Homeostatic coordination of cellular phosphate uptake and efflux requires an organelle-based receptor for the inositol pyrophosphate IP8.

Phosphate (Pi) serves countless metabolic pathways and is involved in macromolecule synthesis, energy storage, cellular signaling, and bone maintenance. Herein, we describe the coordination of Pi uptake and efflux pathways to maintain mammalian cell Pi homeostasis. We discover that XPR1, the presumed Pi efflux transporter, separately supervises rates of Pi uptake. This direct, regulatory interplay arises from XPR1 being a binding partner for the Pi uptake transporter PiT1, involving a predicted transmembrane helix/extramembrane loop in XPR1, and its hitherto unknown localization in a subset of intracellular LAMP1-positive puncta (named "XLPVs"). A pharmacological mimic of Pi homeostatic challenge is sensed by the inositol pyrophosphate IP8, which functionalizes XPR1 to respond in a temporally hierarchal manner, initially adjusting the rate of Pi efflux, followed subsequently by independent modulation of PiT1 turnover to reset the rate of Pi uptake. These observations generate a unifying model of mammalian cellular Pi homeostasis, expanding opportunities for therapeutic intervention.

Suppression of inositol pyrophosphate toxicosis and hyper-repression of the fission yeast PHO regulon by loss-of-function mutations in chromatin remodelers Snf22 and Sol1.

Inositol pyrophosphates are signaling molecules that regulate cellular phosphate homeostasis in eukaryal taxa. In fission yeast, where the phosphate regulon (comprising phosphate acquisition genes pho1, pho84, and tgp1) is repressed under phosphate-replete conditions by lncRNA-mediated transcriptional interference, mutations of inositol pyrophosphatases that increase IP8 levels derepress the PHO regulon by eliciting precocious termination of lncRNA transcription. Asp1 pyrophosphatase mutations resulting in too much IP8 are cytotoxic in YES medium owing to overexpression of glycerophosphodiester transporter Tgp1. IP8 toxicosis is ameliorated by mutations in cleavage/polyadenylation and termination factors, perturbations of the Pol2 CTD code, and mutations in SPX domain proteins that act as inositol pyrophosphate sensors. Here, we show that IP8 toxicity is alleviated by deletion of snf22+, the gene encoding the ATPase subunit of the SWI/SNF chromatin remodeling complex, by an ATPase-inactivating snf22-(D996A-E997A) allele, and by deletion of the gene encoding SWI/SNF subunit Sol1. Deletion of snf22+ hyper-repressed pho1 expression in phosphate-replete cells; suppressed the pho1 derepression elicited by mutations in Pol2 CTD, termination factor Seb1, Asp1 pyrophosphatase, and 14-3-3 protein Rad24 (that favor precocious prt lncRNA termination); and delayed pho1 induction during phosphate starvation. RNA analysis and lack of mutational synergies suggest that Snf22 is not impacting 3'-processing/termination. Using reporter assays, we find that Snf22 is important for the activity of the tgp1 and pho1 promoters, but not for the promoters that drive the synthesis of the PHO-repressive lncRNAs. Transcription profiling of snf22∆ and snf22-(D996A-E997A) cells identified an additional set of 66 protein-coding genes that were downregulated in both mutants.IMPORTANCERepression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to inositol pyrophosphate dynamics. Cytotoxic asp1-STF alleles derepress the PHO genes via the action of IP8 as an agonist of precocious lncRNA 3'-processing/termination. IP8 toxicosis is alleviated by mutations of the Pol2 CTD and the 3'-processing/termination machinery that dampen the impact of toxic IP8 levels on termination. In this study, a forward genetic screen revealed that IP8 toxicity is suppressed by mutations of the Snf22 and Sol1 subunits of the SWI/SNF chromatin remodeling complex. Genetic and biochemical evidence indicates that the SWI/SNF is not affecting 3'-processing/termination or lncRNA promoter activity. Rather, SWI/SNF is critical for firing the PHO mRNA promoters. Our results implicate the ATP-dependent nucleosome remodeling activity of SWI/SNF as necessary to ensure full access of PHO-activating transcription factor Pho7 to its binding sites in the PHO mRNA promoters.

