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Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

ABSTRACT: Inositol pyrophosphate 1,5-IP₈ regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP₈ levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP₇ to 1,5-IP₈ and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP₇, 1-IP₇, and 1,5-IP₈. Aps1 displays a ~twofold preference for hydrolysis of 1-IP₇ versus 5-IP₇ and aps1∆ cells have twofold higher levels of 1-IP₇ vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP₈ toxicosis, whereby excessive 1,5-IP₈ drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP₈ levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity.

Importance

Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP₈, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP₈ is formed by phosphorylation of 5-IP₇ and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP₇, 1-IP₇, and 1,5-IP₈ in vitro, with a ~twofold preference for 1-IP₇ over 5-IP₇. aps1∆ cells have twofold higher levels of 1-IP₇ than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP₈ triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

SUBMITTER: Ghosh S

PROVIDER: S-EPMC11323792 | biostudies-literature | 2024 Aug

REPOSITORIES: biostudies-literature

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Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

Ghosh Shreya S Sanchez Ana M AM Schwer Beate B Prucker Isabel I Jork Nikolaus N Jessen Henning J HJ Shuman Stewart S

mBio 20240628 8

Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol py ...[more]

PMID: 38940614

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Project description:Inositol pyrophosphate 1,5-IP8 is a signaling molecule that regulates phosphate and polyphosphate homeostasis in the fission yeast Schizosaccharomyces pombe. 1,5-IP8 levels are dictated by a balance between the Asp1 kinase domain that converts 5-IP7 to 1,5-IP8 and two pyrophosphatases-the Asp1 pyrophosphatase domain (histidine acid phosphatase family) and the Aps1 pyrophosphatase enzyme (Nudix family)-that hydrolyze the β-phosphates of 1,5-IP8. Here, we characterize S. pombe Siw14 (SpSiw14), a cysteinyl-phosphatase family member and a homolog of Saccharomyces cerevisiae Siw14, as a third fission yeast pyrophosphatase implicated in inositol pyrophosphate catabolism. We find that SpSiw14's substrate repertoire embraces inorganic pyrophosphate, inorganic polyphosphate, and the inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8, in addition to the generic substrate p-nitrophenylphosphate. Genetic analyses revealed that (i) elimination of the SpSiw14 protein or inactivation of the SpSiw14 pyrophosphatase by the C189S mutation had no effect on S. pombe growth but was lethal in the absence of Aps1 and (ii) the synthetic lethality of siw14∆ aps1∆ depended on the synthesis of 1,5-IP8 by the Asp1 kinase. We conclude that SpSiw14 and Aps1 pyrophosphatases have essential but redundant functions in fission yeast, and that their synthetic lethality is a consequence of the toxic effects of too much 1,5-IP8. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of components of the fission yeast 3'-processing/termination machinery fortifies the case for overzealous transcription termination as the basis for 1,5-IP8 toxicosis. 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 1,5-IP8 levels are determined by a balance between the activities of the inositol polyphosphate kinase Asp1 and several inositol pyrophosphatase enzymes. Here, we characterize Schizosaccharomyces pombe Siw14 (SpSiw14) as a cysteinyl-phosphatase-family pyrophosphatase enzyme capable of hydrolyzing the phosphoanhydride substrates inorganic pyrophosphate, inorganic polyphosphate, and inositol pyrophosphates 5-IP7, 1-IP7, and 1,5-IP8. Genetic analyses implicate SpSiw14 in 1,5-IP8 catabolism in vivo, insofar as: loss of SpSiw14 activity is lethal in the absence of the Nudix-type inositol pyrophosphatase enzyme Aps1; and siw14∆ aps1∆ lethality depends on synthesis of 1,5-IP8 by the Asp1 kinase. Suppression of siw14∆ aps1∆ lethality by loss-of-function mutations of 3'-processing/termination factors points to precocious transcription termination as the cause of 1,5-IP8 toxicosis.

Project description:Inositol pyrophosphates (IPPs) are signaling molecules that regulate cellular phosphate homeostasis in diverse eukaryal taxa. In fission yeast, mutations that increase 1,5-IP8 derepress the PHO regulon while mutations that ablate IP8 synthesis are PHO hyper-repressive. Fission yeast Asp1, the principal agent of 1,5-IP8 dynamics, is a bifunctional enzyme composed of an N-terminal IPP kinase domain and a C-terminal IPP pyrophosphatase domain. Here we conducted a biochemical characterization and mutational analysis of the autonomous Asp1 kinase domain (aa 1-385). Reaction of Asp1 kinase with IP6 and ATP resulted in both IP6 phosphorylation to 1-IP7 and hydrolysis of the ATP γ-phosphate, with near-equal partitioning between productive 1-IP7 synthesis and unproductive ATP hydrolysis under optimal kinase conditions. By contrast, reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus ATP hydrolysis. Alanine scanning identified essential constituents of the active site. We deployed the Ala mutants to show that derepression of pho1 expression correlated with Asp1's kinase activity. In the case of full-length Asp1, the activity of the C-terminal pyrophosphatase domain stifled net phosphorylation of the 1-position during reaction of Asp1 with ATP and either IP6 or 5-IP7. We report that inorganic phosphate is a concentration-dependent enabler of net IP8 synthesis by full-length Asp1 in vitro, by virtue of its antagonism of IP8 turnover. IMPORTANCE Expression of the fission yeast phosphate regulon is sensitive to the intracellular level of the inositol pyrophosphate (IPP) signaling molecule 1,5-IP8. IP8 dynamics are determined by Asp1, a bifunctional enzyme comprising N-terminal IPP 1-kinase and C-terminal IPP 1-pyrophosphatase domains that catalyze IP8 synthesis and catabolism, respectively. Here, we interrogated the activities and specificities of the Asp1 kinase domain and full length Asp1. We find that reaction of Asp1 kinase with 5-IP7 is 22-fold faster than with IP6 and is strongly biased in favor of IP8 synthesis versus the significant unproductive ATP hydrolysis seen during its reaction with IP6. We report that full-length Asp1 catalyzes futile cycles of 1-phosphate phosphorylation by its kinase component and 1-pyrophosphate hydrolysis by its pyrophosphatase component that result in unproductive net consumption of the ATP substrate. Net synthesis of 1,5-IP8 is enabled by physiological concentrations of inorganic phosphate that selectively antagonize IP8 turnover.

Dataset Information

Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

Importance

Publications

Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

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