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

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 <i>asp1-H397A aps1</i>∆ and <i>siw14</i>∆ <i>aps1</i>∆ 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 <i>aps1</i>∆ <i>siw14</i>∆ cells. In the absence of all three pyrophosphatases, cells accrue high levels of 1,5-IP₈ and 1-IP₇ while IP₆ declines.