Kcs1 and Vip1: The Key Enzymes behind Inositol Pyrophosphate Signaling in Saccharomyces cerevisiae.

The inositol pyrophosphate pathway, a complex cell signaling network, plays a pivotal role in orchestrating vital cellular processes in the budding yeast, where it regulates cell cycle progression, growth, endocytosis, exocytosis, apoptosis, telomere elongation, ribosome biogenesis, and stress responses. This pathway has gained significant attention in pharmacology and medicine due to its role in generating inositol pyrophosphates, which serve as crucial signaling molecules not only in yeast, but also in higher eukaryotes. As targets for therapeutic development, genetic modifications within this pathway hold promise for disease treatment strategies, offering practical applications in biotechnology. The model organism Saccharomyces cerevisiae, renowned for its genetic tractability, has been instrumental in various studies related to the inositol pyrophosphate pathway. This review is focused on the Kcs1 and Vip1, the two enzymes involved in the biosynthesis of inositol pyrophosphate in S. cerevisiae, highlighting their roles in various cell processes, and providing an up-to-date overview of their relationship with phosphate homeostasis. Moreover, the review underscores the potential applications of these findings in the realms of medicine and biotechnology, highlighting the profound implications of comprehending this intricate signaling network.

Functions, Mechanisms, and therapeutic applications of the inositol pyrophosphates 5PP-InsP5 and InsP8 in mammalian cells.

Water-soluble myo-inositol phosphates have long been characterized as second messengers. The signaling properties of these compounds are determined by the number and arrangement of phosphate groups on the myo-inositol backbone. Recently, higher inositol phosphates with pyrophosphate groups were recognized as signaling molecules. 5-Diphosphoinositol 1,2,3,4,6-pentakisphosphate (5PP-InsP5) is the most abundant isoform, constituting more than 90% of intracellular inositol pyrophosphates. 5PP-InsP5 can be further phosphorylated to 1,5-bisdiphosphoinositol 2,3,4,6-tetrakisphosphate (InsP8). These two molecules, 5PP-InsP5 and InsP8, are present in various subcellular compartments, where they participate in regulating diverse cellular processes such as cell death, energy homeostasis, and cytoskeletal dynamics. The synthesis and metabolism of inositol pyrophosphates are subjected to tight regulation, allowing for their highly specific functions. Blocking the 5PP-InsP5/InsP8 signaling pathway by inhibiting the biosynthesis of 5PP-InsP5 demonstrates therapeutic benefits in preclinical studies, and thus holds promise as a therapeutic approach for certain diseases treatment, such as metabolic disorders.

Genetic suppressor screen identifies Tgp1 (glycerophosphocholine transporter), Kcs1 (IP6 kinase), and Plc1 (phospholipase C) as determinants of inositol pyrophosphate toxicosis in fission yeast.

