Tandem inactivation of inositol pyrophosphatases Asp1, Siw14, and Aps1 illuminates functional redundancies in inositol pyrophosphate catabolism in fission yeast
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ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE285739 | GEO | 2025/03/26
REPOSITORIES: GEO
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Project description:Tandem inactivation of inositol pyrophosphatases Asp1, Siw14, and Aps1 illuminates functional redundancies in inositol pyrophosphate catabolism in fission yeast
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Project description:The maintenance of cellular phosphate (Pi) homeostasis is of paramount importance in living organisms. Little is known about how cells sense intracellular Pi status in multi-cellular eukaryotes. Here, we report the identification of inositol pyrophosphate InsP8, a ligand that binds to the Pi sensor protein SPX1 to inhibit PHR1, the central regulator of Pi starvation responses in Arabidopsis. The concentration of InsP8, synthesized by diphosphoinositol pentakisphosphate kinases VIH1 and VIH2, depends on cellular Pi level and the double mutants accumulated higher amounts of Pi. The paradox of Pi toxicity and induction of Pi starvation inducible genes indicated that the cells could not sense cellular Pi without InsP8. Our study reveals that InsP8 acts as a Pi signaling molecule and binds to the receptor SPX1 to regulate Pi homeostasis.
2022-02-22 | GSE126927 | GEO
Project description:Fission yeast phosphate acquisition genes pho1, pho84, and tgp1 are repressed in phosphate-rich medium by transcription of upstream lncRNAs. Here we show phosphate homeostasis is subject to metabolite control by inositol pyrophosphates (IPPs), exerted through the 3'-processing/termination machinery and the Pol2 CTD code. Increasing IP8 (via Asp1 IPP pyrophosphatase mutation) de-represses the PHO regulon and leads to precocious termination of prt lncRNA synthesis. pho1 de-repression by IP8 depends on cleavage-polyadenylation factor (CPF) subunits, termination factor Rhn1, and the Thr4 letter of the CTD code. pho1 de-repression by mutation of the Ser7 CTD letter depends on IP8. Simultaneous inactivation of Asp1 and Aps1 pyrophosphatases (i.e., too much IP8) is lethal, but this lethality is suppressed by mutations of CPF subunits Ppn1, Swd22, Ssu72, and Ctf1 and CTD mutation T4A. Failure to synthesize IP8 (via Asp1 IPP kinase mutation) results in pho1 hyper-repression. Synthetic lethality of asp1∆ with Ppn1, Swd22, and Ssu72 mutations argues that IP8 plays an important role in essential 3'-processing/termination events, albeit in a manner genetically redundant to CPF. Transcriptional profiling delineates an IPP-responsive regulon composed of genes overexpressed when IP8 levels are increased. Our results establish a novel role for IPPs in cell physiology.
2019-06-16 | GSE127550 | GEO
Project description:Inositol pyrophosphates (PP-InsPs) are nutrient messengers whose cellular levels are precisely regulated. Diphosphoinositol pentakisphosphate kinases (PPIP5Ks) generate the active signaling molecule 1,5-InsP8. PPIP5Ks harbor phosphatase domains that hydrolyze PP-InsPs. Plant and Fungi Atypical Dual Specificity Phosphatases (PFA-DSPs) and NUDIX phosphatases (NUDTs) are also involved in PP-InsP degradation. Here, we analyze the relative contributions of the three different phosphatase families to plant PP-InsP catabolism. We report the biochemical characterization of inositol pyrophosphate phosphatases from Arabidopsis and Marchantia polymorpha. Overexpression of different PFA-DSP and NUDT enzymes affects PP-InsP levels and leads to stunted growth phenotypes in Arabidopsis. nudt17/18/21 knock-out mutants have altered PP-InsP pools and gene expression patterns, but no apparent growth defects. In contrast, Marchantia polymorpha Mppfa-dsp1ge, Mpnudt1ge and Mpvip1ge mutants display severe growth and developmental phenotypes and associated changes in cellular PP-InsP levels. Analysis of Mppfa-dsp1geand Mpvip1ge mutants supports a role for PP-InsPs in Marchantia phosphate signaling, and additional functions in nitrate homeostasis and cell wall biogenesis. Simultaneous elimination of two phosphatase activities enhanced the observed growth phenotypes. Taken together, PPIP5K, PFA-DSP and NUDT inositol pyrophosphate phosphatases regulate growth and development by collectively shaping plant PP-InsP pools.
