Tandem inactivation of inositol pyrophosphatases Asp1, Siw14, and Aps1 illuminates functional redundancies in inositol pyrophosphate catabolism in fission yeast

Abstract

Inositol pyrophosphates 5-IP₇, 1-IP₇, and 1,5-IP₈ are eukaryal signaling molecules that influence cell physiology, especially phosphate homeostasis. In fission yeast, 1,5-IP₈ and 1-IP₇ impact gene expression by acting as agonists of RNA 3'-processing and transcription termination. 1,5-IP₈ is synthesized by position-specific kinases Kcs1 and Asp1 that convert IP₆ to 5-IP₇ and 5-IP₇ to 1,5-IP₈, respectively. Inositol pyrophosphatase enzymes Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase) are agents of inositol pyrophosphate catabolism in fission yeast. Whereas Asp1, Siw14, and Aps1 are individually inessential, double pyrophosphatase mutants asp1-H397A aps1∆ and siw14∆ aps1∆ display severe growth defects caused by overzealous 3'-processing/termination. By applying CE-ESI-MS to profile the inositol pyrophosphate content of fission yeast mutants in which inositol pyrophosphate toxicity is genetically suppressed, we elucidated the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Asp1, which exclusively cleaves the 1-β-phosphate, and Aps1, which prefers to cleave the 1-β-phosphate, play essential overlapping roles in guarding against the accumulation of toxic levels of 1-IP₇. Aps1 and Siw14 together catabolize the inositol-5-pyrophosphates, and their simultaneous inactivation results in overaccumulation of 5-IP₇. Cells lacking all three pyrophosphatases amass high levels of 1,5-IP₈ and 1-IP₇, with concomitant depletion of IP₆. A genetic screen identified three missense mutations in the catalytic domain of Kcs1 kinase that suppressed inositol-1-pyrophosphate toxicosis. The screen also implicated the 3'-processing factor Swd22, the inositol pyrophosphate sensor Spx1, and the nuclear poly(A)-binding protein Nab2 as mediators of inositol-1-pyrophosphate toxicity.IMPORTANCEInositol pyrophosphates are key effectors of eukaryal cellular phosphate homeostasis. They are synthesized by kinases that add a β-phosphate to the 5- or 1-phosphate groups of IP₆ and catabolized by three classes of pyrophosphatases that hydrolyze the β-phosphates of 5-IP₇, 1-IP₇, or 1,5-IP₈. Whereas the fission yeast inositol pyrophosphatases-Asp1 (histidine acid phosphatase), Siw14 (cysteinyl phosphatase), and Aps1 (Nudix hydrolase)-are inessential for growth, Asp1/Aps1 and Aps1/Siw14 double mutations and Asp1/Siw14/Aps1 triple mutations elicit severe or lethal growth defects. By profiling the inositol pyrophosphate content of pyrophosphatase mutants in which this toxicity is genetically suppressed, we reveal the functional redundancies of the Asp1, Siw14, and Aps1 pyrophosphatases. Their synergies are manifested as excess accumulation of 1-IP₇ upon dual inactivation of Asp1 and Aps1 or an excess of 5-IP₇ in aps1∆ siw14∆ cells. In the absence of all three pyrophosphatases, cells accrue high levels of 1,5-IP₈ and 1-IP₇ while IP₆ declines.

Keywords: Schizosaccharomyces pombe; inositol pyrophosphates; pyrophosphatases.

Fig 1

Inositol pyrophosphate metabolism in fission yeast. The chemical structures of IP₆, 5-IP₇, 1,5-IP₈, and 1-IP₇ are shown, with “P” denoting phosphate. The positions of the myo-inositol ring are indicated for IP₆. The fission yeast enzymes that add (Kcs1 and Asp1-kinase) or remove (Siw14, Asp1-pyrophosphatase, Aps1) β-phosphate groups are indicated.