The inositol pyrophosphate signaling molecule 1,5-IP8 is an agonist of RNA 3'-processing and transcription termination in fission yeast that regulates the expression of phosphate acquisition genes pho1, pho84, and tgp1. IP8 is synthesized from 5-IP7 by the Asp1 N-terminal kinase domain and catabolized by the Asp1 C-terminal pyrophosphatase domain. asp1-STF mutations that delete or inactivate the Asp1 pyrophosphatase domain elicit growth defects in yeast extract with supplements (YES) medium ranging from severe sickness to lethality. We now find that the toxicity of asp1-STF mutants is caused by a titratable constituent of yeast extract. Via a genetic screen for spontaneous suppressors, we identified a null mutation of glycerophosphodiester transporter tgp1 that abolishes asp1-STF toxicity in YES medium. This result, and the fact that tgp1 mRNA expression is increased by >40-fold in asp1-STF cells, prompted discovery that: (i) glycerophosphocholine (GPC) recapitulates the toxicity of yeast extract to asp1-STF cells in a Tgp1-dependent manner, and (ii) induced overexpression of tgp1 in asp1+ cells also elicits toxicity dependent on GPC. asp1-STF suppressor screens yielded a suite of single missense mutations in the essential IP6 kinase Kcs1 that generates 5-IP7, the immediate precursor to IP8. Transcription profiling of the kcs1 mutants in an asp1+ background revealed the downregulation of the same phosphate acquisition genes that were upregulated in asp1-STF cells. The suppressor screen also returned single missense mutations in Plc1, the fission yeast phospholipase C enzyme that generates IP3, an upstream precursor for the synthesis of inositol pyrophosphates.IMPORTANCEThe inositol pyrophosphate metabolite 1,5-IP8 governs repression of fission yeast phosphate homeostasis genes pho1, pho84, and tgp1 by lncRNA-mediated transcriptional interference. Asp1 pyrophosphatase mutations that increase IP8 levels elicit precocious lncRNA termination, leading to derepression of the PHO genes. Deletions of the Asp1 pyrophosphatase domain result in growth impairment or lethality via IP8 agonism of transcription termination. It was assumed that IP8 toxicity ensues from dysregulation of essential genes. In this study, a suppressor screen revealed that IP8 toxicosis of Asp1 pyrophosphatase mutants is caused by: (i) a >40-fold increase in the expression of the inessential tgp1 gene encoding a glycerophosphodiester transporter and (ii) the presence of glycerophosphocholine in the growth medium. The suppressor screen yielded missense mutations in two upstream enzymes of inositol polyphosphate metabolism: the phospholipase C enzyme Plc1 that generates IP3 and the essential Kcs1 kinase that converts IP6 to 5-IP7, the immediate precursor of IP8.

Evolutionary perspective on mammalian inorganic polyphosphate (polyP) biology.

Inorganic polyphosphate (polyP), the polymeric form of phosphate, is attracting ever-growing attention due to the many functions it appears to perform within mammalian cells. This essay does not aim to systematically review the copious mammalian polyP literature. Instead, we examined polyP synthesis and functions in various microorganisms and used an evolutionary perspective to theorise key issues of this field and propose solutions. By highlighting the presence of VTC4 in distinct species of very divergent eucaryote clades (Opisthokonta, Viridiplantae, Discoba, and the SAR), we propose that whilst polyP synthesising machinery was present in the ancestral eukaryote, most lineages subsequently lost it during evolution. The analysis of the bacteria-acquired amoeba PPK1 and its unique polyP physiology suggests that eukaryote cells must have developed mechanisms to limit cytosolic polyP accumulation. We reviewed the literature on polyP in the mitochondria from the perspective of its endosymbiotic origin from bacteria, highlighting how mitochondria could possess a polyP physiology reminiscent of their 'bacterial' beginning that is not yet investigated. Finally, we emphasised the similarities that the anionic polyP shares with the better-understood negatively charged polymers DNA and RNA, postulating that the nucleus offers an ideal environment where polyP physiology might thrive.

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

Eukaryotic 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 and SPX receptor domains. Since cells synthesize various inositol pyrophosphates and SPX domains bind them promiscuously, it is unclear whether a specific inositol pyrophosphate regulates SPX domains in vivo, or whether multiple inositol pyrophosphates act as a pool. In contrast to previous models, which postulated that phosphate starvation is signaled by increased production of the inositol pyrophosphate 1-IP7, we now show that the levels of all detectable inositol pyrophosphates 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.

Itraconazole inhibits endothelial cell migration by disrupting inositol pyrophosphate-dependent focal adhesion dynamics and cytoskeletal remodeling.

