became available that allows to separate the entire spectrum of different InsPPs. We now apply this technique for the comprehensive analysis of InsPP dynamics under a variety of starvation and stress conditions. These experiments reveal that Pi sufficiency is signaled through the accumulation of a specific isomer of InsPPs, which is called 5-InsP7. To compensate for the non-functionality of the available Pi biosensors, we now try develop new sensors based on a different principle.
Studies of cytosolic Pi homeostasis would greatly benefit from the capacity to acutely change the Pi content of the cytosol. To develop this capacity, we have created a series of mutated Pi transporters, hoping to obtain an "open" yeast, i.e. a yeast cell carrying a Pi-permeable channel that it cannot control. Preliminary results obtained are very encouraging and suggest that we may be able to obtain such "open" yeast.
The complexity and redundancy of mechanisms that stabilize cytosolic Pi pose a challenge. Yeast cells dispose of 7 protein systems that are regulated through INPHORS and that either bring Pi into the cytosol or withdraw it from there. All 7 systems carry SPX domains, which are key constituents of INPHORS. The resulting high degrees of redundancy and resilience of the system can explanain why mutations in any one of these systems show disappointingly weak phenotypes, which are not at all in accord with the essential role of Pi homeostasis that we postulate. Therefore, we generated yeast cells lacking all INPHORS-responsive (SPX-carrying) protein systems. To our relief, this is lethal for the cells, confirming the essential nature of INPHORS signaling. We have thus generated simplified "minimal" yeast, which express only a single SPX protein. This leads to a viable cell that allows us to study the properties of a single INPHORS-responsive system at a time, circumventing the problems of redundancy and resilience.
Concentration of cytosolic Pi and of inositol pyrophosphates (InsPPs)n must also 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. At the end of 2020, a novel technique for InsPP separation and quantification became available that allows to separate the entire spectrum of different InsPPs. We now apply this technique for the comprehensive analysis of InsPP dynamics under a variety of starvation and stress conditions. These experiments reveal that Pi sufficiency is signaled through the accumulation of a specific isomer of InsPPs, which is called 5-InsP7. To compensate for the non-functionality of the available Pi biosensors, we now try develop new sensors based on a different principle.
Studies of cytosolic Pi homeostasis would greatly benefit from the capacity to acutely change the Pi content of the cytosol. To develop this capacity, we have created a series of mutated Pi transporters, hoping to obtain an "open" yeast, i.e. a yeast cell carrying a Pi-permeable channel that it cannot control. Preliminary results obtained are very encouraging and suggest that we may be able to obtain such "open" yeast.
Our systematic InsPP profiling led us to revise the paradigm on how the transcriptional response to Pi starvation is regulated by the cells. We assign the triggering of the Pi starvation response to a different isomer of InsPP (5-InsP7 instead of 1-InsP7) and to a different mechanism (through an SPX domain instead of direct binding to the kinase domain that regulates a Pi-controlled transcription factor).
Metabolomic analyses put us on track to discover a second branch of regulation by which transcription is controlled as a function of Pi availability. This novel mechanism operates by changing chromatin structure.
In a third line of investigation, we obtained novel insight into how acidocalcisomes can operate as Pi buffers for the cytosol. We discovered a novel feedback loop, which leads acidocalcisomes to degrade the polyP that they synthesize and store in their lumen until a certain free lumenal Pi concentration is established. Then, Pi can be released from these organelles through an SPX-controlled Pi transporter, which is activated by its cytosolic SPX domain when Pi in the cytosol is low.
Our analyses of binding partners and co-regulators of SPX domains is in progress. As a first result, we found that the SPL2 protein may be a general cofactor of SPX domains. It competes with InsPPs for binding to SPX. We expect these analyses to provide several other cofactors of SPX domains in the further course of the project.
Metabolomic analyses put us on track to discover a second branch of regulation by which transcription is controlled as a function of Pi availability. This novel mechanism operates by changing chromatin structure.
In a third line of investigation, we obtained novel insight into how acidocalcisomes can operate as Pi buffers for the cytosol. We discovered a novel feedback loop, which leads acidocalcisomes to degrade the polyP that they synthesize and store in their lumen until a certain free lumenal Pi concentration is established. Then, Pi can be released from these organelles through an SPX-controlled Pi transporter, which is activated by its cytosolic SPX domain when Pi in the cytosol is low.
Our analyses of binding partners and co-regulators of SPX domains is in progress. As a first result, we found that the SPL2 protein may be a general cofactor of SPX domains. It competes with InsPPs for binding to SPX. We expect these analyses to provide several other cofactors of SPX domains in the further course of the project.