Fig 2

Mutational effects on inositol pyrophosphate levels as gauged by CE-ESI-MS. The amounts (pmol) of 1,5-IP₈, 5-IP₇, 1-IP₇, and IP₆ in equal volumes of extracts of the indicated fission yeast strains (from 10 A₆₀₀ units of cells) are plotted. The bar heights depict the mean of three individual biological replicates (denoted by dots) ±SEM.

Fig 3

Effect of inactivating all three inositol pyrophosphatases. (A) Serial fivefold dilutions of fission yeast strains (as specified on the left) were spot-tested for growth on YES agar at the indicated temperatures. (B) The amounts (pmol) of 1,5-IP₈, 1-IP₇, 5-IP₇, and IP₆ in equal volumes of extracts of the indicated fission yeast are plotted. The bar heights depict the mean of three individual biological replicates (denoted by dots) ±SEM.

Fig 4

SPX domain and RING domain mutations echo spx1∆ with respect to inositol pyrophosphate changes. The amounts (pmol) of 1,5-IP₈, 5-IP₇, 1-IP₇, and IP₆ in equal volumes of extracts of fission yeast strains—wild-type (WT), asp1-H397A aps1∆ spx1-Y26A/K30A/K34A, and asp1-H397A aps1∆ spx1-C374A/C377A—are plotted. The bar heights depict the mean of three individual biological replicates (denoted by dots) ± SEM. Note that the inositol pyrophosphate and IP₆ data reported in Fig. 4 and 3B are from the same experiment; consequently, the wild-type control values are the same in both figures.

Fig 5

Isolation of spontaneous suppressors of asp1-H397A aps1∆ tgp1∆ toxicosis. (A) Serial fivefold dilutions of fission yeast strains—wild-type (WT), asp1-H397A aps1∆ tgp1∆ (Hat), and eight independent Hat-suppressor strains (Hat-S series)—were spot-tested for growth on YES agar at the indicated temperatures. (B) The indicated strains were grown to A₆₀₀ of 0.5 to 0.8 in liquid culture in ePMGT medium at 30°C. Cells were then harvested, washed with water, and assayed for Pho1 acid phosphatase activity. (C) Whole-genome sequencing of the Hat-S strains revealed the indicated mutations.

Fig 6

Predicted role of Spx1 Phe393 in the Zn-binding RING domain. Stereo view of an AlphaFold 3 model of the Spx1 RING domain in complex with two zinc atoms (green spheres). The atomic contacts between the Cys and His side chains that tetrahedrally coordinate the Zn atoms are denoted by black dashed lines. Phe393, the residue mutated to Val in the Hat-S4 strain, is situated midway between the two Zn complexes. van der Waals contacts between Phe293 and nearby amino acids are denoted by green dashed lines.

Fig 7

Kcs1 mutations that suppress the inositol pyrophosphate toxicity cluster within the IP₆ kinase domain. (A) AlphaFold 3 model of the Kcs1 kinase domain in complex with ATP (stick model) and Mg²⁺ (green sphere). The amino acids that are mutated in the Hat-S suppressor strains colocalize within the region flanking the ATP site, demarcated by the box. (B) Stereo view of the predicted atomic interactions of the amino acids that are mutated in kcs1 alleles that suppress inositol pyrophosphate toxicosis.

Fig 8

nab2∆ suppresses asp1-H397A aps1∆ tgp1∆ toxicosis. (A) Serial fivefold dilutions of the indicated fission yeast strains were spot-tested for growth on YES agar at the indicated temperatures. (B) The indicated yeast strains were grown to A₆₀₀ of 0.5 to 0.8 in liquid culture in YES medium at 30°C. Cells were then harvested, washed with water, and assayed for Pho1 acid phosphatase activity.

Fig 9

Summary of changes in inositol pyrophosphate metabolism in cells lacking two or more inositol pyrophosphatases. (A) Cells lacking Asp1 and Aps1 pyrophosphatases. (B) Cells lacking Siw14 and Aps1 pyrophosphatases. (C) Cells lacking Asp1, Siw14, and Aps1 pyrophosphatases.