The antifungal drug itraconazole has been repurposed to anti-angiogenic agent, but the mechanisms of action have been elusive. Here we report that itraconazole disrupts focal adhesion dynamics and cytoskeletal remodeling, which requires 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7). We find that inositol hexakisphosphate kinase 1 (IP6K1) binds Arp2 and generates 5-InsP7 to recruit coronin, a negative regulator of the Arp2/3 complex. IP6K1 also produces focal adhesion-enriched 5-InsP7, which binds focal adhesion kinase (FAK) at the FERM domain to promote its dimerization and phosphorylation. Itraconazole treatment elicits displacement of IP6K1/5-InsP7, thus augments 5-InsP7-mediated inhibition of Arp2/3 complex and reduces 5-InsP7-mediated FAK dimerization. Itraconazole-treated cells display reduced focal adhesion dynamics and actin cytoskeleton remodeling. Accordingly, itraconazole severely disrupts cell motility, an essential component of angiogenesis. These results demonstrate critical roles of IP6K1-generated 5-InsP7 in regulating focal adhesion dynamics and actin cytoskeleton remodeling and reveal functional mechanisms by which itraconazole inhibits cell motility.

Duf89 abets lncRNA control of fission yeast phosphate homeostasis via its antagonism of precocious lncRNA transcription termination.

Fission yeast phosphate homeostasis gene pho1 is actively repressed during growth in phosphate-rich medium by transcription in cis of a long noncoding (lnc) RNA from the 5' flanking prt(nc-pho1) gene. Pho1 expression is: (i) derepressed by genetic maneuvers that favor precocious lncRNA 3'-processing and termination, in response to DSR and PAS signals in prt; and (ii) hyperrepressed in genetic backgrounds that dampen 3'-processing/termination efficiency. Governors of 3'-processing/termination include the RNA polymerase CTD code, the CPF (cleavage and polyadenylation factor) complex, termination factors Seb1 and Rhn1, and the inositol pyrophosphate signaling molecule 1,5-IP8 Here, we present genetic and biochemical evidence that fission yeast Duf89, a metal-dependent phosphatase/pyrophosphatase, is an antagonist of precocious 3'-processing/termination. We show that derepression of pho1 in duf89Δ cells correlates with squelching the production of full-length prt lncRNA and is erased or attenuated by: (i) DSR/PAS mutations in prt; (ii) loss-of-function mutations in components of the 3'-processing and termination machinery; (iii) elimination of the CTD Thr4-PO4 mark; (iv) interdicting CTD prolyl isomerization by Pin1; (v) inactivating the Asp1 kinase that synthesizes IP8; and (vi) loss of the putative IP8 sensor Spx1. The findings that duf89Δ is synthetically lethal with pho1-derepressive mutations CTD-S7A and aps1Δ-and that this lethality is rescued by CTD-T4A, CPF/Rhn1/Pin1 mutations, and spx1Δ-implicate Duf89 more broadly as a collaborator in cotranscriptional regulation of essential fission yeast genes. The duf89-D252A mutation, which abolishes Duf89 phosphohydrolase activity, phenocopied duf89 +, signifying that duf89Δ phenotypes are a consequence of Duf89 protein absence, not absence of Duf89 catalysis.

Flux regulation through glycolysis and respiration is balanced by inositol pyrophosphates in yeast.

Although many prokaryotes have glycolysis alternatives, it's considered as the only energy-generating glucose catabolic pathway in eukaryotes. Here, we managed to create a hybrid-glycolysis yeast. Subsequently, we identified an inositol pyrophosphatase encoded by OCA5 that could regulate glycolysis and respiration by adjusting 5-diphosphoinositol 1,2,3,4,6-pentakisphosphate (5-InsP7) levels. 5-InsP7 levels could regulate the expression of genes involved in glycolysis and respiration, representing a global mechanism that could sense ATP levels and regulate central carbon metabolism. The hybrid-glycolysis yeast did not produce ethanol during growth under excess glucose and could produce 2.68 g/L free fatty acids, which is the highest reported production in shake flask of Saccharomyces cerevisiae. This study demonstrated the significance of hybrid-glycolysis yeast and determined Oca5 as an inositol pyrophosphatase controlling the balance between glycolysis and respiration, which may shed light on the role of inositol pyrophosphates in regulating eukaryotic metabolism.