| S-EPMC11611267 | biostudies-literature
Project description:Expression of the fission yeast Schizosaccharomyces pombe phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme consisting of an N-terminal kinase domain and a C-terminal pyrophosphatase domain that catalyze IP8 synthesis and catabolism, respectively. Here, we report structures of the Asp1 kinase domain, crystallized with two protomers in the asymmetric unit, one of which was complexed with ligands (ADPNP, ADP, or ATP; Mg2+ or Mn2+; IP6, 5-IP7, or 1,5-IP8) and the other which was ligand-free. The ligand-free enzyme adopts an "open" conformation that allows ingress of substrates and egress of products. ADPNP, ADP, and ATP and associated metal ions occupy a deep phospho-donor pocket in the active site. IP6 or 5-IP7 engagement above the nucleotide favors adoption of a "closed" conformation, in which surface protein segments undergo movement and a disordered-to-ordered transition to form an inositol polyphosphate-binding site. In a structure mimetic of the kinase Michaelis complex, the anionic 5-IP7 phosphates are encaged by an ensemble of nine cationic amino acids: Lys43, Arg223, Lys224, Lys260, Arg274, Arg285, Lys290, Arg293, and Lys341. Alanine mutagenesis of amino acids that contact the adenosine nucleoside of the ATP donor underscored the contributions of Asp258 interaction with the ribose 3'-OH and of Glu248 with adenine-N6. Changing Glu248 to Gln elicited a gain of function whereby the kinase became adept at using GTP as phosphate donor. Wild-type Asp1 kinase can utilize N6-benzyl-ATP as phosphate donor. IMPORTANCE The inositol pyrophosphate signaling molecule 1,5-IP8 modulates fission yeast phosphate homeostasis via its action as an agonist of RNA 3'-processing and transcription termination. Cellular IP8 levels are determined by Asp1, a bifunctional enzyme composed of an N-terminal kinase and a C-terminal pyrophosphatase domain. Here, we present a series of crystal structures of the Asp1 kinase domain, in a ligand-free state and in complexes with nucleotides ADPNP, ADP, and ATP, divalent cations magnesium and manganese, and inositol polyphosphates IP6, 5-IP7, and 1,5-IP8. Substrate binding elicits a switch from open to closed conformations, entailing a disordered-to-ordered transition and a rearrangement or movement of two peptide segments that form a binding site for the phospho-acceptor. Our structures, along with structure-guided mutagenesis, fortify understanding of the mechanism and substrate specificity of Asp1 kinase, and they extend and complement structural and functional studies of the orthologous human kinase PPIP5K2.
| S-EPMC9765450 | biostudies-literature
Project description: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.
| S-EPMC8749416 | biostudies-literature
Project description:Using a consensus sequence in inositol phosphate kinase, we have identified and cloned a 44-kDa mammalian inositol phosphate kinase with broader catalytic capacities than any other member of the family and which we designate mammalian inositol phosphate multikinase (mIPMK). By phosphorylating inositol 4,5-bisphosphate, mIPMK provides an alternative biosynthesis for inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)]. mIPMK also can form the pyrophosphate disphosphoinositol tetrakisphosphate (PP-InsP(4)) from InsP(5). Additionally, mIPMK forms InsP(4) from Ins(1,4,5)P(3) and InsP(5) from Ins(1,3,4,5)P(4).
| S-EPMC30134 | biostudies-literature