Nucleolar Architecture Is Modulated by a Small Molecule, the Inositol Pyrophosphate 5-InsP7.

Inositol pyrophosphates (PP-InsPs); are a functionally diverse family of eukaryotic molecules that deploy a highly-specialized array of phosphate groups as a combinatorial cell-signaling code. One reductive strategy to derive a molecular-level understanding of the many actions of PP-InsPs is to individually characterize the proteins that bind them. Here, we describe an alternate approach that seeks a single, collective rationalization for PP-InsP binding to an entire group of proteins, i.e., the multiple nucleolar proteins previously reported to bind 5-InsP7 (5-diphospho-inositol-1,2,3,4,6-pentakisphosphate). Quantitative confocal imaging of the outer nucleolar granular region revealed its expansion when cellular 5-InsP7 levels were elevated by either (a) reducing the 5-InsP7 metabolism by a CRISPR-based knockout (KO) of either NUDT3 or PPIP5Ks; or (b), the heterologous expression of wild-type inositol hexakisphosphate kinase, i.e., IP6K2; separate expression of a kinase-dead IP6K2 mutant did not affect granular volume. Conversely, the nucleolar granular region in PPIP5K KO cells shrank back to the wild-type volume upon attenuating 5-InsP7 synthesis using either a pan-IP6K inhibitor or the siRNA-induced knockdown of IP6K1+IP6K2. Significantly, the inner fibrillar volume of the nucleolus was unaffected by 5-InsP7. We posit that 5-InsP7 acts as an 'electrostatic glue' that binds together positively charged surfaces on separate proteins, overcoming mutual protein-protein electrostatic repulsion the latter phenomenon is a known requirement for the assembly of a non-membranous biomolecular condensate.

Inositol pyrophosphate profiling reveals regulatory roles of IP6K2-dependent enhanced IP7 metabolism in the enteric nervous system.

Inositol pyrophosphates regulate diverse physiological processes; to better understand their functional roles, assessing their tissue-specific distribution is important. Here, we profiled inositol pyrophosphate levels in mammalian organs using an originally designed liquid chromatography-mass spectrometry (LC-MS) protocol and discovered that the gastrointestinal tract (GIT) contained the highest levels of diphosphoinositol pentakisphosphate (IP7) and its precursor inositol hexakisphosphate (IP6). Although their absolute levels in the GIT are diet dependent, elevated IP7 metabolism still exists under dietary regimens devoid of exogenous IP7. Of the major GIT cells, enteric neurons selectively express the IP7-synthesizing enzyme IP6K2. We found that IP6K2-knockout mice exhibited significantly impaired IP7 metabolism in the various organs including the proximal GIT. In addition, our LC-MS analysis displayed that genetic ablation of IP6K2 significantly impaired IP7 metabolism in the gut and duodenal muscularis externa containing myenteric plexus. Whole transcriptome analysis of duodenal muscularis externa further suggested that IP6K2 inhibition significantly altered expression levels of the gene sets associated with mature neurons, neural progenitor/stem cells, and glial cells, as well as of certain genes modulating neuronal differentiation and functioning, implying critical roles of the IP6K2-IP7 axis in developmental and functional regulation of the enteric nervous system. These results collectively reveal an unexpected role of mammalian IP7-a highly active IP6K2-IP7 pathway is conducive to the enteric nervous system.

Elevation of inositol pyrophosphate IP7 in the mammalian spinal cord of amyotrophic lateral sclerosis.

Background: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder associated with progressive impairment of spinal motor neurons. Continuous research endeavor is underway to fully understand the molecular mechanisms associating with this disorder. Although several studies have implied the involvement of inositol pyrophosphate IP7 in ALS, there is no direct experimental evidence proving this notion. In this study, we analyzed inositol pyrophosphate IP7 and its precursor IP6 in the mouse and human ALS biological samples to directly assess whether IP7 level and/or its metabolism are altered in ALS disease state. Methods: We used a liquid chromatography-mass spectrometry (LC-MS) protocol originally-designed for mammalian IP6 and IP7 analysis. We measured the abundance of these molecules in the central nervous system (CNS) of ALS mouse model SOD1(G93A) transgenic (TG) mice as well as postmortem spinal cord of ALS patients. Cerebrospinal fluid (CSF) and peripheral blood mononuclear cells (PBMCs) from ALS patients were also analyzed to assess if IP7 status in these biofluids is associated with ALS disease state. Results: SOD1(G93A) TG mice showed significant increase of IP7 level in the spinal cord compared with control mice at the late stage of disease progression, while its level in cerebrum and cerebellum remains constant. We also observed significantly elevated IP7 level and its product-to-precursor ratio (IP7/IP6) in the postmortem spinal cord of ALS patients, suggesting enhanced enzymatic activity of IP7-synthesizing kinases in the human ALS spinal cord. In contrast, human CSF did not contain detectable level of IP6 and IP7, and neither the IP7 level nor the IP7/IP6 ratio in human PBMCs differentiated ALS patients from age-matched healthy individuals. Conclusion: By directly analyzing IP7 in the CNS of ALS mice and humans, the findings of this study provide direct evidence that IP7 level and/or the enzymatic activity of IP7-generating kinases IP6Ks are elevated in ALS spinal cord. On the other hand, this study also showed that IP7 is not suitable for biofluid-based ALS diagnosis. Further investigation is required to elucidate a role of IP7 in ALS pathology and utilize IP7 metabolism on the diagnostic application of ALS.

Regulation of plant biotic interactions and abiotic stress responses by inositol polyphosphates.

Inositol pyrophosphates (PP-InsPs), derivatives of inositol hexakisphosphate (phytic acid, InsP6) or lower inositol polyphosphates, are energy-rich signaling molecules that have critical regulatory functions in eukaryotes. In plants, the biosynthesis and the cellular targets of these messengers are not fully understood. This is because, in part, plants do not possess canonical InsP6 kinases and are able to synthesize PP-InsP isomers that appear to be absent in yeast or mammalian cells. This review will shed light on recent discoveries in the biosynthesis of these enigmatic messengers and on how they regulate important physiological processes in response to abiotic and biotic stresses in plants.

Versatile signaling mechanisms of inositol pyrophosphates.

Inositol pyrophosphates (PP-InsPs) constitute a group of highly charged messengers, which regulate central biological processes in health and disease, such as cellular phosphate and general energy homeostasis. Deciphering the molecular mechanisms underlying PP-InsP-mediated signaling remains a challenge due to the unique properties of these molecules, the different modes of action they can access, and a somewhat limited chemical and analytical toolset. Herein, we summarize the most recent mechanistic insights into PP-InsP signaling, which illustrate our progress in connecting mechanism and function of PP-InsPs.

The Inositol Phosphate System-A Coordinator of Metabolic Adaptability.

All cells rely on nutrients to supply energy and carbon building blocks to support cellular processes. Over time, eukaryotes have developed increasingly complex systems to integrate information about available nutrients with the internal state of energy stores to activate the necessary processes to meet the immediate and ongoing needs of the cell. One such system is the network of soluble and membrane-associated inositol phosphates that coordinate the cellular responses to nutrient uptake and utilization from growth factor signaling to energy homeostasis. In this review, we discuss the coordinated interactions of the inositol polyphosphates, inositol pyrophosphates, and phosphoinositides in major metabolic signaling pathways to illustrate the central importance of the inositol phosphate signaling network in nutrient responses.

Structural and catalytic analyses of the InsP6 kinase activities of higher plant ITPKs.

Inositol phosphate signaling in plants is of substantial agricultural interest, with a considerable focus on the inositol tris/tetrakisphosphate kinase (ITPK) family of inositol phosphate kinases. Historically, the 4-6 isoforms of ITPKs that higher plants each express have been studied for their multiplexing a metabolic pathway to synthesize inositol hexakisphosphate (ie InsP6 or phytate), through the phosphorylation and dephosphorylation of multiple inositol phosphates, including Ins(1,3,4,5,6)P5 (inositol-1,3,4,5,6-pentakisphosphate). A more recent discovery is ITPK-catalyzed phosphorylation of InsP6 to inositol pyrophosphates, which regulate plant immunity and phosphate homeostasis. However, a molecular-based explanation for these alternate catalytic activities has been missing, because no plant ITPK structure has previously been solved. Herein, we provide biochemical and structural analyses of ITPKs from Zea mays and Glycine max. For this work we introduce a simple, enzyme-coupled microplate-based assay of InsP6  kinase activity that should promote more general access to this important field. Furthermore, a ZmITPK1/InsP6 crystal complex is described at a resolution of 2.6 Å, which identifies a number of catalytically important residues; their functionality is confirmed by mutagenesis. We further demonstrate that ZmITPK1 adds a ß-phosphate to the 3-position of Ins(1,2,3,4,5)P5 , yielding a candidate signal for regulating phosphate homeostasis. An impactful discovery is our description of a 29-residue catalytic specificity element; by interchanging this element between GmITPK1 and GmITPK2, we demonstrate how its isoform-specific sequence specifically determines whether the host protein phosphorylates InsP6 , without substantially affecting Ins(1,3,4,5,6)P5  metabolism. Our structural rationalization of key catalytic differences between alternate ITPK isoforms will complement future research into their functional diversity.

Cleavage-Polyadenylation Factor Cft1 and SPX Domain Proteins Are Agents of Inositol Pyrophosphate Toxicosis in Fission Yeast.

Inositol pyrophosphate (IPP) dynamics govern expression of the fission yeast phosphate homeostasis regulon via their effects on lncRNA-mediated transcription interference. The growth defects (ranging from sickness to lethality) elicited by fission yeast mutations that inactivate IPP pyrophosphatase enzymes are exerted via the agonistic effects of too much 1,5-IP8 on RNA 3'-processing and transcription termination. To illuminate determinants of IPP toxicosis, we conducted a genetic screen for spontaneous mutations that suppressed the sickness of Asp1 pyrophosphatase mutants. We identified a missense mutation, C823R, in the essential Cft1 subunit of the cleavage and polyadenylation factor complex that suppresses even lethal Asp1 IPP pyrophosphatase mutations, thereby fortifying the case for 3'-processing/termination as the target of IPP toxicity. The suppressor screen also identified Gde1 and Spx1 (SPAC6B12.07c), both of which have an IPP-binding SPX domain and both of which are required for lethality elicited by Asp1 mutations. A survey of other SPX proteins in the proteome identified the Vtc4 and Vtc2 subunits of the vacuolar polyphosphate polymerase as additional agents of IPP toxicosis. Gde1, Spx1, and Vtc4 contain enzymatic modules (glycerophosphodiesterase, RING finger ubiquitin ligase, and polyphosphate polymerase, respectively) fused to their IPP-sensing SPX domains. Structure-guided mutagenesis of the IPP-binding sites and the catalytic domains of Gde1 and Spx1 indicated that both modules are necessary to elicit IPP toxicity. Whereas Vtc4 polymerase catalytic activity is required for IPP toxicity, its IPP-binding site is not. Epistasis analysis, transcriptome profiling, and assays of Pho1 expression implicate Spx1 as a transducer of IP8 signaling to the 3'-processing/transcription termination machinery. IMPORTANCE Impeding the catabolism of the inositol pyrophosphate (IPP) signaling molecule IP8 is cytotoxic to fission yeast. Here, by performing a genetic suppressor screen, we identified several cellular proteins required for IPP toxicosis. Alleviation of IPP lethality by a missense mutation in the essential Cft1 subunit of the cleavage and polyadenylation factor consolidates previous evidence that toxicity results from IP8 action as an agonist of RNA 3'-processing and transcription termination. Novel findings are that IP8 toxicity depends on IPP-sensing SPX domain proteins with associated enzymatic functions: Gde1 (glycerophosphodiesterase), Spx1 (ubiquitin ligase), and Vtc2/4 (polyphosphate polymerase). The effects of Spx1 deletion on phosphate homeostasis imply a role for Spx1 in communicating an IP8-driven signal to the transcription and RNA processing apparatus.

Plant adaptation to low phosphorus availability: Core signaling, crosstalks, and applied implications.

Phosphorus (P) is an essential nutrient for plant growth and reproduction. Plants preferentially absorb P as orthophosphate (Pi), an ion that displays low solubility and that is readily fixed in the soil, making P limitation a condition common to many soils and Pi fertilization an inefficient practice. To cope with Pi limitation, plants have evolved a series of developmental and physiological responses, collectively known as the Pi starvation rescue system (PSR), aimed to improve Pi acquisition and use efficiency (PUE) and protect from Pi-starvation-induced stress. Intensive research has been carried out during the last 20 years to unravel the mechanisms underlying the control of the PSR in plants. Here we review the results of this research effort that have led to the identification and characterization of several core Pi starvation signaling components, including sensors, transcription factors, microRNAs (miRNAs) and miRNA inhibitors, kinases, phosphatases, and components of the proteostasis machinery. We also refer to recent results revealing the existence of intricate signaling interplays between Pi and other nutrients and antagonists, N, Fe, Zn, and As, that have changed the initial single-nutrient-centric view to a more integrated view of nutrient homeostasis. Finally, we discuss advances toward improving PUE and future research priorities.

Wheat inositol pyrophosphate kinase TaVIH2-3B modulates cell-wall composition and drought tolerance in Arabidopsis.

BACKGROUND: Inositol pyrophosphates (PP-InsPs) are high-energy derivatives of inositol, involved in different signalling and regulatory responses of eukaryotic cells. Distinct PP-InsPs species are characterized by the presence of phosphate at a variable number of the 6-carbon inositol ring backbone, and two distinct classes of inositol phosphate kinases responsible for their synthesis have been identified in Arabidopsis, namely ITPKinase (inositol 1,3,4 trisphosphate 5/6 kinase) and PP-IP5Kinase (diphosphoinositol pentakisphosphate kinases). Plant PP-IP5Ks are capable of synthesizing InsP8 and were previously shown to control defense against pathogens and phosphate response signals. However, other potential roles of plant PP-IP5Ks, especially towards abiotic stress, remain poorly understood. RESULTS: Here, we characterized the physiological functions of two Triticum aestivum L. (hexaploid wheat) PPIP5K homologs, TaVIH1 and TaVIH2. We demonstrate that wheat VIH proteins can utilize InsP7 as the substrate to produce InsP8, a process that requires the functional VIH-kinase domains. At the transcriptional level, both TaVIH1 and TaVIH2 are expressed in different wheat tissues, including developing grains, but show selective response to abiotic stresses during drought-mimic experiments. Ectopic overexpression of TaVIH2-3B in Arabidopsis confers tolerance to drought stress and rescues the sensitivity of Atvih2 mutants. RNAseq analysis of TaVIH2-3B-expressing transgenic lines of Arabidopsis shows genome-wide reprogramming with remarkable effects on genes involved in cell-wall biosynthesis, which is supported by the observation of enhanced accumulation of polysaccharides (arabinogalactan, cellulose, and arabinoxylan) in the transgenic plants. CONCLUSIONS: Overall, this work identifies a novel function of VIH proteins, implicating them in modulation of the expression of cell-wall homeostasis genes, and tolerance to water-deficit stress. This work suggests that plant VIH enzymes may be linked to drought tolerance and opens up the possibility of future research into using plant VIH-derived products to generate drought-resistant plants.